U.S. patent application number 15/057308 was filed with the patent office on 2016-06-23 for methods of inhibiting tumorigenesis in colon adenocarcinoma and compositions therefor.
This patent application is currently assigned to The Ohio State University Research Foundation. The applicant listed for this patent is The Government of the USA, as represented by the Secretary of the Department of Health and Human Se, The Ohio State University Research Foundation, The Government of the USA, as represented by the Secretary of the Department of Health and Human Se. Invention is credited to Carlo M. Croce, Curtis C. Harris, Aaron J. Schetter.
Application Number | 20160177305 15/057308 |
Document ID | / |
Family ID | 38923889 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160177305 |
Kind Code |
A1 |
Croce; Carlo M. ; et
al. |
June 23, 2016 |
Methods of Inhibiting Tumorigenesis in Colon Adenocarcinoma and
Compositions Therefor
Abstract
Described herein are methods and compositions for the treatment
of colon cancers.
Inventors: |
Croce; Carlo M.; (Columbus,
OH) ; Harris; Curtis C.; (Garrett Park, MD) ;
Schetter; Aaron J.; (Silver Spring, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Ohio State University Research Foundation
The Government of the USA, as represented by the Secretary of the
Department of Health and Human Se |
Columbus
Rockville |
OH
MD |
US
US |
|
|
Assignee: |
The Ohio State University Research
Foundation
Columbus
OH
The Government of the USA, as represented by the Secretary of
the Department of Health and Human Se
Rockville
MD
|
Family ID: |
38923889 |
Appl. No.: |
15/057308 |
Filed: |
March 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13296545 |
Nov 15, 2011 |
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15057308 |
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12373358 |
Feb 11, 2009 |
8084199 |
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PCT/US2007/015892 |
Jul 12, 2007 |
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13296545 |
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60807304 |
Jul 13, 2006 |
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60932736 |
Jun 1, 2007 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 2320/30 20130101;
C12Q 1/6886 20130101; C12Q 2600/118 20130101; A61P 1/04 20180101;
Y10T 436/143333 20150115; C12Q 1/6809 20130101; A61P 35/00
20180101; C12Q 2600/112 20130101; C12Q 2600/178 20130101; C12Q
2600/106 20130101; C12N 2310/141 20130101; C12Q 2525/207 20130101;
C12N 15/113 20130101; C12N 2310/113 20130101; C12Q 2600/136
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. PO1-CA76259 and No. PO1-CA81534 awarded by the National Cancer
Institute. The government has certain rights in the invention.
Claims
1. A method of inhibiting tumorigenesis in a subject who has, or is
suspected of having, a colon cancer-related disease in which at
least one miR gene product selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof, is down-regulated or up-regulated in the cancer cells of
the subject, relative to control cells, comprising: (1) when the at
least one miR gene product is down-regulated in the cancer cells,
administering to the subject an effective amount of at least one
isolated miR gene product selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof, such that tumorigenesis is inhibited in the subject; or
(2) when the at least one miR gene product is up-regulated in the
cancer cells, administering to the subject an effective amount of
at least one compound for inhibiting expression of the at least one
miR gene product selected from the group consisting of miR-20a,
miR-21, miR-106a, miR-181b, miR-203 and combinations thereof, such
that tumorigenesis is inhibited in the subject.
2. The method of claim 1, wherein the at least one isolated miR
gene product in step (1) and/or in step (2) is miR-21 or an
isolated variant or biologically-active fragment or functional
equivalent thereof, or an antibody that binds thereto.
3. A method of inhibiting tumorigenesis in a subject who has a
colon cancer, comprising: (1) determining the amount of at least
one miR gene product in cancer cells from the subject, relative to
control cells; and (2) altering the amount of miR gene product
expressed in the cancer cells by: (i) administering to the subject
an effective amount of at least one isolated miR gene product
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof, if the amount of the
miR gene product expressed in the cancer cells is less than the
amount of the miR gene product expressed in control cells; or (ii)
administering to the subject an effective amount of at least one
compound for inhibiting expression of the at least one miR gene
product, if the amount of the miR gene product expressed in the
cancer cells is greater than the amount of the miR gene product
expressed in control cells, such that tumorigenesis is inhibited in
the subject.
4. The method of claim 3, wherein the at least one isolated miR
gene product in step (i) is miR-21 or an isolated variant or
biologically-active fragment thereof.
5. The method of claim 3, wherein the at least one miR gene product
in step (ii) is selected from the group consisting of miR-20a,
miR-21, miR-106a, miR-181b, miR-203 and combinations thereof, or an
isolated variant or biologically-active fragment thereof.
6. A pharmaceutical composition for treating a colon cancer-related
disease, comprising: at least one miR gene product selected from
the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof; and, a
pharmaceutically-acceptable carrier.
7. The pharmaceutical composition of claim 6, wherein the at least
one miR gene product corresponds to a miR gene product that is up-
or down-regulated in cancer cells relative to suitable control
cells.
8. The pharmaceutical composition of claim 6, wherein the miR gene
product is miR-21.
9. The pharmaceutical composition of claim 6, wherein the colon
cancer-related disease is adenocarcinoma.
10. A pharmaceutical composition for treating a colon cancer,
comprising at least one miR expression-inhibition compound, and a
pharmaceutically-acceptable carrier, wherein the at least one miR
expression-inhibition compound is specific for a miR gene product
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
11. The pharmaceutical composition of claim 10, wherein the at
least one miR expression-inhibition compound is specific for a miR
gene product that is up- or down-regulated in cancer cells relative
to suitable control cells.
12. A method for treating, preventing, reversing or limiting the
severity of a colon cancer-related disease complication in an
individual in need thereof, comprising: administering to the
individual an agent that interferes with at least one colon
cancer-related disease response signaling pathway, in an amount
sufficient to interfere with such signaling, wherein the agent
comprises at least one miR gene product selected from the group
consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and
combinations thereof.
13. The method of claim 12, wherein the at least one miR gene
product is miR-21.
14. An agent that interferes with at least one colon cancer-related
disease response signaling pathway, for the manufacture of a
medicament for treating, preventing, reversing or limiting the
severity of a colon cancer-related disease complication in an
individual, wherein the agent comprises at least one miR gene
product selected from the group consisting of miR-20a, miR-21,
miR-106a, miR-181b, miR-203 and combinations thereof.
15. The agent of claim 14, wherein the at least one miR gene
product is miR-21.
16. A method of treating, preventing, reversing or limiting the
severity of a colon cancer-related disease complication in an
individual in need thereof, comprising administering to the
individual an agent that interferes with at least one colon
cancer-related disease response cascade, wherein the agent
comprises at least one miR gene product selected from the group
consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and
combinations thereof.
17. The method of claim 16, wherein the at least one miR gene
product is miR-21.
18. An agent that interferes with at least one colon cancer-related
disease response cascade, for the manufacture of a medicament for
treating, preventing, reversing or limiting the severity of a colon
cancer-related disease complication in an individual, wherein the
agent comprises at least one miR gene product selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof.
19. The agent of claim 18, wherein the at least one miR gene
product is miR-21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 13/296,545 filed Nov. 15, 2011, now abandoned, which is a
divisional application of Ser. No. 12/373,358 filed Feb. 11, 2009,
now U.S. Pat. No. 8,084,199 issued Dec. 27, 2011, which claims
priority to PCT/US2007/015892 filed Jul. 12, 2007, which the
benefit of U.S. Provisional Application Ser. No. 60/807,304 filed
Jul. 13, 2006 and Ser. No. 60/932,736 filed Jun. 1, 2007, the
disclosures of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Colon adenocarcinoma is a major cause of cancer mortality
worldwide. Colorectal cancer is the third most common and second
leading cause of cancer death in the United States. Sporadic colon
adenocarcinomas initiate as adenomas and evolve through a
progression of molecular, cellular and histologic changes. While
five-year mortality rates have modestly declined over the last
three decades, there is still a need to identify new prognostic
biomarkers and therapeutic targets for this disease. Currently,
chemotherapy has significant therapeutic value but surgery is the
only curative form of treatment.
[0004] Ideal therapeutic targets should be causally associated with
disease and amenable to designing therapeutic interventions;
whereas ideal biomarkers should be easy to measure and have strong
associations with clinical outcomes. MicroRNAs could match both
criteria.
[0005] MicroRNAs are 18-25 nucleotide, non-coding RNA molecules
that regulate the translation of many genes. Since their discovery,
they have been found to regulate a variety of cellular processes
including apoptosis, differentiation and cell proliferation.
MicroRNAs may also have a causal role in carcinogenesis. MicroRNA
expression levels are altered in most tumor types, including colon
tumors. The microRNAs miR-15 and miR-16a are deleted or
downregulated in the majority of chronic lymphocytic leukemias.
Experimental manipulation of specific microRNAs modulates tumor
development in mouse model systems. The prognostic potential of
microRNAs has also been demonstrated for chronic lymphocytic
leukemia, lung cancer.sup.8 and neuroblastomas.
[0006] Aberrant microRNAs expression may be causal to
carcinogenesis, inhibiting specific microRNAs may have therapeutic
implications. Modified antisense oligonucleotides can be designed
to specifically inhibit microRNA function. Antagomirs are one type
of antisense oligonucleotide that has proven effective at
inhibiting microRNA function in vivo in mice.
SUMMARY OF THE INVENTION
[0007] In one broad aspect, there is provided herein a method of
diagnosing whether a subject has, is at risk for developing, or has
a decrease survival prognosis for, a colon cancer-related disease.
The method includes measuring the level of at least one miR gene
product in a test sample from the subject, wherein an alteration in
the level of the miR gene product in the test sample, relative to
the level of a corresponding miR gene product in a control sample,
is indicative of the subject either having, or being at risk for
developing, the colon cancer-related disease. In a particular
aspect, the at least one miR gene product is selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof. In one embodiment, the one miR gene
product is miR-21.
[0008] In another broad aspect, there is provided herein a method
of testing for at least an initiation of, predisposition to, or
decreased survival prognosis for, a colon cancer-related disease
response, which comprises:
[0009] (1) determining an expression level of at least one marker
in a sample from a test subject; the at least one marker including
at least one miR gene product selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof;
[0010] (2) comparing the expression level determined in step (1)
with a control expression level of the marker in a sample from a
healthy subject; and
[0011] (3) judging the subject to have a colon cancer-related
disease when the result of the comparison in step (2) indicates
that: i) the expression level of the at least marker in the test
subject is higher than that in the control, or ii) the expression
level of the at least one marker in the test subject is lower than
that in the control.
[0012] The sample can comprise one or more of tissue, blood,
plasma, serum, urine, and feces. Also, all method steps can be
performed in vitro.
[0013] In another broad aspect, there is provided herein a method
of diagnosing whether a subject has, is at risk for developing, or
has a decrease survival prognosis for, a colon cancer-related
disease, comprising:
[0014] (1) reverse transcribing RNA from a test sample obtained
from the subject to provide a set of target
oligodeoxynucleotides;
[0015] (2) hybridizing the target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides to
provide a hybridization profile for the test sample; and
[0016] (3) comparing the test sample hybridization profile to a
hybridization profile generated from a control sample, wherein an
alteration in the signal of at least one miRNA is indicative of the
subject either having, being at risk for developing, or having a
decreased survival prognosis for, a colon cancer-related
disease.
[0017] In a particular aspect, the signal of at least one miRNA,
relative to the signal generated from the control sample, is up- or
down-regulated. Also, the microarray can comprise miRNA-specific
probe oligonucleotides for one or more miRNAs selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof.
[0018] In another broad aspect, there is provided herein a method
of inhibiting tumorigenesis in a subject who has, or is suspected
of having, a colon cancer-related disease in which at least one miR
gene product selected from the group consisting of miR-20a, miR-21,
miR-106a, miR-181b, miR-203 and combinations thereof, is
down-regulated or up-regulated in the cancer cells of the subject,
relative to control cells, comprising:
[0019] (1) when the at least one miR gene product is down-regulated
in the cancer cells, administering to the subject an effective
amount of at least one isolated miR gene product selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof, such that tumorigenesis is inhibited in
the subject; or
[0020] (2) when the at least one miR gene product is up-regulated
in the cancer cells, administering to the subject an effective
amount of at least one compound for inhibiting expression of the at
least one miR gene product selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof, such that tumorigenesis is inhibited in the subject.
[0021] In a particular aspect, at least one isolated miR gene
product in step (1) and/or in step (2) is miR-21 or an isolated
variant or biologically-active fragment or functional equivalent
thereof, or an antibody that binds thereto.
[0022] In another broad aspect, there is provided herein a method
of inhibiting tumorigenesis in a subject who has a colon cancer,
comprising:
[0023] (1) determining the amount of at least one miR gene product
in cancer cells from the subject, relative to control cells;
and
[0024] (2) altering the amount of miR gene product expressed in the
cancer cells by: [0025] (i) administering to the subject an
effective amount of at least one isolated miR gene product selected
from the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof, if the amount of the miR gene
product expressed in the cancer cells is less than the amount of
the miR gene product expressed in control cells; or [0026] (ii)
administering to the subject an effective amount of at least one
compound for inhibiting expression of the at least one miR gene
product, if the amount of the miR gene product expressed in the
cancer cells is greater than the amount of the miR gene product
expressed in control cells,
[0027] such that tumorigenesis is inhibited in the subject.
[0028] In a particular aspect, the at least one isolated miR gene
product in step (i) is miR-21 or an isolated variant or
biologically-active fragment thereof. Also, in certain embodiments,
the at least one miR gene product in step (ii) is selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof, or an isolated variant or
biologically-active fragment thereof.
[0029] In another broad aspect, there is provided herein a method
of identifying an inhibitor of tumorigenesis, comprising providing
a test agent to a cell and measuring the level of at least one miR
gene product associated with an altered expression levels in a
colon cancer-related disease, wherein an increase or decrease in
the level of the miR gene product in the cell, relative to a
suitable control cell, is indicative of the test agent being an
inhibitor of tumorigenesis.
[0030] In another broad aspect, there is provided herein a method
of identifying an inhibitor of tumorigenesis, comprising providing
a test agent to a cell and measuring the level of at least one miR
gene product associated with an altered expression level in a colon
cancer-related disease, wherein a decrease in the level of the miR
gene product in the cell, relative to a suitable control cell, is
indicative of the test agent being an inhibitor of
tumorigenesis.
[0031] In another broad aspect, there is provided herein a marker
for assessing one or more metabolic pathways that contribute to at
least one of initiation, progression, severity, pathology,
aggressiveness, grade, activity, disability, mortality, morbidity,
disease sub-classification or other underlying pathogenic or
pathological feature of at least one colon cancer-related disease,
wherein the marker comprises one or more miR gene products selected
from the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof.
[0032] In another broad aspect, there is provided herein a
composition comprising one or more of the markers described
herein.
[0033] In another broad aspect, there is provided herein a method
of identifying a potential for the initiation or development of at
least one colon cancer-related disease in a subject, the method
providing measuring one or more of the markers described herein. In
certain embodiments, one or more markers are present in an isolated
sample and all method steps are performed in vitro.
[0034] In another broad aspect, there is provided herein a reagent
for testing for a colon cancer-related disease, wherein the reagent
comprises a polynucleotide comprising the nucleotide sequence of at
least one marker described herein or a nucleotide sequence
complementary to the nucleotide sequence of the marker.
[0035] In another broad aspect, there is provided herein a reagent
for testing for a colon cancer-related disease, wherein the reagent
comprises an antibody that recognizes a protein encoded by at least
one marker described herein.
[0036] In another broad aspect, there is provided herein a DNA chip
for testing for a colon cancer-related disease, on which a probe
has been immobilized to assay at least one marker described
herein.
[0037] In another broad aspect, there is provided herein a method
of assessing the effectiveness of a therapy to prevent, diagnose
and/or treat at least one colon cancer-related disease
comprising:
[0038] 1) subjecting an animal to a therapy whose effectiveness is
being assessed, and
[0039] 2) determining the level of effectiveness of the treatment
being tested in treating or preventing the colon cancer-related
disease by evaluating at least one marker described herein.
[0040] In certain embodiments, the candidate therapeutic agent
comprises one or more of: pharmaceutical compositions,
nutraceutical compositions, and homeopathic compositions. Also, the
therapy being assessed can be for use in a human subject. In
certain embodiments, the method is not a method of treatment of the
human or animal body by surgery or therapy.
[0041] In another broad aspect, there is provided herein a method
of assessing the potential of at least one material for an ability
to initiate a colon cancer-related disease response in an animal
model, the method providing:
[0042] 1) measuring one or more of up- or down-regulated markers
described herein after exposure of the animal to one or more
materials in amounts sufficient to initiate a colon cancer-related
disease response in the animal; and
[0043] 2) determining whether at least one of the up- or
down-regulated markers has the ability to initiate a colon
cancer-related disease response.
[0044] In another broad aspect, there is provided herein a
pharmaceutical composition for treating a colon cancer-related
disease, comprising: at least one miR gene product selected from
the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof; and, a
pharmaceutically-acceptable carrier.
[0045] In another broad aspect, there is provided herein a
pharmaceutical composition for treating a colon cancer, comprising
at least one miR expression-inhibition compound and a
pharmaceutically-acceptable carrier, wherein the at least one miR
expression-inhibition compound is specific for a miR gene product
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
[0046] In another broad aspect, there is provided herein an article
of manufacture comprising: at least one capture reagent that binds
to a marker for a colon cancer-related disease selected from at
least one of the markers described herein.
[0047] In another broad aspect, there is provided herein a kit for
screening for a candidate compound for a therapeutic agent to treat
a colon cancer-related disease, wherein the kit comprises: one or
more reagents of at least one marker described herein, and a cell
expressing at least one marker. In certain embodiments, the
presence of the marker is detected using a reagent comprising an
antibody or an antibody fragment which specifically binds with at
least one marker. Also, in certain embodiments, the reagent is
labeled, radio-labeled, or biotin-labeled, and/or the antibody or
antibody fragment is radio-labeled, chromophore-labeled,
fluorophore-labeled, or enzyme-labeled. In a particular embodiment,
the kit further includes a container comprising at least one of the
markers. Also, the reagent can comprise one or more of: an
antibody, a probe to which the reagent is attached or is
attachable, and an immobilized metal chelate.
[0048] In another broad aspect, there is provided herein a
screening test for a colon cancer-related disease comprising:
[0049] contacting one or more of the markers with a substrate for
such marker and with a test agent, and
[0050] determining whether the test agent modulates the activity of
the marker.
[0051] In certain embodiments, all method steps can be performed in
vitro.
[0052] In another broad aspect, there is provided herein a
microarray for predicting the presence of a colon cancer-related
disease in a subject comprising an antibody directed to at least
one marker.
[0053] In another broad aspect, there is provided herein methods,
compositions and the like, where a level of expression of the
marker is assessed by detecting the presence of a transcribed
polynucleotide or portion thereof, wherein the transcribed
polynucleotide comprises a coding region of the marker. Also, the
sample can be a colon cancer-associated body fluid or tissue. In a
particular embodiment, the sample comprises cells obtained from the
patient.
[0054] In another broad aspect, there is provided herein a method
for treating, preventing, reversing or limiting the severity of a
colon cancer-related disease complication in an individual in need
thereof, comprising:
[0055] administering to the individual an agent that interferes
with at least one colon cancer-related disease response signaling
pathway, in an amount sufficient to interfere with such signaling,
wherein the agent comprises at least one miR gene product selected
from the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof.
[0056] In another broad aspect, there is provided herein the use of
an agent that interferes with at least one colon cancer-related
disease response signaling pathway, for the manufacture of a
medicament for treating, preventing, reversing or limiting the
severity of a colon cancer-related disease complication in an
individual, wherein the agent comprises at least one miR gene
product selected from the group consisting of miR-20a, miR-21,
miR-106a, miR-181b, miR-203 and combinations thereof.
[0057] In another broad aspect, there is provided herein a method
of treating, preventing, reversing or limiting the severity of a
colon cancer-related disease complication in an individual in need
thereof, comprising administering to the individual an agent that
interferes with at least one colon cancer-related disease response
cascade, wherein the agent comprises at least one miR gene product
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
[0058] In another broad aspect, there is provided herein the use of
an agent that interferes with at least one colon cancer-related
disease response cascade, for the manufacture of a medicament for
treating, preventing, reversing or limiting the severity of a colon
cancer-related disease complication in an individual, wherein the
agent comprises at least one miR gene product selected from the
group consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203
and combinations thereof.
[0059] In another broad aspect, there is provided herein a
computer-readable medium comprising a database having a plurality
of digitally-encoded reference profiles, wherein at least a first
reference profile represents a level of at least a first marker in
one or more samples from one or more subjects exhibiting an indicia
of a colon cancer-related disease response,
[0060] wherein the marker comprises one or more miR gene products
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
[0061] In certain embodiments, the computer readable medium
includes at least a second reference profile that represents a
level of at least a second marker in one or more samples from one
or more subjects exhibiting indicia of a colon cancer-related
disease response; or subjects having a colon cancer-related
disease.
[0062] In another broad aspect, there is provided herein a computer
system for determining whether a subject has, is predisposed to
having, or has a poor survival prognosis for, a colon
cancer-related disease, comprising the database described herein,
and a server comprising a computer-executable code for causing the
computer to receive a profile of a subject, identify from the
database a matching reference profile that is diagnostically
relevant to the subject profile, and generate an indication of
whether the subject has, or is predisposed to having, a colon
cancer-related disease.
[0063] In another broad aspect, there is provided herein a
computer-assisted method for evaluating the presence, absence,
nature or extent of a colon cancer-related disease in a subject,
comprising:
[0064] 1) providing a computer comprising a model or algorithm for
classifying data from a sample obtained from the subject, wherein
the classification includes analyzing the data for the presence,
absence or amount of at least one marker, wherein the marker
comprises one or more miR gene products selected from the group
consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and
combinations thereof;
[0065] 2) inputting data from the biological sample obtained from
the subject; and,
[0066] 3) classifying the biological sample to indicate the
presence, absence, nature or extent of a colon cancer-related
disease.
[0067] In another broad aspect, at least one miR gene product and
combinations thereof includes isolated variants or
biologically-active fragments.
[0068] In another broad aspect, there is provided herein an animal
model for colon cancer wherein at least one of the following
biological or chemical processes occurs in the animal model up- or
down regulation of one or more miR gene products is selected from
the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof. In certain embodiments, the
animal model is a nonhuman vertebrate. In particular embodiments,
the animal model is a mouse, rat, rabbit, or primate.
[0069] In another broad aspect, there are provided methods of
determining chemotherapy effectiveness in patients with colon
adenocarcinoma, comprising: measuring the level of at least one
miR-21 gene product in a test sample from a patient with colon
adenocarcinoma, determining poor chemotherapy effectiveness in the
patient with colon adenocarcinoma when an increase in at least the
level of the miR-21 gene product in the test sample, relative to
the level of a corresponding miR-21 gene product in a control
sample, is present.
[0070] In another broad aspect, there are provided methods of
testing for poor response to adjuvant chemotherapy in a patient
with colon adenocarcinoma, which comprises:
[0071] (1) determining an expression level of at least one marker
in a sample from a patient having colon adenocarcinoma; the at
least one marker including at least one miR-21 gene product;
[0072] (2) comparing the expression level determined in step (1)
with a control expression level of the marker in a sample from a
subject who does not have colon adenocarcinoma; and
[0073] (3) judging the patient as having poor response to adjuvant
chemotherapy when the result of the comparison in step (2)
indicates that:
[0074] the expression level of at least miR-21 in the test subject
is higher than that in the control.
[0075] The present invention therefore provides methods as
described, wherein the sample comprises one or more of tissue;
tumor tissue; blood, plasma; serum, urine; and feces.
[0076] The present invention therefore provides methods as
described, wherein all method steps are performed in vitro.
[0077] In another broad aspect, there are provided methods of
diagnosing whether a subject has poor response to adjuvant
chemotherapy in a patient with colon adenocarcinoma,
comprising:
[0078] (1) reverse transcribing RNA from a test sample obtained
from a patient with adenocarcimoa to provide a set of target
oligodeoxynucleotides;
[0079] (2) hybridizing the target oligodeoxynucleotides to a
microarray comprising miR-21 specific probe oligonucleotides to
provide a hybridization profile for the test sample; and
[0080] (3) comparing the test sample hybridization profile to a
hybridization profile generated from a control sample,
[0081] wherein an increase in the signal of the miR-21 is
indicative of the subject having poor response to adjuvant
chemotherapy in the patient with colon adenocarcinoma.
[0082] The present invention therefore provides methods as
described, wherein a level of expression of miR-21 gene product is
assessed by detecting the presence of a transcribed polynucleotide
or portion thereof, wherein the transcribed polynucleotide
comprises a coding region of miR-21 gene product.
[0083] The present invention therefore provides methods as
described, wherein the sample is a colon cancer-associated body
fluid or tissue.
[0084] The present invention therefore provides methods as
described, wherein the sample comprises cells obtained from the
patient.
[0085] The present invention therefore provides methods as
described, wherein the at least one miR-21 gene product includes
isolated variants or biologically-active fragments thereof.
[0086] The present invention therefore provides methods as
described, wherein the chemotherapy is selected from:
5-fluorouracil; tegafur with uracil; fluorouracil drug;
fluorouracil drug-levamisole regimen; and fluorouracil
drug-leucovorin regimen.
[0087] Various objects and advantages of this invention will become
apparent to those skilled in the art from the following detailed
description of the preferred embodiment, when read in light of the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0089] FIGS. 1a-1g: MiR-21 is expressed at higher levels in colon
adenocarcinomas with increasing expression in more advanced
tumors.
[0090] (FIG. 1a) In situ hybridization for miR-21 was optimized to
distinguish high and low expression of miR-21. Colonic epithelial
cells in human tumor (T) express higher levels of miR-21 compared
to adjacent nontumorous tissue (N). (FIG. 1c) Nuclei and cytoplasm
of colonic epithelial cells in tumor tissue express significant
amounts of miR-21 in tumor tissue, at high magnification. (FIG. 1e)
Non-tumor tissue shows no significant expression of miR-21 at the
same magnification.
[0091] (FIG. 1b, FIG. 1d, FIG. 1f) The scramble control probe shows
no significant staining at low or high magnification in serial
sections of tumor and non-tumor tissue, as expected. Scale bars
(FIGS. 1c-f) indicate 500 .mu.M (g) miR-21 is expressed at higher
levels in more advanced tumors. Dot plots represent miR-21 relative
Ct values (from quantitative RT-PCR) for adenoma and tumor
expression levels that have been normalized to paired non-adenoma
or nontumorous tissue, respectively. Tissue types have been ordered
from adenoma to stage I-IV tumors. Bars indicate median value.
There is a significant trend that more advanced tumors have higher
expression of miR-21 (nonparametric test for trend across ordered
groups).
[0092] FIG. 2: miR-21 is expressed at higher levels in more
advanced tumors. MicroRNA microarrays were used to measure miR-21
expression levels. Dot plots represent miR-21 log.sub.2
(tumor/nontumor ratios) as calculated from microRNA microarrays
from the original cohort. The probe hsa-miR-21-prec 17No 1 from the
microarray was used to measure miR-21 expression. Tissues with
undetectable expression of miR-21 based on microarray data were
excluded. Tissue types have been ordered from TNM stage I to stage
IV tumors. Bars indicate median value. There is a significant trend
that more advanced tumors have higher expression of miR-21 (p=0.04;
nonparametric test for trend across ordered groups).
[0093] FIGS. 3a and 3b: High miR-21 expression in tumors predicts a
poor survival in subjects with typical adenocarcinoma histology in
both independent cohorts. This analysis excludes subjects with
either mucinous adenocarcinoma or adenosquamous carcinoma
histology.
[0094] (FIG. 3a) MicroRNA microarrays were used in the Maryland
test cohort to measure microRNA expression levels of tumors and
nontumorous tissues. Tissues with undetectable expression of miR-21
based on microarray data were excluded. High miR-21 expression was
classified based on highest tertile. Red lines indicate individuals
with high expression while green lines correspond to low
expression. For nontumorous tissue, 24/69 tissues were classified
as high while 26/72 tumors were classified as high. High miR-21
expression in tumors (right) is associated with poor survival while
it is not associated in nontumorous tissue.
[0095] (FIG. 3b) Validation of the association with high miR-21
expression in tumors and poor prognosis in an independent cohort.
Expression levels of miR-21 were measured by quantitative RT-PCR.
High expression is based on the highest tertile. 35/103 nontumorous
tissues were classified as high and 34/103 tumor tissues were
classified as high. P-values are log rank p-values from
Kaplan-Meier analysis. X's on all lines indicate the time at which
an individual was censored.
[0096] FIGS. 4a and 4b: High miR-21 expression in tumors predicts
poor survival in both independent cohorts. This analysis includes
all subjects regardless of adenocarcinoma histology.
[0097] (FIG. 4a) MicroRNA microarrays were used in the Maryland
test cohort to measure microRNA expression levels of tumors and
nontumorous tissues. Tissues with undetectable expression of miR-21
based on microarray data were excluded. High miR21 expression was
classified based on highest tertile. Red lines indicate individuals
with high expression while green lines correspond to low
expression. For nontumorous tissue, 26/74 tissues were classified
as high while 28/79 tumors were classified as high. High miR-21
expression in tumors (right) is associated with poor survival while
it is not associated in nontumorous tissue.
[0098] (FIG. 4b) Validation of the association with high miR-21
expression in tumors and poor prognosis in an independent cohort.
Expression levels of miR-21 were measured by quantitative RT-PCR.
High expression is based on the highest tertile. 37/111 nontumorous
tissues were classified as high and 37/111 tumor tissues were
classified as high. All p-values are log rank p-values from
Kaplan-Meier analysis. X's on all lines indicate the time at which
an individual was censored.
[0099] FIGS. 5a, 5b and 5c: High miR-21 expression is associated
with a poor response to adjuvant chemotherapy for cases with
conventional adenocarcinoma histology. This analysis includes
subjects from the validation cohort, excluding subjects with
mucinous adenocarcinoma or adenosquamous carcinoma histologies.
[0100] (FIG. 5a) Comparison of survival rates for TNM stage II/III
subjects with conventional adenocarcinoma histology by miR-21
expression levels and receipt of adjuvant chemotherapy. For the 77
stage II/III subjects, 25 were classified as low miR-21 receiving
therapy, 28 as low miR-21 and not receiving therapy, 11 as high
miR-21 receiving therapy, and 13 as high miR-21 and not receiving
therapy. For stage II/III subjects who received adjuvant
chemotherapy, high miR-21 expression in tumors is associated with a
poor survival (p=0.03).
[0101] (FIG. 5b) Comparison of TNM stage II subjects with
conventional adenocarcinoma histology. For the 33 stage II
subjects, 8 were classified as low miR-21 receiving therapy, 15 as
low miR-21 and not receiving therapy, 3 as high miR-21 receiving
therapy, and 7 as high miR-21 and not receiving therapy. All stage
II subjects who received chemotherapy survived for the duration of
this study.
[0102] (FIG. 5c) Comparison of TNM stage III subjects with
conventional adenocarcinoma histology. For the 44 stage III
subjects, 17 were classified as low miR-21 receiving therapy, 13 as
low miR-21 and not receiving therapy, 8 as high miR-21 receiving
therapy, and 6 as high miR-21 and not receiving therapy. For stage
III subjects who received adjuvant chemotherapy, high miR-21
expression in tumors is associated with a poor survival (p=0.02).
X's on all lines indicate the time at which an individual was
censored.
[0103] FIGS. 6a, 6b and 6c: Combined analysis of Maryland test
cohort and Hong Kong validation cohort examining associations
between miR-21 expression in tumors and receipt of adjuvant
chemotherapy with prognosis. This analysis includes all TNM stage
II/III subjects from both cohorts. Excluded were individuals with
mucinous adenocarcinoma or adenosquamous carcinoma histologies. The
left column includes Kaplan-Meier plots analyzing the association
between receipt of adjuvant therapy and prognosis. The center
column includes analysis of the association between high miR-21
expression in tumors and prognosis, and the right column subdivides
individuals based on both chemotherapy and miR-21 expression
status.
[0104] (FIG. 6a) All TNM stage II/III subjects. For the 119 stage
II/III subjects, 40 were classified as low miR-21 receiving
therapy, 41 as low miR-21 and not receiving therapy, 16 as high
miR-21 receiving therapy, and 22 as high miR-21 and not receiving
therapy. High miR-21 expression is associated with a poor survival
for those who receive chemotherapy (p=0.003) as well as those who
do not receive therapy (p=0.04).
[0105] (FIG. 6b) All TNM stage II subjects. For the 52 stage II/III
subjects, 10 were classified as low miR-21 receiving therapy, 25 as
low miR-21 and not receiving therapy, 4 as high miR-21 receiving
therapy, and 13 as high miR-21 and not receiving therapy.
Associations between high miR-21 expression and prognosis was not
statistically significant in individuals who received chemotherapy
(p=0.11) or those who did not receive chemotherapy (p=0.06).
[0106] (FIG. 6c) All TNM stage III subjects. For the 67 stage III
subjects, 30 were classified as low miR-21 receiving therapy, 16 as
low miR-21 and not receiving therapy, 12 as high miR-21 receiving
therapy, and 9 as high miR-21 and not receiving therapy. High
miR-21 expression is significantly associated with poor survival in
stage III subjects who received chemotherapy (p=0.007), but not in
subjects who did not receive chemotherapy (p=0.30). X's on all
lines indicate the time at which an individual was censored.
[0107] FIGS. 7a-7c. Global miRNA profiles are associated with
clinical TNM staging and survival prognosis. Hierarchical
clustering of miRNA TIN ratios resulted in forming two groups
arbitrarily named group A and group B. The resulting HEAT map and
cluster assignments are shown in FIG. 7a. These two groups were
composed of individuals with significantly different survival
prognoses for TNM staging with group B individuals more likely to
be diagnosed as either stage III or IV compared to group A
individuals (FIG. 7b). Kaplan-Meier analysis shows that Group B
individuals also have a worse survival prognosis (FIG. 7c).
[0108] FIGS. 8a-8i. TIN ratios of individual miRNAs are predictive
of survival prognosis. Displayed here are graphs showing TIN ratios
by TNM staging (left) and Kaplan-Meier analysis (right) for each of
these 9 miRNAs. The Y axis (TIN ratio by TNM staging graphs)
indicates the log(2) transformed TIN ratio for each individual
while the Y axis groups individuals by TNM staging (I, II, III or
IV). The significance values shown are the result of a
nonparametric test for trend of average TIN ratio values across
individuals grouped by staging. Kaplan-Meier plots include all
individuals with TIN ratio data for that particular miRNA. We found
that TIN ratios were associated with both clinical staging and
survival prognosis.
[0109] FIGS. 9a and 9b. A miRNA signature of 9 miRNAs predicts risk
of dying of colon cancer. TIN ratios of miR-21, miR-106a, miR181b,
miR-16b, miR-203, let-7g, miR-29a, miR-103-2 and miR-10a were each
shown to be predictive of colon cancer prognosis. Hierarchical
clustering of TIN ratios of these 9 miRNAs resulted in dividing
individuals into two groups (1A) with significantly different
survival prognoses (1B). Group B individuals were at a
significantly higher risk for dying from colon cancer than group A.
Individuals were excluded from this analysis if they were missing
greater than 2 of the 9 TIN ratios making up the miRNA
signature.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0110] In one broad aspect, there is provided herein the
identification of particular microRNAs whose expression is altered
in cancer cells associated with different colon cancers, relative
to normal control cells.
[0111] As used herein interchangeably, a "miR gene product,"
"microRNA," "miR," or "miRNA" refers to the unprocessed (e.g.,
precursor) or processed (e.g., mature) RNA transcript from a miR
gene. As the miR gene products are not translated into protein, the
term "miR gene products" does not include proteins. The unprocessed
miR gene transcript is also called a "miR precursor" or "miR prec"
and typically comprises an RNA transcript of about 70-100
nucleotides in length. The miR precursor can be processed by
digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III
(e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA
molecule. This active 19-25 nucleotide RNA molecule is also called
the "processed" miR gene transcript or "mature" miRNA.
[0112] The active 19-25 nucleotide RNA molecule can be obtained
from the miR precursor through natural processing routes (e.g.,
using intact cells or cell lysates) or by synthetic processing
routes (e.g., using isolated processing enzymes, such as isolated
Dicer, Argonaut, or RNAse III). It is understood that the active
19-25 nucleotide RNA molecule can also be produced directly by
biological or chemical synthesis, without having been processed
from the miR precursor. When a microRNA is referred to herein by
name, the name corresponds to both the precursor and mature forms,
unless otherwise indicated.
[0113] In one aspect, there is provided herein methods of
diagnosing whether a subject has, or is at risk for developing, a
colon cancer, comprising measuring the level of at least one miR
gene product in a test sample from the subject and comparing the
level of the miR gene product in the test sample to the level of a
corresponding miR gene product in a control sample. As used herein,
a "subject" can be any mammal that has, or is suspected of having,
a solid cancer. In a preferred embodiment, the subject is a human
who has, or is suspected of having, a colon cancer.
[0114] In one embodiment, the at least one miR gene product
measured in the test sample is selected from the group consisting
of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof. In a particular embodiment, the miR gene product is
miR-21.
[0115] The colon cancer-related disease can be any disorder or
cancer that arises from the colon tissues. Such cancers are
typically associated with the formation and/or presence of tumor
masses and can be, for example, adenocarcinomas.
[0116] In one embodiment, the colon is an adenocarcinoma and the at
least one miR gene product measured in the test sample is selected
from the group consisting of miR-20a, miR-21, miR-106a, miR-181b,
miR-203 and combinations thereof.
[0117] In a further embodiment, the at least one miR gene product
measured in the test sample is miR-21.
[0118] The level of at least one miR gene product can be measured
in a biological sample (e.g., cells, tissues) obtained from the
subject. For example, a tissue sample (e.g., from a tumor) can be
removed from a subject suspected of having a colon cancer-related
disease by conventional biopsy techniques. In another embodiment, a
blood sample can be removed from the subject, and blood cells
(e.g., white blood cells) can be isolated for DNA extraction by
standard techniques. The blood or tissue sample is preferably
obtained from the subject prior to initiation of radiotherapy,
chemotherapy or other therapeutic treatment. A corresponding
control tissue or blood sample can be obtained from unaffected
tissues of the subject, from a normal human individual or
population of normal individuals, or from cultured cells
corresponding to the majority of cells in the subject's sample. The
control tissue or blood sample is then processed along with the
sample from the subject, so that the levels of miR gene product
produced from a given miR gene in cells from the subject's sample
can be compared to the corresponding miR gene product levels from
cells of the control sample. A reference miR expression standard
for the biological sample can also be used as a control.
[0119] An alteration (e.g., an increase or decrease) in the level
of a miR gene product in the sample obtained from the subject,
relative to the level of a corresponding miR gene product in a
control sample, is indicative of the presence of a colon
cancer-related disease in the subject.
[0120] In one embodiment, the level of the at least one miR gene
product in the test sample is greater than the level of the
corresponding miR gene product in the control sample (i.e.,
expression of the miR gene product is "up-regulated"). As used
herein, expression of a miR gene product is "up-regulated" when the
amount of miR gene product in a cell or tissue sample from a
subject is greater than the amount of the same gene product in a
control cell or tissue sample.
[0121] In another embodiment, the level of the at least one miR
gene product in the test sample is less than the level of the
corresponding miR gene product in the control sample (i.e.,
expression of the miR gene product is "down-regulated"). As used
herein, expression of a miR gene is "down-regulated" when the
amount of miR gene product produced from that gene in a cell or
tissue sample from a subject is less than the amount produced from
the same gene in a control cell or tissue sample.
[0122] The relative miR gene expression in the control and normal
samples can be determined with respect to one or more RNA
expression standards. The standards can comprise, for example, a
zero miR gene expression level, the miR gene expression level in a
standard cell line, the miR gene expression level in unaffected
tissues of the subject, or the average level of miR gene expression
previously obtained for a population of normal human controls.
[0123] The level of a miR gene product in a sample can be measured
using any technique that is suitable for detecting RNA expression
levels in a biological sample. Suitable techniques (e.g., Northern
blot analysis, RT-PCR, in situ hybridization) for determining RNA
expression levels in a biological sample (e.g., cells, tissues) are
well known to those of skill in the art. In a particular
embodiment, the level of at least one miR gene product is detected
using Northern blot analysis. For example, total cellular RNA can
be purified from cells by homogenization in the presence of nucleic
acid extraction buffer, followed by centrifugation. Nucleic acids
are precipitated, and DNA is removed by treatment with DNase and
precipitation. The RNA molecules are then separated by gel
electrophoresis on agarose gels according to standard techniques,
and transferred to nitrocellulose filters. The RNA is then
immobilized on the filters by heating. Detection and quantification
of specific RNA is accomplished using appropriately labeled DNA or
RNA probes complementary to the RNA in question. See, for example,
Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds.,
2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7,
the entire disclosure of which is incorporated by reference.
[0124] Suitable probes for Northern blot hybridization of a given
miR gene product can be produced from the nucleic acid sequences
and include, but are not limited to, probes having at least about
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarity
to a miR gene product of interest. Methods for preparation of
labeled DNA and RNA probes, and the conditions for hybridization
thereof to target nucleotide sequences, are described in Molecular
Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd
edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and
11, the disclosures of which are incorporated herein by
reference.
[0125] In one non-limiting example, the nucleic acid probe can be
labeled with, e.g., a radionuclide, such as .sup.3H, .sup.32P,
.sup.33P, .sup.14C, or .sup.35S; a heavy metal; a ligand capable of
functioning as a specific binding pair member for a labeled ligand
(e.g., biotin, avidin or an antibody); a fluorescent molecule; a
chemiluminescent molecule; an enzyme or the like.
[0126] Probes can be labeled to high specific activity by either
the nick translation method of Rigby et al. (1977), J. Mol. Biol.
113:237-251 or by the random priming method of Fienberg et al.
(1983), Anal. Biochem. 132:6-13, the entire disclosures of which
are incorporated herein by reference. The latter is the method of
choice for synthesizing .sup.32P-labeled probes of high specific
activity from single-stranded DNA or from RNA templates. For
example, by replacing preexisting nucleotides with highly
radioactive nucleotides according to the nick translation method,
it is possible to prepare .sup.32P-labeled nucleic acid probes with
a specific activity well in excess of 10.sup.8 cpm/microgram.
Autoradiographic detection of hybridization can then be performed
by exposing hybridized filters to photographic film. Densitometric
scanning of the photographic films exposed by the hybridized
filters provides an accurate measurement of miR gene transcript
levels. Using another approach, miR gene transcript levels can be
quantified by computerized imaging systems, such as the Molecular
Dynamics 400-B 2D Phosphorimager available from Amersham
Biosciences, Piscataway, N.J.
[0127] Where radionuclide labeling of DNA or RNA probes is not
practical, the random-primer method can be used to incorporate an
analogue, for example, the dTTP analogue
5-(N--(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl) deoxyuridine
triphosphate, into the probe molecule. The biotinylated probe
oligonucleotide can be detected by reaction with biotin-binding
proteins, such as avidin, streptavidin, and antibodies (e.g.,
anti-biotin antibodies) coupled to fluorescent dyes or enzymes that
produce color reactions.
[0128] In addition to Northern and other RNA hybridization
techniques, determining the levels of RNA transcripts can be
accomplished using the technique of in situ hybridization. This
technique requires fewer cells than the Northern blotting
technique, and involves depositing whole cells onto a microscope
cover slip and probing the nucleic acid content of the cell with a
solution containing radioactive or otherwise labeled nucleic acid
(e.g., cDNA or RNA) probes. This technique is particularly
well-suited for analyzing tissue biopsy samples from subjects. The
practice of the in situ hybridization technique is described in
more detail in U.S. Pat. No. 5,427,916, the entire disclosure of
which is incorporated herein by reference.
[0129] In one non-limiting example, suitable probes for in situ
hybridization of a given miR gene product can be produced from the
nucleic acid sequences, and include, but are not limited to, probes
having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or
complete complementarity to a miR gene product of interest, as
described above.
[0130] The relative number of miR gene transcripts in cells can
also be determined by reverse transcription of miR gene
transcripts, followed by amplification of the reverse-transcribed
transcripts by polymerase chain reaction (RT-PCR). The levels of
miR gene transcripts can be quantified in comparison with an
internal standard, for example, the level of mRNA from a
"housekeeping" gene present in the same sample. A suitable
"housekeeping" gene for use as an internal standard includes, e.g.,
myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods
for performing quantitative and semi-quantitative RT-PCR, and
variations thereof, are well known to those of skill in the
art.
[0131] In some instances, it may be desirable to simultaneously
determine the expression level of a plurality of different miR gene
products in a sample. In other instances, it may be desirable to
determine the expression level of the transcripts of all known miR
genes correlated with a cancer. Assessing cancer-specific
expression levels for hundreds of miR genes or gene products is
time consuming and requires a large amount of total RNA (e.g., at
least 20 .mu.g for each Northern blot) and autoradiographic
techniques that require radioactive isotopes.
[0132] To overcome these limitations, an oligolibrary, in microchip
format (i.e., a microarray), may be constructed containing a set of
oligonucleotide (e.g., oligodeoxynucleotides) probes that are
specific for a set of miR genes. Using such a microarray, the
expression level of multiple microRNAs in a biological sample can
be determined by reverse transcribing the RNAs to generate a set of
target oligodeoxynucleotides, and hybridizing them to probe the
oligonucleotides on the microarray to generate a hybridization, or
expression, profile. The hybridization profile of the test sample
can then be compared to that of a control sample to determine which
microRNAs have an altered expression level in solid cancer
cells.
[0133] As used herein, "probe oligonucleotide" or "probe
oligodeoxynucleotide" refers to an oligonucleotide that is capable
of hybridizing to a target oligonucleotide. "Target
oligonucleotide" or "target oligodeoxynucleotide" refers to a
molecule to be detected (e.g., via hybridization). By "miR-specific
probe oligonucleotide" or "probe oligonucleotide specific for a
miR" is meant a probe oligonucleotide that has a sequence selected
to hybridize to a specific miR gene product, or to a reverse
transcript of the specific miR gene product.
[0134] An "expression profile" or "hybridization profile" of a
particular sample is essentially a fingerprint of the state of the
sample; while two states may have any particular gene similarly
expressed, the evaluation of a number of genes simultaneously
allows the generation of a gene expression profile that is unique
to the state of the cell. That is, normal tissue may be
distinguished from cancerous (e.g., tumor) tissue, and within
cancerous tissue, different prognosis states (for example, good or
poor long term survival prospects) may be determined. By comparing
expression profiles of the colon cancer tissue in different states,
information regarding which genes are important (including both up-
and down-regulation of genes) in each of these states is obtained.
The identification of sequences that are differentially expressed
in colon cancer tissue, as well as differential expression
resulting in different prognostic outcomes, allows the use of this
information in a number of ways.
[0135] In one non-limiting example, a particular treatment regime
may be evaluated (e.g., to determine whether a chemotherapeutic
drug acts to improve the long-term prognosis in a particular
patient). Similarly, diagnosis may be done or confirmed by
comparing patient samples with known expression profiles.
Furthermore, these gene expression profiles (or individual genes)
allow screening of drug candidates that suppress the colon cancer
expression profile or convert a poor prognosis profile to a better
prognosis profile.
[0136] Accordingly, there is also provided herein methods of
diagnosing whether a subject has, or is at risk for developing, a
colon cancer, comprising reverse transcribing RNA from a test
sample obtained from the subject to provide a set of target
oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides
to a microarray comprising miRNA-specific probe oligonucleotides to
provide a hybridization profile for the test sample, and comparing
the test sample hybridization profile to a hybridization profile
generated from a control sample or reference standard, wherein an
alteration in the signal of at least one miRNA is indicative of the
subject either having, or being at risk for developing, a solid
cancer.
[0137] In one embodiment, the microarray comprises miRNA-specific
probe oligonucleotides for a substantial portion of all known human
miRNAs. In a particular embodiment, the microarray comprises
miRNA-specific probe oligonucleotides for one or more miRNAs
selected from the group consisting of miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
[0138] The microarray can be prepared from gene-specific
oligonucleotide probes generated from known miRNA sequences. The
array may contain two different oligonucleotide probes for each
miRNA, one containing the active, mature sequence and the other
being specific for the precursor of the miRNA. The array may also
contain controls, such as one or more mouse sequences differing
from human orthologs by only a few bases, which can serve as
controls for hybridization stringency conditions. tRNAs or other
RNAs (e.g., rRNAs, mRNAs) from both species may also be printed on
the microchip, providing an internal, relatively stable, positive
control for specific hybridization. One or more appropriate
controls for non-specific hybridization may also be included on the
microchip. For this purpose, sequences are selected based upon the
absence of any homology with any known miRNAs.
[0139] The microarray may be fabricated using techniques known in
the art. For example, probe oligonucleotides of an appropriate
length, e.g., 40 nucleotides, are 5'-amine modified at position C6
and printed using commercially available microarray systems, e.g.,
the GeneMachine OmniGrid.TM. 100 Microarrayer and Amersham
CodeLink.TM. activated slides. Labeled cDNA oligomer corresponding
to the target RNAs is prepared by reverse transcribing the target
RNA with labeled primer. Following first strand synthesis, the
RNA/DNA hybrids are denatured to degrade the RNA templates. The
labeled target cDNAs thus prepared are then hybridized to the
microarray chip under hybridizing conditions, e.g.,
6.times.SSPE/30% formamide at 25.degree. C. for 18 hours, followed
by washing in 0.75.times.TNT (Tris HCl/NaCl/Tween 20) at 37.degree.
C. for 40 minutes. At positions on the array where the immobilized
probe DNA recognizes a complementary target cDNA in the sample,
hybridization occurs. The labeled target cDNA marks the exact
position on the array where binding occurs, allowing automatic
detection and quantification. The output consists of a list of
hybridization events, indicating the relative abundance of specific
cDNA sequences, and therefore the relative abundance of the
corresponding complementary miRs, in the patient sample.
[0140] According to one embodiment, the labeled cDNA oligomer is a
biotin-labeled cDNA, prepared from a biotin-labeled primer. The
microarray is then processed by direct detection of the
biotin-containing transcripts using, e.g., Streptavidin-Alexa647
conjugate, and scanned utilizing conventional scanning methods.
Image intensities of each spot on the array are proportional to the
abundance of the corresponding miR in the patient sample.
[0141] The use of the array has several advantages for miRNA
expression detection. First, the global expression of several
hundred genes can be identified in the same sample at one time
point. Second, through careful design of the oligonucleotide
probes, expression of both mature and precursor molecules can be
identified. Third, in comparison with Northern blot analysis, the
chip requires a small amount of RNA, and provides reproducible
results using 2.5 .mu.g of total RNA. The relatively limited number
of miRNAs (a few hundred per species) allows the construction of a
common microarray for several species, with distinct
oligonucleotide probes for each. Such a tool allows for analysis of
trans-species expression for each known miR under various
conditions.
[0142] In addition to use for quantitative expression level assays
of specific miRs, a microchip containing miRNA-specific probe
oligonucleotides corresponding to a substantial portion of the
miRNome, preferably the entire miRNome, may be employed to carry
out miR gene expression profiling, for analysis of miR expression
patterns. Distinct miR signatures can be associated with
established disease markers, or directly with a disease state.
[0143] According to the expression profiling methods described
herein, total RNA from a sample from a subject suspected of having
a colon cancer-related disease quantitatively reverse transcribed
to provide a set of labeled target oligodeoxynucleotides
complementary to the RNA in the sample. The target
oligodeoxynucleotides are then hybridized to a microarray
comprising miRNA-specific probe oligonucleotides to provide a
hybridization profile for the sample. The result is a hybridization
profile for the sample representing the expression pattern of miRNA
in the sample. The hybridization profile comprises the signal from
the binding of the target oligodeoxynucleotides from the sample to
the miRNA-specific probe oligonucleotides in the microarray. The
profile may be recorded as the presence or absence of binding
(signal vs. zero signal).
[0144] More preferably, the profile recorded includes the intensity
of the signal from each hybridization. The profile is compared to
the hybridization profile generated from a normal, i.e.,
noncancerous, control sample. An alteration in the signal is
indicative of the presence of, or propensity to develop, cancer in
the subject.
[0145] Other techniques for measuring miR gene expression are also
within the skill in the art, and include various techniques for
measuring rates of RNA transcription and degradation.
[0146] There is also provided herein methods of determining the
prognosis of a subject with a colon cancer, comprising measuring
the level of at least one miR gene product, which is associated
with a particular prognosis in a colon cancer-related disease
(e.g., a good or positive prognosis, a poor or adverse prognosis),
in a test sample from the subject.
[0147] According to these methods, an alteration in the level of a
miR gene product that is associated with a particular prognosis in
the test sample, as compared to the level of a corresponding miR
gene product in a control sample, is indicative of the subject
having a solid cancer with a particular prognosis. In one
embodiment, the miR gene product is associated with an adverse
(i.e., poor) prognosis. Examples of an adverse prognosis include,
but are not limited to, low survival rate and rapid disease
progression. In certain embodiments, the level of the at least one
miR gene product is measured by reverse transcribing RNA from a
test sample obtained from the subject to provide a set of target
oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides
to a microarray that comprises miRNA-specific probe
oligonucleotides to provide a hybridization profile for the test
sample, and comparing the test sample hybridization profile to a
hybridization profile generated from a control sample.
[0148] Without wishing to be bound by any one theory, it is
believed that alterations in the level of one or more miR gene
products in cells can result in the deregulation of one or more
intended targets for these miRs, which can lead to the formation of
solid cancers. Therefore, altering the level of the miR gene
product (e.g., by decreasing the level of a miR gene product that
is up-regulated in solid cancer cells, by increasing the level of a
miR gene product that is down-regulated in solid cancer cells) may
successfully treat the solid cancer.
[0149] Accordingly, there is further provided herein methods of
inhibiting tumorigenesis in a subject who has, or is suspected of
having, a solid cancer wherein at least one miR gene product is
deregulated (e.g., down-regulated, up-regulated) in the cancer
cells of the subject. When the at least one isolated miR gene
product is down-regulated in the cancer cells (e.g., miR-21), the
method comprises administering an effective amount of the at least
one isolated miR gene product, or an isolated variant or
biologically-active fragment thereof, such that proliferation of
cancer cells in the subject is inhibited.
[0150] For example, when a miR gene product is down-regulated in a
cancer cell in a subject, administering an effective amount of an
isolated miR gene product to the subject can inhibit proliferation
of the cancer cell. The isolated miR gene product that is
administered to the subject can be identical to the endogenous
wild-type miR gene product (e.g., a miR gene product) that is
down-regulated in the cancer cell or it can be a variant or
biologically-active fragment thereof.
[0151] As defined herein, a "variant" of a miR gene product refers
to a miRNA that has less than 100% identity to a corresponding
wild-type miR gene product and possesses one or more biological
activities of the corresponding wild-type miR gene product.
Examples of such biological activities include, but are not limited
to, inhibition of expression of a target RNA molecule (e.g.,
inhibiting translation of a target RNA molecule, modulating the
stability of a target RNA molecule, inhibiting processing of a
target RNA molecule) and inhibition of a cellular process
associated with solid cancer (e.g., cell differentiation, cell
growth, cell death). These variants include species variants and
variants that are the consequence of one or more mutations (e.g., a
substitution, a deletion, an insertion) in a miR gene. In certain
embodiments, the variant is at least about 95%, 98%, or 99%
identical to a corresponding wild-type miR gene product.
[0152] As defined herein, a "biologically-active fragment" of a miR
gene product refers to an RNA fragment of a miR gene product that
possesses one or more biological activities of a corresponding
wild-type miR gene product. As described above, examples of such
biological activities include, but are not limited to, inhibition
of expression of a target RNA molecule and inhibition of a cellular
process associated with a colon cancer. In certain embodiments, the
biologically-active fragment is at least about 15 or 17 nucleotides
in length. In a particular embodiment, an isolated miR gene product
can be administered to a subject in combination with one or more
additional anti-cancer treatments. Suitable anti-cancer treatments
include, but are not limited to, chemotherapy, radiation therapy
and combinations thereof (e.g., chemoradiation).
[0153] When the at least one isolated miR gene product is
up-regulated in the cancer cells, the method comprises
administering to the subject an effective amount of at least one
compound for inhibiting expression of the at least one miR gene
product, referred to herein as miR gene expression-inhibition
compounds, such that proliferation of solid cancer cells is
inhibited. In a particular embodiment, the at least one miR
expression-inhibition compound is specific for a miR gene product
selected from the group consisting miR-20a, miR-21, miR-106a,
miR-181b, miR-203 and combinations thereof.
[0154] A miR gene expression-inhibiting compound can be
administered to a subject in combination with one or more
additional anti-cancer treatments. Suitable anti-cancer treatments
include, but are not limited to, chemotherapy, radiation therapy
and combinations thereof (e.g., chemoradiation).
[0155] The terms "treat", "treating" and "treatment", as used
herein, refer to ameliorating symptoms associated with a disease or
condition, for example, a solid cancer, including preventing or
delaying the onset of the disease symptoms, and/or lessening the
severity or frequency of symptoms of the disease or condition. The
terms "subject", "patient" and "individual" are defined herein to
include animals, such as mammals, including, but not limited to,
primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea
pigs, rats, mice or other bovine, ovine, equine, canine, feline,
rodent, or murine species. In a preferred embodiment, the animal is
a human.
[0156] As used herein, an "effective amount" of an isolated miR
gene product is an amount sufficient to inhibit proliferation of a
cancer cell in a subject suffering from a solid cancer. One skilled
in the art can readily determine an effective amount of a miR gene
product to be administered to a given subject, by taking into
account factors, such as the size and weight of the subject; the
extent of disease penetration; the age, health and sex of the
subject; the route of administration; and whether the
administration is regional or systemic.
[0157] For example, an effective amount of an isolated miR gene
product can be based on the approximate weight of a tumor mass to
be treated. The approximate weight of a tumor mass can be
determined by calculating the approximate volume of the mass,
wherein one cubic centimeter of volume is roughly equivalent to one
gram. An effective amount of the isolated miR gene product based on
the weight of a tumor mass can be in the range of about 10-500
micrograms/gram of tumor mass. In certain embodiments, the tumor
mass can be at least about 10 micrograms/gram of tumor mass, at
least about 60 micrograms/gram of tumor mass or at least about 100
micrograms/gram of tumor mass.
[0158] An effective amount of an isolated miR gene product can also
be based on the approximate or estimated body weight of a subject
to be treated. Preferably, such effective amounts are administered
parenterally or enterally, as described herein. For example, an
effective amount of the isolated miR gene product is administered
to a subject can range from about 5 to about 3000 micrograms/kg of
body weight, from about 700-1000 micrograms/kg of body weight, or
greater than about 1000 micrograms/kg of body weight.
[0159] One skilled in the art can also readily determine an
appropriate dosage regimen for the administration of an isolated
miR gene product to a given subject. For example, a miR gene
product can be administered to the subject once (e.g., as a single
injection or deposition). Alternatively, a miR gene product can be
administered once or twice daily to a subject for a period of from
about three to about twenty-eight days, more particularly from
about seven to about ten days. In a particular dosage regimen, a
miR gene product is administered once a day for seven days. Where a
dosage regimen comprises multiple administrations, it is understood
that the effective amount of the miR gene product administered to
the subject can comprise the total amount of gene product
administered over the entire dosage regimen.
[0160] As used herein, an "isolated" miR gene product is one that
is synthesized, or altered or removed from the natural state
through human intervention. For example, a synthetic miR gene
product, or a miR gene product partially or completely separated
from the coexisting materials of its natural state, is considered
to be "isolated." An isolated miR gene product can exist in
substantially-purified form, or can exist in a cell into which the
miR gene product has been delivered. Thus, a miR gene product that
is deliberately delivered to, or expressed in, a cell is considered
an "isolated" miR gene product. A miR gene product produced inside
a cell from a miR precursor molecule is also considered to be an
"isolated" molecule. According to one particular embodiment, the
isolated miR gene products described herein can be used for the
manufacture of a medicament for treating a solid cancer in a
subject (e.g., a human).
[0161] Isolated miR gene products can be obtained using a number of
standard techniques. For example, the miR gene products can be
chemically synthesized or recombinantly produced using methods
known in the art. In one embodiment, miR gene products are
chemically synthesized using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. Commercial
suppliers of synthetic RNA molecules or synthesis reagents include,
e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,
Colo., U.S.A.), Pierce Chemical (part of Perbio Science, Rockford,
Ill., U.S.A.), Glen Research (Sterling, Va., U.S.A.), ChemGenes
(Ashland, Mass., U.S.A.) and Cruachem (Glasgow, UK).
[0162] Alternatively, the miR gene products can be expressed from
recombinant circular or linear DNA plasmids using any suitable
promoter. Suitable promoters for expressing RNA from a plasmid
include, e.g., the U6 or H1 RNA pol III promoter sequences, or the
cytomegalovirus promoters. Selection of other suitable promoters is
within the skill in the art. The recombinant plasmids of the
invention can also comprise inducible or regulatable promoters for
expression of the miR gene products in cancer cells.
[0163] The miR gene products that are expressed from recombinant
plasmids can be isolated from cultured cell expression systems by
standard techniques. The miR gene products that are expressed from
recombinant plasmids can also be delivered to, and expressed
directly in, the cancer cells. The use of recombinant plasmids to
deliver the miR gene products to cancer cells is discussed in more
detail below.
[0164] The miR gene products can be expressed from a separate
recombinant plasmid, or they can be expressed from the same
recombinant plasmid. In one embodiment, the miR gene products are
expressed as RNA precursor molecules from a single plasmid, and the
precursor molecules are processed into the functional miR gene
product by a suitable processing system, including, but not limited
to, processing systems extant within a cancer cell. Other suitable
processing systems include, e.g., the in vitro Drosophila cell
lysate system (e.g., as described in U.S. Published Patent
Application No. 2002/0086356 to Tuschl et al., the entire
disclosure of which is incorporated herein by reference) and the E.
coli RNAse III system (e.g., as described in U.S. Published Patent
Application No. 2004/0014113 to Yang et al., the entire disclosure
of which is incorporated herein by reference).
[0165] Selection of plasmids suitable for expressing the miR gene
products, methods for inserting nucleic acid sequences into the
plasmid to express the gene products, and methods of delivering the
recombinant plasmid to the cells of interest are within the skill
in the art. See, for example, Zeng et al. (2002), Molecular Cell
9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448;
Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al.
(2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes
Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505;
and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire
disclosures of which are incorporated herein by reference.
[0166] In one embodiment, a plasmid expressing the miR gene
products comprises a sequence encoding a miR precursor RNA under
the control of the CMV intermediate-early promoter. As used herein,
"under the control" of a promoter means that the nucleic acid
sequences encoding the miR gene product are located 3' of the
promoter, so that the promoter can initiate transcription of the
miR gene product coding sequences.
[0167] The miR gene products can also be expressed from recombinant
viral vectors. It is contemplated that the miR gene products can be
expressed from two separate recombinant viral vectors, or from the
same viral vector. The RNA expressed from the recombinant viral
vectors can either be isolated from cultured cell expression
systems by standard techniques, or can be expressed directly in
cancer cells. The use of recombinant viral vectors to deliver the
miR gene products to cancer cells is discussed in more detail
below.
[0168] The recombinant viral vectors of the invention comprise
sequences encoding the miR gene products and any suitable promoter
for expressing the RNA sequences. Suitable promoters include, but
are not limited to, the U6 or H1 RNA pol III promoter sequences, or
the cytomegalovirus promoters. Selection of other suitable
promoters is within the skill in the art. The recombinant viral
vectors of the invention can also comprise inducible or regulatable
promoters for expression of the miR gene products in a cancer
cell.
[0169] Any viral vector capable of accepting the coding sequences
for the miR gene products can be used; for example, vectors derived
from adenovirus (AV); adeno-associated virus (AAV); retroviruses
(e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus);
herpes virus, and the like. The tropism of the viral vectors can be
modified by pseudotyping the vectors with envelope proteins or
other surface antigens from other viruses, or by substituting
different viral capsid proteins, as appropriate.
[0170] For example, lentiviral vectors of the invention can be
pseudotyped with surface proteins from vesicular stomatitis virus
(VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the
invention can be made to target different cells by engineering the
vectors to express different capsid protein serotypes. For example,
an AAV vector expressing a serotype 2 capsid on a serotype 2 genome
is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2
vector can be replaced by a serotype 5 capsid gene to produce an
AAV 2/5 vector. Techniques for constructing AAV vectors that
express different capsid protein serotypes are within the skill in
the art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.
76:791-801, the entire disclosure of which is incorporated herein
by reference.
[0171] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing RNA into the vector, methods of delivering the viral
vector to the cells of interest, and recovery of the expressed RNA
products are within the skill in the art. See, for example,
Dornburg (1995), Gene Therapy 2:301-310; Eglitis (1988),
Biotechniques 6:608-614; Miller (1990), Hum. Gene Therapy 1:5-14;
and Anderson (1998), Nature 392:25-30, the entire disclosures of
which are incorporated herein by reference.
[0172] Particularly suitable viral vectors are those derived from
AV and AAV. A suitable AV vector for expressing the miR gene
products, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia et al. (2002), Nat. Biotech. 20:1006-1010, the entire
disclosure of which is incorporated herein by reference. Suitable
AAV vectors for expressing the miR gene products, methods for
constructing the recombinant AAV vector, and methods for delivering
the vectors into target cells are described in Samulski et al.
(1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J. Virol.,
70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.
Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent
Application No. WO 94/13788; and International Patent Application
No. WO 93/24641, the entire disclosures of which are incorporated
herein by reference. In one embodiment, the miR gene products are
expressed from a single recombinant AAV vector comprising the CMV
intermediate early promoter.
[0173] In a certain embodiment, a recombinant AAV viral vector of
the invention comprises a nucleic acid sequence encoding a miR
precursor RNA in operable connection with a polyT termination
sequence under the control of a human U6 RNA promoter. As used
herein, "in operable connection with a polyT termination sequence"
means that the nucleic acid sequences encoding the sense or
antisense strands are immediately adjacent to the polyT termination
signal in the 5' direction. During transcription of the miR
sequences from the vector, the polyT termination signals act to
terminate transcription.
[0174] In other embodiments of the treatment methods of the
invention, an effective amount of at least one compound that
inhibits miR expression can be administered to the subject. As used
herein, "inhibiting miR expression" means that the production of
the precursor and/or active, mature form of miR gene product after
treatment is less than the amount produced prior to treatment. One
skilled in the art can readily determine whether miR expression has
been inhibited in a cancer cell, using, for example, the techniques
for determining miR transcript level discussed above for the
diagnostic method. Inhibition can occur at the level of gene
expression (i.e., by inhibiting transcription of a miR gene
encoding the miR gene product) or at the level of processing (e.g.,
by inhibiting processing of a miR precursor into a mature, active
miR).
[0175] As used herein, an "effective amount" of a compound that
inhibits miR expression is an amount sufficient to inhibit
proliferation of a cancer cell in a subject suffering from a cancer
(e.g., a colon cancer). One skilled in the art can readily
determine an effective amount of a miR expression-inhibition
compound to be administered to a given subject, by taking into
account factors, such as the size and weight of the subject; the
extent of disease penetration; the age, health and sex of the
subject; the route of administration; and whether the
administration is regional or systemic.
[0176] For example, an effective amount of the
expression-inhibition compound can be based on the approximate
weight of a tumor mass to be treated, as described herein. An
effective amount of a compound that inhibits miR expression can
also be based on the approximate or estimated body weight of a
subject to be treated, as described herein.
[0177] One skilled in the art can also readily determine an
appropriate dosage regimen for administering a compound that
inhibits miR expression to a given subject.
[0178] Suitable compounds for inhibiting miR gene expression
include double-stranded RNA (such as short- or small-interfering
RNA or "siRNA"), antisense nucleic acids, and enzymatic RNA
molecules, such as ribozymes. Each of these compounds can be
targeted to a given miR gene product and interfere with the
expression of (e.g., inhibit translation of, induce cleavage or
destruction of) the target miR gene product.
[0179] For example, expression of a given miR gene can be inhibited
by inducing RNA interference of the miR gene with an isolated
double-stranded RNA ("dsRNA") molecule which has at least 90%, for
example at least 95%, at least 98%, at least 99%, or 100%, sequence
homology with at least a portion of the miR gene product. In a
particular embodiment, the dsRNA molecule is a "short or small
interfering RNA" or "siRNA."
[0180] siRNA useful in the present methods comprise short
double-stranded RNA from about 17 nucleotides to about 29
nucleotides in length, preferably from about 19 to about 25
nucleotides in length. The siRNA comprise a sense RNA strand and a
complementary antisense RNA strand annealed together by standard
Watson-Crick base-pairing interactions (hereinafter "base-paired").
The sense strand comprises a nucleic acid sequence that is
substantially identical to a nucleic acid sequence contained within
the target miR gene product.
[0181] As used herein, a nucleic acid sequence in an siRNA which is
"substantially identical" to a target sequence contained within the
target mRNA is a nucleic acid sequence that is identical to the
target sequence, or that differs from the target sequence by one or
two nucleotides. The sense and antisense strands of the siRNA can
comprise two complementary, single-stranded RNA molecules, or can
comprise a single molecule in which two complementary portions are
base-paired and are covalently linked by a single-stranded
"hairpin" area.
[0182] The siRNA can also be altered RNA that differs from
naturally-occurring RNA by the addition, deletion, substitution
and/or alteration of one or more nucleotides. Such alterations can
include addition of non-nucleotide material, such as to the end(s)
of the siRNA or to one or more internal nucleotides of the siRNA,
or modifications that make the siRNA resistant to nuclease
digestion, or the substitution of one or more nucleotides in the
siRNA with deoxyribonucleotides.
[0183] One or both strands of the siRNA can also comprise a 3'
overhang. As used herein, a "3' overhang" refers to at least one
unpaired nucleotide extending from the 3'-end of a duplexed RNA
strand. Thus, in certain embodiments, the siRNA comprises at least
one 3' overhang of from 1 to about 6 nucleotides (which includes
ribonucleotides or deoxyribonucleotides) in length, from 1 to about
5 nucleotides in length, from 1 to about 4 nucleotides in length,
or from about 2 to about 4 nucleotides in length. In a particular
embodiment, the 3' overhang is present on both strands of the
siRNA, and is 2 nucleotides in length. For example, each strand of
the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or
diuridylic acid ("uu").
[0184] The siRNA can be produced chemically or biologically, or can
be expressed from a recombinant plasmid or viral vector, as
described above for the isolated miR gene products. Exemplary
methods for producing and testing dsRNA or siRNA molecules are
described in U.S. Published Patent Application No. 2002/0173478 to
Gewirtz and in U.S. Published Patent Application No. 2004/0018176
to Reich et al., the entire disclosures of both of which are
incorporated herein by reference.
[0185] Expression of a given miR gene can also be inhibited by an
antisense nucleic acid. As used herein, an "antisense nucleic acid"
refers to a nucleic acid molecule that binds to target RNA by means
of RNA-RNA, RNA-DNA or RNA-peptide nucleic acid interactions, which
alters the activity of the target RNA. Antisense nucleic acids
suitable for use in the present methods are single-stranded nucleic
acids (e.g., RNA, DNA, RNA-DNA chimeras, peptide nucleic acid
(PNA)) that generally comprise a nucleic acid sequence
complementary to a contiguous nucleic acid sequence in a miR gene
product. The antisense nucleic acid can comprise a nucleic acid
sequence that is 50-100% complementary, 75-100% complementary, or
95-100% complementary to a contiguous nucleic acid sequence in a
miR gene product.
[0186] Without wishing to be bound by any theory, it is believed
that the antisense nucleic acids activate RNase H or another
cellular nuclease that digests the miR gene product/antisense
nucleic acid duplex.
[0187] Antisense nucleic acids can also contain modifications to
the nucleic acid backbone or to the sugar and base moieties (or
their equivalent) to enhance target specificity, nuclease
resistance, delivery or other properties related to efficacy of the
molecule. Such modifications include cholesterol moieties, duplex
intercalators, such as acridine, or one or more nuclease-resistant
groups.
[0188] Antisense nucleic acids can be produced chemically or
biologically, or can be expressed from a recombinant plasmid or
viral vector, as described above for the isolated miR gene
products. Exemplary methods for producing and testing are within
the skill in the art; see, e.g., Stein and Cheng (1993), Science
261:1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entire
disclosures of which are incorporated herein by reference.
[0189] Expression of a given miR gene can also be inhibited by an
enzymatic nucleic acid. As used herein, an "enzymatic nucleic acid"
refers to a nucleic acid comprising a substrate binding region that
has complementarity to a contiguous nucleic acid sequence of a miR
gene product, and which is able to specifically cleave the miR gene
product. The enzymatic nucleic acid substrate binding region can
be, for example, 50-100% complementary, 75-100% complementary, or
95-100% complementary to a contiguous nucleic acid sequence in a
miR gene product. The enzymatic nucleic acids can also comprise
modifications at the base, sugar, and/or phosphate groups. An
exemplary enzymatic nucleic acid for use in the present methods is
a ribozyme.
[0190] The enzymatic nucleic acids can be produced chemically or
biologically, or can be expressed from a recombinant plasmid or
viral vector, as described above for the isolated miR gene
products. Exemplary methods for producing and testing dsRNA or
siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl.
Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic
Acid Drug Dev. 9:25-31; and U.S. Pat. No. 4,987,071 to Cech et al,
the entire disclosures of which are incorporated herein by
reference.
[0191] Administration of at least one miR gene product, or at least
one compound for inhibiting miR expression, will inhibit the
proliferation of cancer cells in a subject who has a solid cancer.
As used herein, to "inhibit the proliferation of a cancer cell"
means to kill the cell, or permanently or temporarily arrest or
slow the growth of the cell. Inhibition of cancer cell
proliferation can be inferred if the number of such cells in the
subject remains constant or decreases after administration of the
miR gene products or miR gene expression-inhibition compounds. An
inhibition of cancer cell proliferation can also be inferred if the
absolute number of such cells increases, but the rate of tumor
growth decreases.
[0192] The number of cancer cells in the body of a subject can be
determined by direct measurement, or by estimation from the size of
primary or metastatic tumor masses. For example, the number of
cancer cells in a subject can be measured by immunohistological
methods, flow cytometry, or other techniques designed to detect
characteristic surface markers of cancer cells.
[0193] The size of a tumor mass can be ascertained by direct visual
observation, or by diagnostic imaging methods, such as X-ray,
magnetic resonance imaging, ultrasound, and scintigraphy.
Diagnostic imaging methods used to ascertain size of the tumor mass
can be employed with or without contrast agents, as is known in the
art. The size of a tumor mass can also be ascertained by physical
means, such as palpation of the tissue mass or measurement of the
tissue mass with a measuring instrument, such as a caliper.
[0194] The miR gene products or miR gene expression-inhibition
compounds can be administered to a subject by any means suitable
for delivering these compounds to cancer cells of the subject. For
example, the miR gene products or miR expression-inhibition
compounds can be administered by methods suitable to transfect
cells of the subject with these compounds, or with nucleic acids
comprising sequences encoding these compounds.
[0195] In one embodiment, the cells are transfected with a plasmid
or viral vector comprising sequences encoding at least one miR gene
product or miR gene expression-inhibition compound.
[0196] Transfection methods for eukaryotic cells are well known in
the art, and include, e.g., direct injection of the nucleic acid
into the nucleus or pronucleus of a cell; electroporation; liposome
transfer or transfer mediated by lipophilic materials;
receptor-mediated nucleic acid delivery, bioballistic or particle
acceleration; calcium phosphate precipitation, and transfection
mediated by viral vectors.
[0197] For example, cells can be transfected with a liposomal
transfer compound, e.g., DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium
methylsulfate, Boehringer-Mannheim) or an equivalent, such as
LIPOFECTIN. The amount of nucleic acid used is not critical to the
practice of the invention; acceptable results may be achieved with
0.1-100 micrograms of nucleic acid/10.sup.5 cells. For example, a
ratio of about 0.5 micrograms of plasmid vector in 3 micrograms of
DOTAP per 10.sup.5 cells can be used.
[0198] A miR gene product or miR gene expression-inhibition
compound can also be administered to a subject by any suitable
enteral or parenteral administration route. Suitable enteral
administration routes for the present methods include, e.g., oral,
rectal, or intranasal delivery. Suitable parenteral administration
routes include, e.g., intravascular administration (e.g.,
intravenous bolus injection, intravenous infusion, intra-arterial
bolus injection, intra-arterial infusion and catheter instillation
into the vasculature); peri- and intra-tissue injection (e.g.,
peri-tumoral and intra-tumoral injection, intra-retinal injection,
or subretinal injection); subcutaneous injection or deposition,
including subcutaneous infusion (such as by osmotic pumps); direct
application to the tissue of interest, for example by a catheter or
other placement device (e.g., a retinal pellet or a suppository or
an implant comprising a porous, non-porous, or gelatinous
material); and inhalation. Particularly suitable administration
routes are injection, infusion and direct injection into the
tumor.
[0199] In the present methods, a miR gene product or miR gene
product expression-inhibition compound can be administered to the
subject either as naked RNA, in combination with a delivery
reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral
vector) comprising sequences that express the miR gene product or
miR gene product expression-inhibition compound. Suitable delivery
reagents include, e.g., the Mirus Transit TKO lipophilic reagent;
lipofectin; lipofectamine; cellfectin; polycations (e.g.,
polylysine), and liposomes.
[0200] Recombinant plasmids and viral vectors comprising sequences
that express the miR gene products or miR gene
expression-inhibition compounds, and techniques for delivering such
plasmids and vectors to cancer cells, are discussed herein and/or
are well known in the art.
[0201] In a particular embodiment, liposomes are used to deliver a
miR gene product or miR gene expression-inhibition compound (or
nucleic acids comprising sequences encoding them) to a subject.
Liposomes can also increase the blood half-life of the gene
products or nucleic acids. Suitable liposomes for use in the
invention can be formed from standard vesicle-forming lipids, which
generally include neutral or negatively charged phospholipids and a
sterol, such as cholesterol. The selection of lipids is generally
guided by consideration of factors, such as the desired liposome
size and half-life of the liposomes in the blood stream. A variety
of methods are known for preparing liposomes, for example, as
described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467;
and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369,
the entire disclosures of which are incorporated herein by
reference.
[0202] The liposomes for use in the present methods can comprise a
ligand molecule that targets the liposome to cancer cells. Ligands
that bind to receptors prevalent in cancer cells, such as
monoclonal antibodies that bind to tumor cell antigens, are
preferred.
[0203] The liposomes for use in the present methods can also be
modified so as to avoid clearance by the mononuclear macrophage
system ("MMS") and reticuloendothelial system ("RES"). Such
modified liposomes have opsonization-inhibition moieties on the
surface or incorporated into the liposome structure. In a
particularly preferred embodiment, a liposome of the invention can
comprise both an opsonization-inhibition moiety and a ligand.
[0204] Opsonization-inhibiting moieties for use in preparing the
liposomes of the invention are typically large hydrophilic polymers
that are bound to the liposome membrane. As used herein, an
opsonization-inhibiting moiety is "bound" to a liposome membrane
when it is chemically or physically attached to the membrane, e.g.,
by the intercalation of a lipid-soluble anchor into the membrane
itself, or by binding directly to active groups of membrane lipids.
These opsonization-inhibiting hydrophilic polymers form a
protective surface layer that significantly decreases the uptake of
the liposomes by the MMS and RES; e.g., as described in U.S. Pat.
No. 4,920,016, the entire disclosure of which is incorporated
herein by reference.
[0205] Opsonization-inhibiting moieties suitable for modifying
liposomes are preferably water-soluble polymers with a
number-average molecular weight from about 500 to about 40,000
daltons, and more preferably from about 2,000 to about 20,000
daltons. Such polymers include polyethylene glycol (PEG) or
polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG,
and PEG or PPG stearate; synthetic polymers, such as polyacrylamide
or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric
polyamidoamines; polyacrylic acids; polyalcohols, e.g.,
polyvinylalcohol and polyxylitol to which carboxylic or amino
groups are chemically linked, as well as gangliosides, such as
ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or
derivatives thereof, are also suitable. In addition, the
opsonization-inhibiting polymer can be a block copolymer of PEG and
either a polyamino acid, polysaccharide, polyamidoamine,
polyethyleneamine, or polynucleotide. The opsonization-inhibiting
polymers can also be natural polysaccharides containing amino acids
or carboxylic acids, e.g., galacturonic acid, glucuronic acid,
mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid,
alginic acid, carrageenan; aminated polysaccharides or
oligosaccharides (linear or branched); or carboxylated
polysaccharides or oligosaccharides, e.g., reacted with derivatives
of carbonic acids with resultant linking of carboxylic groups.
Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or a
derivative thereof. Liposomes modified with PEG or PEG-derivatives
are sometimes called "PEGylated liposomes."
[0206] The opsonization-inhibiting moiety can be bound to the
liposome membrane by any one of numerous well-known techniques. For
example, an N-hydroxysuccinimide ester of PEG can be bound to a
phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a
membrane. Similarly, a dextran polymer can be derivatized with a
stearylamine lipid-soluble anchor via reductive amination using
Na(CN)BH.sub.3 and a solvent mixture, such as tetrahydrofuran and
water in a 30:12 ratio at 60.degree. C.
[0207] Liposomes modified with opsonization-inhibition moieties
remain in the circulation much longer than unmodified liposomes.
For this reason, such liposomes are sometimes called "stealth"
liposomes. Stealth liposomes are known to accumulate in tissues fed
by porous or "leaky" microvasculature. Thus, tissue characterized
by such microvasculature defects, for example, solid tumors, will
efficiently accumulate these liposomes; see Gabizon, et al. (1988),
Proc. Natl. Acad. Sci., U.S.A., 18:6949-53. In addition, the
reduced uptake by the RES lowers the toxicity of stealth liposomes
by preventing significant accumulation of the liposomes in the
liver and spleen. Thus, liposomes that are modified with
opsonization-inhibition moieties are particularly suited to deliver
the miR gene products or miR gene expression-inhibition compounds
(or nucleic acids comprising sequences encoding them) to tumor
cells.
[0208] The miR gene products or miR gene expression-inhibition
compounds can be formulated as pharmaceutical compositions,
sometimes called "medicaments," prior to administering them to a
subject, according to techniques known in the art. Accordingly, the
invention encompasses pharmaceutical compositions for treating a
solid cancer. In one embodiment, the pharmaceutical composition
comprises at least one isolated miR gene product, or an isolated
variant or biologically-active fragment thereof, and a
pharmaceutically-acceptable carrier. In a particular embodiment,
the at least one miR gene product corresponds to a miR gene product
that has a decreased level of expression in solid cancer cells
relative to suitable control cells. In certain embodiments the
isolated miR gene product is selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof.
[0209] In other embodiments, the pharmaceutical compositions of the
invention comprise at least one miR expression-inhibition compound.
In a particular embodiment, the at least one miR gene
expression-inhibition compound is specific for a miR gene whose
expression is greater in colon cancer cells than control cells. In
certain embodiments, the miR gene expression-inhibition compound is
specific for one or more miR gene products selected from the group
consisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and
combinations thereof.
[0210] Pharmaceutical compositions of the present invention are
characterized as being at least sterile and pyrogen-free. As used
herein, "pharmaceutical compositions" include formulations for
human and veterinary use. Methods for preparing pharmaceutical
compositions of the invention are within the skill in the art, for
example as described in Remington's Pharmaceutical Science, 17th
ed., Mack Publishing Company, Easton, Pa. (1985), the entire
disclosure of which is incorporated herein by reference.
[0211] The present pharmaceutical compositions comprise at least
one miR gene product or miR gene expression-inhibition compound (or
at least one nucleic acid comprising sequences encoding them)
(e.g., 0.1 to 90% by weight), or a physiologically-acceptable salt
thereof, mixed with a pharmaceutically-acceptable carrier. In
certain embodiments, the pharmaceutical compositions of the
invention additionally comprise one or more anti-cancer agents
(e.g., chemotherapeutic agents). The pharmaceutical formulations of
the invention can also comprise at least one miR gene product or
miR gene expression-inhibition compound (or at least one nucleic
acid comprising sequences encoding them), which are encapsulated by
liposomes and a pharmaceutically-acceptable carrier. In one
embodiment, the pharmaceutical composition comprises a miR gene or
gene product that is miR-21.
[0212] Especially suitable pharmaceutically-acceptable carriers are
water, buffered water, normal saline, 0.4% saline, 0.3% glycine,
hyaluronic acid and the like.
[0213] In a particular embodiment, the pharmaceutical compositions
of the invention comprise at least one miR gene product or miR gene
expression-inhibition compound (or at least one nucleic acid
comprising sequences encoding them) that is resistant to
degradation by nucleases. One skilled in the art can readily
synthesize nucleic acids that are nuclease resistant, for example,
by incorporating one or more ribonucleotides that is modified at
the 2'-position into the miR gene product. Suitable 2'-modified
ribonucleotides include those modified at the 2'-position with
fluoro, amino, alkyl, alkoxy, and O-allyl.
[0214] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include, e.g., physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (such as, for example, calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0215] For solid pharmaceutical compositions of the invention,
conventional nontoxic solid pharmaceutically-acceptable carriers
can be used; for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0216] For example, a solid pharmaceutical composition for oral
administration can comprise any of the carriers and excipients
listed above and 10-95%, preferably 25%-75%, of the at least one
miR gene product or miR gene expression-inhibition compound (or at
least one nucleic acid comprising sequences encoding them). A
pharmaceutical composition for aerosol (inhalational)
administration can comprise 0.01-20% by weight, preferably 1%-10%
by weight, of the at least one miR gene product or miR gene
expression-inhibition compound (or at least one nucleic acid
comprising sequences encoding them) encapsulated in a liposome as
described above, and a propellant. A carrier can also be included
as desired; e.g., lecithin for intranasal delivery.
[0217] The pharmaceutical compositions of the invention can further
comprise one or more anti-cancer agents. In a particular
embodiment, the compositions comprise at least one miR gene product
or miR gene expression-inhibition compound (or at least one nucleic
acid comprising sequences encoding them) and at least one
chemotherapeutic agent. Chemotherapeutic agents that are suitable
for the methods of the invention include, but are not limited to,
DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic
agents, tubulin stabilizing agents, tubulin destabilizing agents,
hormone antagonist agents, topoisomerase inhibitors, protein kinase
inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors,
caspase inhibitors, metalloproteinase inhibitors, antisense nucleic
acids, triple-helix DNAs, nucleic acids aptamers, and
molecularly-modified viral, bacterial and exotoxic agents. Examples
of suitable agents for the compositions of the present invention
include, but are not limited to, cytidine arabinoside,
methotrexate, vincristine, etoposide (VP-16), doxorubicin
(adriamycin), cisplatin (CDDP), dexamethasone, arglabin,
cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil,
5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D,
mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan,
leucovorin, carmustine, streptozocin, CPT-11, taxol, tamoxifen,
dacarbazine, rituximab, daunorubicin,
1-.beta.-D-arabinofuranosylcytosine, imatinib, fludarabine,
docetaxel, FOLFOX4.
[0218] There is also provided herein methods of identifying an
inhibitor of tumorigenesis, comprising providing a test agent to a
cell and measuring the level of at least one miR gene product in
the cell. In one embodiment, the method comprises providing a test
agent to a cell and measuring the level of at least one miR gene
product associated with decreased expression levels in cancer
cells. An increase in the level of the miR gene product in the cell
after the agent is provided, relative to a suitable control cell
(e.g., agent is not provided), is indicative of the test agent
being an inhibitor of tumorigenesis. In a particular embodiment, at
least one miR gene product associated with decreased expression
levels in cancer cells is selected from the group consisting
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof.
[0219] In other embodiments, the method comprises providing a test
agent to a cell and measuring the level of at least one miR gene
product associated with increased expression levels in cancer
cells. A decrease in the level of the miR gene product in the cell
after the agent is provided, relative to a suitable control cell
(e.g., agent is not provided), is indicative of the test agent
being an inhibitor of tumorigenesis. In a particular embodiment, at
least one miR gene product associated with increased expression
levels in cancer cells is selected from the group consisting of
miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations
thereof.
[0220] Suitable agents include, but are not limited to drugs (e.g.,
small molecules, peptides), and biological macromolecules (e.g.,
proteins, nucleic acids). The agent can be produced recombinantly,
synthetically, or it may be isolated (i.e., purified) from a
natural source. Various methods for providing such agents to a cell
(e.g., transfection) are well known in the art, and several of such
methods are described hereinabove. Methods for detecting the
expression of at least one miR gene product (e.g., Northern
blotting, in situ hybridization, RT-PCR, expression profiling) are
also well known in the art. Several of these methods are also
described hereinabove.
[0221] The invention will now be illustrated by the following
non-limiting examples.
Example 1
MicroRNA Expression Patterns are Altered in Colon Tumors
[0222] We compared microRNA profiles of 84 pairs of colon tumorous
and adjacent nontumorous tissues using microRNA microarrays.sup.30.
These 84 subjects were patients recruited from the greater
Baltimore, Md. area with incident colon adenocarcinoma and are
referred to as the Maryland test cohort (Table 1).
TABLE-US-00001 TABLE 1 Characteristics of Population and Tumors
Maryland Test Hong Kong Cohort Validation Cohort N = 84 N = 113
Baltimore, Hong Recruitment area Maryland, USA Kong, China Age at
enrollment - yr Mean .+-. SD 64.6 .+-. 10.7 55.8 .+-. 15 Range
32-87 32-84 Sex - no. (%) Male 66 (79) 56 (50) Female 18 (21) 57
(50) Race - no. (%) White 52 (62) 0 (0) Black 32 (38) 0 (0) Asian 0
(0) 113 (100) Tumor location - no. (%) Distal 48 (59) 90 (80)
Proximal 34 (41) 23 (20) Adenocarcinoma Histology - no. (%)
Adenocarcinoma 75 (89) 105 (93) Mucinous adenocarcinoma 8 (10) 7
(6) Adenosquamous carcinoma 1 (1) 0 (0) Signet ring cell and
mucinous 0 (0) 1 (1) Adjuvant Chemotherapy2 - no. (%) Received 22
(37) 40 (35) Did not receive 37 (63) 73 (65) TNM Stage - no. (%) 8
(1) 9 (8) II 29 (34) 37 (33) III 36 (43) 48 (42) IV 10 (12) 19 (17)
.sup.1Distal includes tumors located in or distal to the descending
colon. Proximal tumors include tumors in or proximal to the splenic
flexure. Tumor location was available for 82 subjects in the
original cohort and all subjects in the validation cohort.
.sup.2Detailed information pertaining to receipt of chemotherapy
was available for 59 subjects in the test cohort and all subjects
in the validation cohort. Chemotherapy was primarily
fluorouracil-based (in forms of either intravenous 5-fluorouracil
or oral drugs including tegafur with uracil [UFT]) with or without
Levamisole or Leucovorin.
[0223] Tumor microRNA profiles were distinctly different than
nontumor profiles. Thirty-seven independent microRNAs were found to
be differentially expressed in tumors (p<0.001 with false
discovery rate <0.5%; Table 2.
TABLE-US-00002 TABLE 2 Fold Chromosomal Probe mature miR
p-value.sup.1 FDR.sup.2 Change location MicroRNAs the are
Differentially Expressed in Tumors hsa-mir-21No1 miR-21 <1e-07
<1e-07 1.7 17q23.2 hsa-mir-021-prec-17No1 miR-21 <1e-07
<1e-07 1.8 17q23.2 hsa-mir-092-prec-13 = 092-1No2 miR-92
<1e-07 <1e-07 1.4 13g31.3 hsa-mir-222-precNo2 miR-222
1.40E-06 8.05E-05 1.2 Xp11.3 hsa-mir-181b-2No1 miR-181b 1.90E-06
8.74E-05 1.2 9q33.3 hsa-mir-210-prec mIR-210 1.12E-05 0.00032 1.2
11p15.5 hsa-mir-020-prec miR-20a 2.53E-05 0.00057 1.5 13q31.3
hsa-mir-106-prec-X miR-106a 3.30E-05 0.00058 1.4 X26.2
hsa-mir-106aNo1 miR-106a 3.51E-05 0.00058 1.4 X26.2
hsa-mir-093-prec-7.1.sup.=093-1 miR-93 3.52E-05 0.00058 1.2 7q22.1
hsa-mir-335No2 miR-335 3.55E-05 0.00058 1.2 7q32.2
hsa-mir-222-precNol miR-222 4.27E-05 0.00065 1.2 Xpll.3
hsa-mir-338Nol miR-338 5.78E-05 0.00074 1.1 17q25.3 hsa-mir-133bNo2
miR-133b 6.50E-05 0.00079 1.1 6p12.2 hsa-mir-092-prec-X = 092-2
miR-92 7.95E-05 0.00083 1.4 Xq26.2 hsa-mir-346Nol miR-346 8.42E-05
0.00084 1.2 10q23.2 hsa-mir-106bNo1 miR-106b 0.0002091 0.00178 1.2
7q22.1 hsa-mir-135-2-prec miR-153a 0.0002363 0.00194 1.1 12q23.1
hsa-mir-219-lNo2 miR-219 0.0002515 0.00199 1.3 9q34.11
hsa-mir-34aNo1 miR-34a 0.000265 0.00203 1.1 lp36.22
hsa-mir-099b-prec-19No1 miR-99b 0.0003758 0.00259 1.1 19q13.41
hsa-mir-185-precNo2 miR-185 0.0003827 0.00259 1.2 22q11.21
hsa-mir-223-prec miR-223 0.0004038 0.00265 1.4 Xq12
hsa-mir-211-precNo2 miR-211 0.0004338 0.00277 1.1 15q13.3
hsa-mir-135-1-prec miR-135a 0.0004648 0.00287 1.1 3p21.1
hsa-mir-127-prec miR-127 0.0004748 0.00287 1.1 14q32.31
hsa-mir-203-precNol miR-203 0.0004993 0.00294 1.4 14q32.33
hsa-mir-212-precNol miR-212 0.0006339 0.00364 1.1 17p13.3
hsa-mir-095-prec-4 miR-95 0.0006996 0.00392 1.2 4p16.1
hsa-mir-017-precNo2 miR-17-5p 0.0007252 0.00392 1.3 13q31.3
MicroRNAs with reduced Expression in Tumors hsa-mir-342No2 miR-342
4.00E-06 0.00015 0.9 14q32.2 hsa-mir-192-2/3Nol miR-192 8.70E-06
0.00029 0.7 11q13.1 hsa-mir-1-2No2 miR-1 2.22E-05 0.00057 0.9
18g11.2 hsa-mir-34bNo2 miR-34b 4.78E-05 0.00069 0.8 11q23.1
hsa-mir-215-precNol miR-215 5.26E-05 0.00071 0.7 1q41
hsa-mir-192No1 miR-192 7.36E-05 0.00081 0.7 11q13.1 hsa-mir-301 No2
miR-301 7.44E-05 0.00081 0.7 17q23.2 hsa-miR-324-5pNo2 miR-324-5p
1.00E-04 0.00096 0.9 17p13.1 hsa-mir-030a-precNo2 miR-30a-3p
0.0001933 0.00171 0.9 6q13 hsa-mir-1-1 No2 miR-1 0.0002906 0.00216
0.9 20q13.33 hsa-mir-34cNo2 miR-34c 0.0007334 0.00392 0.9 11q23.1
hsa-mir-331 No2 miR-331 0.0008555 0.00446 0.9 12q22 hsa-mir-148bNo2
miR-148b 0.0008726 0.00446 0.9 12q13.13 .sup.1P-values reported are
the result of paired class comparison analysis of microRNA
expression patterns from 84 pairs colon adenocarcinomas and
nontumorus tissue. .sup.2FDR = False Discovery Rate
[0224] Twenty-six microRNAs were expressed at higher levels in
tumors with miR-21 enriched the most at 1.8-fold. Global microRNA
profiles distinguish between tumor and paired nontumorous tissue
with 89% accuracy using either the 3-nearest neighbors or nearest
centroid class prediction algorithms (10-fold cross validation
repeated 100 times), suggesting a systematic change in microRNA
expression patterns during tumor formation.
[0225] We chose miR-20a, miR-21, miR-106a, miR-181b and miR-203 for
validation based on their expression differences between tumor and
paired nontumorous tissue combined with their association to poor
survival. For validation, we measured the expression levels of
these microRNAs with qRT-PCR in tumor and paired nontumorous tissue
from an independent cohort. The validation cohort consists of 113
patients recruited from Hong Kong, China with incident colon cancer
(Table 1).
[0226] MiR-20a (2.3-fold), miR-21 (2.8-fold), miR106a (2.4-fold),
miR-181 1b (1.4-fold) and miR-203 (1.8-fold) were all expressed at
higher levels in tumors (p<0.001, Wilcoxon matched pairs test)
(Table 3a).
[0227] Table 3--MicroRNA Expression in Tumors Vs. Paired
Nontumorous Tissue
TABLE-US-00003 TABLE 3a the Hong Kong Validation Cohort Fold change
in microRNA .DELTA..DELTA.Ct.sup.1 SD (.DELTA..DELTA. Ct)
tumors.sup.2 p-value.sup.3 miR-20a 1.18 0.97 2.3 fold p < 0.001
miR-21 1.47 1.20 2.8 fold p < 0.001 miR-106a 1.25 0.94 2.4 fold
p < 0.001 miR-181 b 0.47 1.03 1.4 fold p < 0.001 miR-203 0.83
1.40 1.8 fold p < 0.001
TABLE-US-00004 TABLE 3b MicroRNA Expression in Adenoma vs. Paired
Non-adenoma Tissue Average Fold change in microRNA .DELTA..DELTA.
Ct.sup.1 SD (.DELTA..DELTA. Ct) adenomas.sup.2 p-value.sup.3
miR-20a -0.11 0.97 0.9 fold p = 0.82 miR-21 0.64 0.90 1.6 fold p =
0.006 miR-106a 0.28 1.22 1.2 fold p = 0.19 miR-181 b 0.30 1.24 1.2
fold p = 0.27 miR-203 0.77 1.98 1.7 fold p = 0.14
[0228] Most tumors (89% for miR-20a, 87% for miR-21, 90% for
miR-106a, 71% for miR-181b and 74% for miR-203) had higher
expression of these microRNAs than paired nontumorous tissue.
Expression patterns for these five microRNAs distinguish tumor
versus paired nontumor status with 96% or 98% accuracy based on
3-nearest neighbors or nearest centroid algorithms, respectively
(10-fold cross validation, repeated 100 times). .sup.1Average
(tumor .DELTA.Ct-paired non-tumor .DELTA.ct) or Average (adenoma
.DELTA.Ct-paired nonadenoma .DELTA.Ct) from qRT-PCR.
.sup.2Calcluated by 2.sup..DELTA..DELTA. 3Wilcoxon matched pairs
test. SD=standard deviation. Bolded numbers are statistically
significant. For the tumor/nontumor comparisons, 113 pairs of
tissues were used for miR-20a and miR-203 while 111 pairs of tissue
were used for miR-21, miR-106a, and miR-181b. For all
adenoma/non-adenoma comparisons, 18 pairs of tissue were used.
[0229] We used in situ hybridization to visualize miR-21 expression
in tumor and adjacent non-tumor tissue (see FIGS. 1a-f).
[0230] MiR-21 is expressed at high levels in both the nuclei and
cytoplasm of colonic epithelial cells in human tumor tissue
compared to adjacent nontumorous tissue. These results are
consistent with the qRT-PCR and microarray data and support a role
for microRNAs in carcinogenesis.
[0231] MiR-21 is Expressed at Higher Levels in Colon Adenomas
[0232] Adenomas represent a precursor stage for colon
adenocarcinomas. We tested miR-20a, miR21, miR-106a, miR-181b and
miR-203 expression levels by qRT-PCR in 18 pairs of adenoma and
adjacent nonadenoma tissue. Although four of five microRNAs showed
increased levels in adenoma tissue, only miR-21 was significantly
enriched at 1.6-fold higher (p=0.006, Wilcoxon matched pairs test)
(see Table 3b).
[0233] Adenoma tissue expressed higher levels of miR-21 in 15/18
matched pairs. More advanced stages of tumors express higher levels
of miR-21. Subjects were stratified based on the diagnosis of
adenoma and TNM staging, where adenoma was considered the least
advanced and TNM Stage IV was most advanced. Adenomas expressed
lower levels of miR-21 expression than tumors from the validation
cohort (p<0.001, Mann-Whitney test). More advanced tumors
expressed higher levels of miR-21 expression (test for trend,
p<0.001) (see FIG. 1g).
[0234] This trend was also observed using microRNA microarray data
from the Maryland test cohort (p=0.04) (see FIG. 2).
[0235] High miR-21 Expression Predicts a Poor Prognosis in Two
Independent Cohorts
[0236] We analyzed individual microRNA tumor/nontumor (T/N)
expression ratios to determine if any were associated with poor
prognosis. T/N microRNA expression ratios were classified as high
based on highest tertile. We searched for any microRNA where high
TIN ratios were associated with cancer survival (p<0.05). From
those, we selected microRNAs that were differentially expressed in
tumors (p<O.001). Five microRNAs satisfied these criteria.
Kaplan-Meier analysis indicated that high T/N ratios for miR-20a
(p=0.02), miR-21 (p=0.004), miR-106a (p=0.01), miR-181b (p=0.04),
and miR-203 (p=0.004) were each associated with a poor
survival.
TABLE-US-00005 TABLE 4a Maryland Test Cohort Univariate analysis
Multivariate analysis2 Characteristic HR (95% CI) p-value HR (95%
CI) p-value miR-21 expression.sup.3 N = 71 Low 1.0 1.0 High 2.5
(1.2-5.2) 0.01 2.9 (1.4-6.1) 0.004 TNM Stage I-II 1.0 1.0 III-IV
3.5 (1.6-7.9) 0.002 3.4 (1.5-7.8) 0.004 Age at enrollment <50
1.0 .gtoreq.50 0.7 (0.2-2.3) 0.52 Sex Female 1.0 Male 1.4 (0.5-3.9)
0.57 Race White 1.0 Black 1.0 (0.5-2.1) 0.97 Tumor Location Distal
1.0 Proximal 0.6 (0.3-1.4) 0.26
TABLE-US-00006 TABLE 4b Hong Kong Validation Cohort Univariate
analysis Multivariate analysis.sup.2 Characteristic HR (95% CI)
p-value HR (95% CI) p-value miR-21 expression.sup.3 n = 103 Low 1.0
1.0 High 2.4 (1.4-3.9) 0.002 2.4 (1.4-4.1) 0.002 TNM Stage I-II 1.0
1.0 III-IV 4.7 (2.4-9.5) <0.001 4.7 (2.4-9.5) <0.001 Age at
enrollment <50 1.0 .gtoreq.50 1.5 (0.9-2.6) 0.14 Sex Female 1.0
Male 1.4 (0.8-2.3) 0.29 Tumor Location Distal 1.0 Proximal 0.7
(0.3-1.4) 0.27 MicroRNA expression was measured with miRNA
microarrays for the Maryland cohort and with qRT-PCR with the Hong
Kong cohort. .sup.1Cases with mucinous adenocarcinoma,
adenosquamous carcinoma or signet ring cell carcinomas were
excluded from this analysis. .sup.2Multivariate analysis used
stepwise addition and removal of clinical covariates found to be
associated with survival in univariate models (p < 0.10) and
final models include only those covariates which were significantly
associated with survival (Wald statistic p < 0.05). .sup.3High
expression in tumors for all miRNAs was defined based on the
highest tertile.
[0237] These five microRNAs were selected for further analysis.
[0238] Colon adenocarcinomas from 89-93% of the subjects in this
study were of a typical histology. A minority of tumors were of
mucinous adenocarcinoma, adenosquamous carcinoma, or signet ring
cell carcinoma histologies (see Table 1). Different subtypes of
adenocarcinomas can be associated with different clinical outcomes,
including survival prognosis. To remove potential confounding
associated with histology, we excluded all subjects with mucinous
adenocarcinomas, adenosquamous carcinomas and signet ring cell
carcinomas from the initial analysis.
[0239] Associations of T/N ratios with poor survival could be due
to microRNA expression levels in the tumor tissue, the surrounding
nontumorous tissue, or a combination of both. To distinguish these
possibilities we analyzed the association of microRNA expression in
tumors and paired nontumors separately. High expression levels in
tumors (based on highest tertile) for miR-20a, miR-21, miR106a,
miR-181b and miR-203 were each associated with a poor survival in
the Maryland test cohort (see FIG. 3a, also from data not shown).
No significant association with microRNA expression in nontumorous
tissue was observed for any of the five microRNAs.
[0240] Univariate and multivariate Cox proportional hazards
analysis was used to evaluate the association of tumor expression
levels with prognosis in individuals with typical adenocarcinoma
(Table 4a).
[0241] Table 4--Univariate and Multivariate Cox Regression Analysis
of miR-21 Expression Levels and Overall Cancer Survival in Subjects
with Colon Adenocarcinoma
[0242] Individuals with tumors expressing high levels of miR-21
were at a significantly higher risk of dying from colon cancer in
both univariate (HR=2.5 [1.2-5.2], p=0.01) and multivariate (HR=2.9
[1.4-6.1], p=0.004) analyses.
[0243] To validate these findings, we used qRT-PCR to measure tumor
and nontumor expression levels for these five microRNAs in the Hong
Kong validation cohort and analyzed associations with prognosis.
High miR-21 tumor expression predicts a poor prognosis in the Hong
Kong validation cohort (p=0.001, Kaplan-Meier log rank test) while
expression in nontumorous tissue does not (see FIG. 3b).
[0244] We did not find statistically significant associations with
prognosis and expression of miR-20a, miR-106a, 181b or miR-203 in
this cohort.
[0245] High miR-21 expression in tumors was not significantly
associated with age, gender, tumor histology, or tumor location
(Fisher's exact test) in the Hong Kong validation cohort. All
covariates were examined by Cox proportional hazards analysis
(Table 4b).
[0246] High miR-21 expression in tumors (HR.sup.=2.4 [1.4-3.9],
p.sup.=0.002) and TNM staging (HR=4.7 [2.4-9.5], p<0.001) were
significantly associated with survival in univariate models.
Multivariate Cox regression analysis demonstrated that high miR-21
expression in tumors predicts poor survival prognosis (HR=2.4
[1.4-4.1], p=0.002) independent of other clinical covariates,
consistent with our findings in the Maryland test cohort.
[0247] We repeated the analysis including all subjects regardless
of tumor histology. In both cohorts, the association with high
miR-21 expression and prognosis remained (See FIG. 4, See Table
5).
[0248] Table 5--Univariate and Multivariate Cox Regression Analysis
of miR-21 Expression Levels and Overall Cancer Survival in Subjects
with all Subjects
TABLE-US-00007 TABLE 5a Maryland Test Cohort Univariate analysis
Multivariate analysis Characteristic HR (95% CI) p-value HR (95%
CI) p-value miR-21 expression.sup.3 N = 79 Low 1.0 1.0 High 2.0
(1.1-4.0) 0.04 2.1 (1.1-4.0) 0.03 TNM Stage I-II 1.0 1.0 III-IV 3.2
(1.5-6.9) 0.002 3.2 (1.5-6.8) 0.003 Age at enrollment <50 1.0
.gtoreq.50 0.7 (0.2-2.4) 0.59 Sex Female 1.0 Male 1.6 (0.7-4.2)
0.33 Race White 1.0 Black 1.0 (0.5-2.0) 0.99 Tumor Location Distal
1.0 Proximal 0.8 (0.3-2.1) 0.65 Histology 1.0 Adenocarcinoma
Mucinous or 0.7 (0.3-2.1) 0.57 Adenosquamous
TABLE-US-00008 TABLE 5b Hong Kong Validation Cohort Multivariate
analysis.sup.2 Univariate analysis Multivariate analysis.sup.2
Characteristic HR (95% CI) p-value HR (95% CI) p-value miR-21
expression.sup.3 n = 111 Low 1.0 1.0 High 2.3 (1.4-3.9) 0.002 2.3
(1.4-3.9) 0.002 TNM Stage I-II 1.0 1.0 III-IV 4.9 (2.5-97)
<0.001 4.9 (2.5-98) <0.001 Age at enrollment <50 1.0
.gtoreq.50 1.4 (0.8-2.4) 0.20 Sex Female 1.0 Male 1.3 (0.8-2.3)
0.27 Tumor Location Distal 1.0 Proximal 0.7 (0.3-1.4) 0.27
Histology 1.0 Adenocarcinoma Mucinous or 1.2 (0.4-3.3) 0.74
Adenosquamous MicroRNA expression was measured with miRNA
microarrays for the Maryland cohort and with qRT-PCR with the Hong
Kong cohort. .sup.1All individuals were included in this analysis
regardless of tumor histology. .sup.2Multivariate analysis used
stepwise addition and removal of clinical covariates found to be
associated with survival in univariate models (p < 0.10) and
final models include only those covariates which were significantly
associated with survival (Wald statistic p < 0.05). .sup.3High
expression in tumors for all miRNAs was defined based on the
highest tertile.
[0249] MiR-21 Expression Levels and Response to Therapy
[0250] Identifying biomarkers associated with a response to
adjuvant chemotherapy will allow physicians to better predict the
benefits of therapy. To this end, we analyzed associations with
miR-21 expression and the response to adjuvant chemotherapy in
stage II and III cancer patients. Information on the administration
of adjuvant chemotherapy was available for 47 of 65 stage II or III
subjects in the Maryland test cohort and all subjects in the Hong
Kong validation cohort.
[0251] In both cohorts, chemotherapy regimens were primarily
fluorouracil-based (in forms of either intravenous 5fluorouracil or
oral drugs including tegafur with uracil [UFT]) with or without
Levamisole or Leucovorin. Only subjects with typical adenocarcinoma
histology were used for this analysis, leaving 20 of 42 stage
II/III individuals who received chemotherapy in the Maryland
cohort. For those who received chemotherapy, high miR-21 expression
in tumors predicted worse overall survival (p=0.01, Kaplan-Meier
log rank test) giving preliminary support that high miR-21 is
associated with poor response to adjuvant chemotherapy.
[0252] For the Hong Kong validation cohort, 77 individuals with
stage II/III cancer with typical adenocarcinoma histology were used
for this analysis. Stage II/III subjects who received adjuvant
chemotherapy had better survival prognosis than those who did not
(p=0.02, Kaplan-Meier log rank test). Among those subjects that
received adjuvant chemotherapy (n=36), high miR-21 expression in
tumors was associated with a poor response to treatment (p=0.03,
Kaplan-Meier log rank test), consistent with observations in the
Maryland cohort (see FIG. 5a).
[0253] In this cohort, all stage II subjects who received adjuvant
chemotherapy (n=11) survived (see FIG. 5b), but for stage III
subjects who received adjuvant chemotherapy (n=25), high miR-21
expression was associated with poor survival (p=0.02, Kaplan-Meier
log rank test) (see FIG. 5c).
[0254] Multivariate Cox regression analysis was used to analyze
these observations to show that high miR-21 expression predicted a
poor prognosis (HR=3.1 [1.5-6.1]; p=0.001) and receiving
chemotherapy was predictive of improved survival outcomes (HR=0.3
[0.1-0.5]; p<0.001) independent of other clinical covariates
(Table 6a).
[0255] Table 6--Univariate and Multivariate Cox Regression Analysis
of miR-21 Expression, Receipt of Adjuvant Chemotherapy and Cancer
Survival in Stage I/III.sup.1 Subjects with Adenocarcinoma
TABLE-US-00009 TABLE 6a Maryland Test Cohort Univariate analysis
Multivariate analysis.sup.2 Characteristic HR (95% CI) p-value HR
(95% CI) p-value miR-21 expression.sup.3 N = 77 Low 1.0 1.0 High
2.6 (1.3-5.1) 0.005 3.1 (1.5-6.1) 0.001 Adjuvant Chemotherapy Did
not receive 1.0 1.0 Received 04. (0.2-0.8) 0.01 0.3 (0.1-0.5)
<0.001 TNM Stage II 1.0 1.0 III 2.8 (1.3-6.0) 0.008 5.4 (2.4-12)
<0.001 Tumor Location Distal 1.0 1.0 Proximal 0.3 (0.1-1.0) 0.04
0.2 (0.1-0.8) 0.02 Age at enrollment <50 1.0 .gtoreq.50 1.6
(0.8-3.1) 0.20 Sex Female 1.0 Male 1.2 (0.6-2.3) 0.61
TABLE-US-00010 TABLE 6b Hong Kong Validation Cohort Multivariate
analysis.sup.2 Univariate analysis Multivariate analysis.sup.2
Characteristic HR (95% CI) p-value HR (95% CI) p-value miR-21
expression.sup.3 N = 119 Low 1.0 1.0 High 2.6 (1.5-4.5) 0.001 3.0
(1.7-5.4) <0.001 Adjuvant Chemotherapy Did not receive 1.0 1.0
Received 07. (0.4-1.2) 0.21 0.4 (0.2-0.8) 0.004 TNM Stage II 1.0
1.0 III 3.2 (1.7-6.1) 0.001 5.2 (2.6-11) <0.001 Tumor Location
Distal 1.0 1.0 Proximal 0.4 (0.2-0.8) 0.02 0.3 (0.1-0.7) 0.007 Age
at enrollment <50 1.0 .gtoreq.50 1.4 (0.7-2.5) 0.32 Sex Female
1.0 Male 1.3 (0.7-2.2) 0.44 Expression of miRNAs was measured with
qRT-PCR. .sup.1TNM stage II/III subjects with typical
adenocarcinoma histology were included in this analysis.
.sup.2Multivariate analysis used stepwise addition and removal of
clinical covariates found to be associated with survival in
univariate models (p < 0.10) and final models include only those
covariates which were significantly associated with survival (Wald
statistic p < 0.05). .sup.3High expression in tumors for all
miRNAs was defined based on the highest tertile. Race was not
associated with poor prognosis.
[0256] Analyses using cancer relapse as an endpoint instead of
cancer death resulted in similar associations with high miR-21
expression in tumors predicting a more rapid disease recurrence
(data not shown).
[0257] An analysis combining both cohorts resulted in similar
associations. Kaplan-Meier analysis demonstrated that high miR-21
expression predicted a poor prognosis in either stage II (p=0.02)
or stage III (p=0.004) subjects (See FIG. 6).
[0258] High miR-21 expression predicted a poor response to
chemotherapy in stage II/III subjects (p=0.003) or in stage III
subjects alone (p=0.007). Multivariate Cox regression demonstrated
that high miR-21 expression predicted poor prognosis (HR=3.0
[1.7-5.4]; p<0.001) and treatment with adjuvant chemotherapy
predicted improved survival (HR=0.4 [0.2-0.8]; p=0.004) independent
of other clinical covariates (Table 6b).
[0259] Discussion
[0260] We analyzed microRNA profiles in colon cancer tissues using
two independent cohorts. Thirty-seven microRNAs were differentially
expressed in tumor tissues by microRNA microarray analysis.
Expression patterns of all five tested microRNAs were validated in
the Hong Kong cohort. The discriminatory power of five microRNAs to
differentiate between tumor and nontumorous tissue indicates that
predictable and systematic changes of microRNA expression patterns
occur during tumorigenesis and are likely representative of the
majority of sporadic colon adenocarcinomas.
[0261] MiR-20a, miR-21, miR-106a, miR-181b and miR-203 were all
found to be expressed at higher levels in colon tumors. These
changes in microRNA expression patterns may be merely associated
with colon cancer or causal to the histologic progression to
cancer. There is strong evidence suggesting that changes in
microRNA expression patterns promote tumor formation, especially
for miR-20a and miR-21. MiR-20a is part of the miR-17-92
polycistronic microRNA cluster.
[0262] Overexpression of this cluster enhances cell proliferation
in vitro and accelerates tumor formation in animal models. Enforced
expression of the miR-17-92 cluster causes increased tumor size and
tumor vascularization in mice by negatively regulating the
anti-angiogenic Tsp1 protein. Experimental evidence also suggests
that increased miR-21 expression promotes tumor development. MiR-21
is expressed at high levels in most solid tumors. Overexpression of
miR-21 acts as an anti-apoptotic factor in human glioblastoma cells
Inhibition of miR-21 inhibits cell growth in vitro and inhibits
tumor growth in xenograft mouse models through an indirect
downregulation of the anti-apoptotic factor Bcl-2. Studies in human
cell lines have shown miR-21 can also target the tumor suppressor
genes PTEN.sup.3 and TPM1. All of these data taken together support
a causal role for altered microRNA expression during
tumorigenesis.
[0263] Adenomas represent a precursor stage of adenocarcinoma.
Adenomas express high levels of miR-21. If increased miR-21
expression promotes colon tumor progression, increased expression
in adenomas may be an early cellular event in the progression to
cancer Inhibiting miR-21 activity may help prevent tumor promotion
in populations at high risk for colon cancer, such as individuals
with familial adenomatous polyposis.
[0264] Thus, there is presented herein evidence that demonstrates
an association with microRNA expression patterns with colon cancer
prognosis and response to adjuvant chemotherapy. More advanced
tumors express higher levels of miR-21. A robust association with
high miR-21 expression in tumors and poor survival was observed in
the Maryland test cohort and the Hong Kong validation cohort,
separately.
[0265] In each cohort, these associations were independent of all
other clinical covariates indicating that miR-21 expression may be
a useful prognostic indicator, in addition to TNM staging and other
clinical parameters, to help identify patients at a higher risk of
terminal cancer. These observations were made in two independent
cohorts with very different racial and geographical compositions.
Therefore, it is likely that our observations are broadly
applicable to other populations.
[0266] High miR-21 expression in tumors was associated with a poor
response to adjuvant chemotherapy in both cohorts. These results
can help predict the benefits of therapy in individuals whose
miR-21 expression status is known. In addition, if high miR-21
expression is causal to the poor survival of colon cancer patients,
antagomirs or other antisense therapeutics that target miR-21 can
have therapeutic benefits in subjects with high miR-21 expressing
tumors. These may be used in addition to current therapies to
improve survival outcomes.
[0267] The inventors herein have found systematic differences in
microRNA expression patterns between colon tumors and paired
nontumorous tissue. High miR-21 expression in tumors predicts poor
survival outcome and poor response to adjuvant chemotherapy in two
independent cohorts, independent of staging and other clinical
covariates suggesting that it may be a useful diagnostic biomarker
for colon adenocarcinomas and survival prognosis including response
to therapy.
[0268] Methods
[0269] Tissue Collection and RNA Isolation:
[0270] Pairs of primary colon tumor and adjacent nontumorous
tissues came from 84 patients recruited from the University of
Maryland Medical Center between 1993 and 2002, and from 113
patients recruited from Queen Mary Hospital in Hong Kong between
1991 and 2000. Detailed backgrounds for each tissue donor,
including age, gender, clinical staging, tumor location, survival
times from diagnosis and receipt of adjuvant chemotherapy have been
collected. Tumor histopathology was classified according to the
World Health Organization Classification of Tumor system'. The
adenoma tissue was obtained from the Cooperative Human Tissue
Network. This study was approved by the Institutional Review Board
of the National Institutes of Health, the Institutional Review
Board of the University of Hong Kong/Hospital Authority Hong Kong
West Cluster and the Institutional Review Board for Human Subject
Research at the University of Maryland.
[0271] RNA Isolation and microRNA Profiling:
[0272] RNA was extracted from tissue using standard TRIZOL
(Invitrogen, Carlsbad) methods. MicroRNA microarray profiling was
performed as previously described. Briefly, 5 lag of total RNA was
labeled and hybridized to each microRNA microarray containing
quadruplicates of approximately 400 human microRNA probes. Slides
were scanned using a PerkinElmer ScanArray LX5K scanner. qRT-PCR of
microRNAs was performed using Taqman MicroRNA assays (Applied
Biosystems, Foster City) according to manufacturer's instructions
with the 7500 real time RT-PCR system (Applied Biosystems, Foster
City). U6B was the normalization control for all qRT-PCR
experiments. All assays were performed in duplicate (miR-20a,
miR-203) or triplicate (miR-21, miR-106a, miR-181b). qRT-PCR for
miR-21, miR-106a and miR-181b was performed by AJS, who was blinded
to the survival outcomes and clinical data for members of the
validation cohort at that time.
[0273] Microarray Analysis:
[0274] The data discussed in this publication have been deposited
in NCBIs Gene Expression Omnibus (GEO, ncbi.nlm.nih.gov/geo/) and
are accessible through GEO Series accession number GSE7828. LOESS
normalized microarray data were imported into BRB array tools 3.5.0
(linus.nci.nih.gov/.BRB-ArrayTools.html) and all subsequent
microarray analyses were performed with this software.
[0275] Microarray analyses were performed. Probes with values
missing from >20% of the arrays were removed from the analysis
leaving 230 probes. Paired, class comparison analysis identified
microRNAs that were differentially expressed in tumors
(p<0.001).
[0276] To initially search for microRNAs associated with poor
survival, tumor/nontumor (T/N) microRNA expression ratios were
analyzed in the Maryland cohort using microarray data. TN
expression ratios for microRNAs were created by subtracting the
log.sub.2 nontumor from the log.sub.2 tumor expression values.
MicroRNAs missing >25% of T/N ratios were filtered out leaving
208. T/N expression ratios were dichotomized with the highest
tertile classified as high and the lower 2 tertiles classified as
low (see Supplemental Methods). This high/low cutoff was used
universally throughout this study. Tumor and nontumor microRNA
expression levels were batch normalized based on the date of
microarray experiments for all analysis of associations with
survival.
[0277] In Situ Hybridization:
[0278] In situ hybridization (ISH) was performed with probes for
human miR-21, scramble, and U6 (Exiqon, Woburn) with a modified
version of the manufacturer's protocol for formalin-fixed
paraffin-embedded (FFPE) tissue written by W. Kloosterman
(exiqon.com/uploads/.LNA 52-FFPE miRNA in situj,rotocol.pdf) on
human colon tissue. Modifications included the use of polyclonal
rabbit anti-DIG/HRP-conjugated antibody and DakoCytomation GenPoint
Tyramide Signal Amplication System (DakoCytomation, Carpinteria),
and VECTOR.RTM. NovaRed.TM. substrate (Vector Laboratories,
Burlingame). Images were taken on an Olympus BX40 microscope using
the Olympus DP70 digital camera and DP controller software
(Olympus, Champaign).
[0279] Statistical Analysis:
[0280] Statistical analyses were performed. Wilcoxon matched pairs
tests were used to analyze differences in microRNA expression
between tumors and paired nontumorous tissue as well as differences
between adenoma and paired non-adenoma tissue for all qRT-PCR data.
All trend tests reported are nonparametric tests for trend across
ordered groups. All Kaplan-Meier analysis was performed with
WINSTAT 2001 (R. Fitch Software). Multivariate Cox regression
analysis was performed using Intercooled Stata 9.2 (StataCorp LP,
College Station). Final multivariate models were based on stepwise
addition and removal of clinical covariates found to be associated
with poor survival in univariate models (p<0.10). A Wald
statistic of p<0.05 was used as criteria for inclusion in final
multivariate models. All p-values reported are 2-sided. Hazards
ratios are reported with 95% confidence intervals in parentheses.
Expression graphs were made using Graphpad Prism 4.0 (Graphpad
Software Inc., San Diego).
[0281] Additional Microarray Analyses
[0282] The microarrays used for this analysis were pin-spotted
microRNA microarrays (from the Ohio State University Comprehensive
Cancer Center, version 2.0). Intensities of each spot were the
median intensities of foreground. Each of the 170 microarrays used
for this study contained 11520 spots. All spots where foreground
intensity was less than background were reassigned as NA (NA marks
missing data spots). All spots flagged as deficient by the scanner
were also reassigned as NA. All blank (no oligo) spots with high
foreground intensity were reassigned as NA. Each microRNA oligo is
represented by quadruplicate spots on these arrays as two distant
pairs of two adjacent spots. If there were 0 or 1 NA for an oligo
quadruple, and the means of the distant oligo pairs differed by
>1 on the log.sub.2 scale, all of the quadruplicate spots were
reassigned as NA. If there were 2 NAs for an oligo quadruple and
the two non-NA spot intensities differed by >1 on the log.sub.2
scale, all of the quadruplicate spots were reassigned NA. If there
were 3 NA spots for a quadruple, the final spot was reassigned as
NA. In total, 1,082,689 of 1,958,400 spots were reassigned as NA
using these methods. LOESS (Locally Weighted Scatterplot Smoothing)
normalization was performed using the R software package. All data
was then imported into BRB array tools version 3.5.0 for analysis
and all replicate spots were averaged. There were originally 85
pairs (tumor and paired nontumorous tissue) of arrays used. One
case that was originally identified as an incident colon carcinoma
patient was later found to have been diagnosed as carcinoma in situ
and was removed from the analysis leaving the study population of
84 subjects. MicroRNA lists were filtered to include only the 389
human hsa-miR probesets. They were further filtered to remove any
probeset missing from more than 25% of the arrays, leaving 230
human microRNA probesets. Paired class comparison analysis was used
to identify microRNAs that were differentially expressed between
tumor and paired nontumorous tissue. For two microRNAs (miR-181b
and miR-338), two independent probes measuring each gave
contradictory results with one probe showing higher expression in
tumors and one probe showing lower expression in tumors for each
microRNA. For each, we discarded the less significant result which
designated both miR-181b and miR-388 as enriched in tumors.
Additionally, qRT-PCR confirmed that miR-181b was enriched in
tumors.
[0283] We initially used tumor/nontumor (T/N) expression profiles
for each microRNA to search for microRNAs that were associated with
poor survival. For this analysis, we decided to dichotomize all
expression data with a universal high and low cutoff to look for
associations with poor survival. To determine what universal
high/low cutoff to use, we dichotomized the T/N expression data
three separate ways and determined which method gave the greatest
number of significant results in the test cohort. High expression
was classified based on higher than median, highest tertile, or
highest quartile and we tested associations with these cutoffs with
a poor survival using univariate Cox regression analysis. Of the 37
microRNAs that were differentially expressed in tumors, high
expression of four were associated with poor survival based on
higher than median, five based on highest tertile, and two based on
highest quartile (p<0.05, data not shown). Dichotomization based
on highest tertile gave the most microRNAs associated with poor
survival based on these criteria in the Maryland test cohort;
therefore, classification based on highest tertile was used
uniformly throughout this study to analyze associations between
microRNA expression levels and a poor prognosis in both the
Maryland test cohort and the Hong Kong validation cohort.
[0284] We used microRNA microarrays to compare miR-21 expression
levels in tumors with prognosis. The microarray probe used for this
analysis was hsa-miR-21-prec17No1. This analysis required batch
normalization of the data based on the date of the microarray
experiment. To normalize by date, arrays expressing the highest 1/3
of a given microRNA were classified as high for each day,
separately. Up to twelve pairs of tissue were profiled on any given
day. For any day in which less than 10 pairs of microarrays were
performed, arrays performed on those days were discarded, resulting
in the loss of 5 pairs of arrays. These data were then combined
together for analysis of associations with survival outcomes. We
checked and found no significant differences in the frequency
distribution of age, sex, race, tumor location, TNM stage, or
cancer survival between groups categorized based on date of
microarray experiment (Fisher's exact test).
[0285] Statistical Analyses
[0286] Cox proportional hazards regression was used to analyze the
effect of mir-21 expression levels and other clinical variable on
patient survival. Clinical variables included were age, sex, race,
tumor location, tumor histology, receipt of adjuvant therapy and
TNM staging. For these models, we chose to dichotomized age as age
>50 versus age <50 as the recommended screening age for colon
cancer is at age 50; tumor location was defined as proximal if
tumor was located within or proximal to the splenic flexure and
distal if tumor was located within or distal to the descending
colon; TNM staging was dichotomized based on metastasic versus
nonmetastasic disease resulting in stage I-II versus III-IV. One
patient in the Maryland cohort died on the day of surgery resulting
in a survival time of 0 months. This case was included in
Kaplan-Meier analysis and removed for Cox regression analysis
causing the difference in cases between miR-21 expression in tumors
for FIG. 2 (n=72) and the number of cases in the Table 4 Cox
regression analysis (n=71). Univariate Cox regression was performed
on each clinical covariate to examine influence of each on patient
survival. Final multivariate models were based on stepwise addition
and removal of clinical covariates found to be associated with poor
survival in univariate models (p<0.10). A Wald statistic of
p<0.05 was used as criteria for inclusion in final multivariate
models. The most parsimonious Cox regression model was used for the
final multivariate model.
Example 2
Initial Results
[0287] MiRNAs are Differentially Expressed in Colon Tumors
[0288] We analyzed miRNA profiles of 85 pairs of cancerous and
adjacent non-cancerous colon tissues using miRNA microarrays. We
found that miRNA expression profiles of tumors were quite different
than normal tissues suggesting that miRNAs may play significant
roles in colon carcinogenesis. Paired class comparison analysis
identified 27 independent miRNAs that were differentially expressed
in these tumors (Table 7).
TABLE-US-00011 TABLE 7 27 miRNAs are differentially expressed in
colon tumors compared to paired, normal tissue. 27 miRNAs were
found to be differentially expressed in tumors using paired class
comparisons analysis in BRB array tools 3.4. A significance value
of p < 0.001 was used as the criteria for differentially
expressed which resulted in an estimated false discovery rate of
0.08%. Up refers to miRNAs that were expressed at higher levels in
tumors while down indicates that miRNA levels were lower in tumors.
Table 7 MicroRNA Up/Downregulated P-Value 1 miR-331 Down 1.00E-07 2
miR-21 Up 1.00E-07 3 miR-34b Down 2.00E-07 4 miR-342 Down 2.00E-07
5 miR-215 Down 2.20E-05 6 miR-371 Down 7.00E-07 7 miR-373 Down
6.30E-06 8 miR-192 Down 7.70E-06 9 miR-148b Down 1.03E-05 10
miR-138 Down 1.49E-05 11 miR-301 Down 1.85E-05 12 miR-338 Down
2.63E-05 13 miR-153 Down 2.67E-05 14 miR-129 Down 3.20E-05 15
miR-222 Up 9.08E-05 16 miR-346 Up 0.000126 17 miR-204 Up 0.000244
18 miR-181 b Up 0.000263 19 let-7a-2 Down 0.000272 20 miR-106a Up
0.000305 21 miR-093 Up 0.000334 22 miR-34c Down 0.000341 23 miR-219
Up 0.000352 24 miR-019b Up 0.000364 25 miR-210 Up 0.000389 26
miR-185 Up 0.000516 27 miR-1 Down 0.00064
[0289] The false discovery rate, to account for the multiple
comparisons testing, was approximately 0.8% indicating that most,
if not all of these miRNAs are differentially expressed and not the
result of multiple comparisons testing. Eleven miRNAs were found to
have elevated expression levels in tumors while 16 miRNAs were
found to be reduced in tumors. Additionally, miRNA profiles could
be used to predict whether or not the tissue was tumor or non-tumor
with 92% accuracy. Based on 2000 random permutations, the
probability of these predictions occurring by random chance was
extremely low (p<0.0005). These results show that there are
systematic differences in mi RNA expression profiles between tumors
and normal tissue indicating that miRNA expression profiles become
altered during colon carcinogenesis.
[0290] Global miRNA Expression Profiles Predict Colon Cancer
Survival Prognosis
[0291] We determined whether miRNA expression profiles predict
patient survival. For this analysis we calculated the tumor versus
normal miRNA expression ratios (TIN ratio) for each miRNA for every
individual. Unsupervised hierarchical clustering of all miRNA TIN
ratios grouped individuals into two groups arbitrarily labeled
group A and group B (FIG. 7).
[0292] These two groups differ significantly in both clinical
staging (p=0.009; FIG. 1b) and survival prognosis (p=0.026; FIG.
7c).
[0293] This indicated that global miRNA profiles are predictive of
clinical staging and more importantly, survival prognosis.
[0294] Univariate and multivariate Cox regression analysis was used
to interrogate this relationship in more detail (Table 8).
TABLE-US-00012 TABLE 8 Cox Regression Analysis of global miRNA
Profiles Univariate (above) and multivariate (below) Cox regression
analyses were performed to show that individuals classified in
miRNA group B were at higher risk of dying of colon cancer. Neither
age, gender or race was significant contributors to survival risk.
For the purposes of these analyses, age was dichotomized into
greater than or less than 50 and race dichotomized into African
American (AA) and Caucasian. Variable HR (95% Cl) p value
Univariate Analysis Cluster B/A 2.6 (1.0-6.3) 0.042 age
.gtoreq.50/age <50 0.62 (0.14--2.7) 0.53 male/female 1.4
(0.48-4.0) 0.54 AA/Caucasian 1.1 (0.83-2.3) 0.83 Multivariate,
adjusting for age, gender and race Cluster B/A 2.7 (1.1-6.8) 0.034
age .gtoreq.50/age <50 0.49 (0.11--2.2) 0.35 male/female 1.5
(0.52-4.4) 0.45 AA/Caucasian 1.0 (0.45-2.2) 0.99
[0295] Group B individuals were to have a significantly higher risk
of dying from colon cancer (hazard ratio [HR]=2.6 (p=0.04). This
risk remained significantly high after adjusting for age, ethnicity
and gender (HR=2.7; p=0.03). These results demonstrate the
potential for using miRNA profiles of colon tumors to predict
prognosis. These results suggest that miRNAs may also play a role
in colon carcinogenesis.
[0296] Profiles of miR-21, miR-106a, miR-181b, miR-16h, miR-203,
let-7g, miR-29a, miR-103-2 and miR-10a Predict Colon Cancer
Prognosis
[0297] We identified individual miRNAs whose expression levels were
predictive of colon cancer prognosis. We used Kaplan Meier survival
plots and multivariate Cox regression analysis on TIN ratios to
identify miRNA expression patterns that were associated with poor
survival prognosis. BRB array tools were used to identify TIN
ratios correlated with poor survival (data not shown). We chose to
analyze these miRNAs in further detail. We also analyzed any miRNA
that was differentially expressed in tumors (p<0.01). TIN ratios
for each individual were dichotomized based on median or highest
quartile TIN ratios. We also removed any miRNA from the analysis
where TIN ratios were missing in greater than 18 individuals. We
identified at least 9 miRNAs, including miR-21, miR-106a, miR-181b,
miR-16h, miR-203, let-7g, miR-29a, miR-103-2 and miR-10a whose TIN
ratios are predictive of colon cancer prognosis (FIG. 8, Table
9).
[0298] Cox Regression Analysis of TIN Ratios for Individual
miRNAs.
[0299] Univariate and multivariate Cox regression analyses were
performed to show that TIN ratios of individual miRNAs could by
used to classify individuals at higher risk of dying of colon
cancer. TIN ratios for these 9 miRNAs were significant predictors
of survival prognosis independent of TNM staging, age, gender and
race. Note that High/Low distinctions for miR-16b, miR-21, miR-29a,
miR-103-2, miR-106a and miR-203 were classified based on median TIN
ratio values while let-7g, miR-10a and miR-181b were classified
based on highest quartile TIN ratios.
TABLE-US-00013 TABLE 9 Cox regression analysis of TIN ratios for
individual miRNAs Variable HR (95% Cl) p = n Univariate analysis
miR-21 High/Low 3.0 (11.3-7.0) 0.01 80 Multivariate analysis miR-21
High/Low 2.8 (1.2-6.8) 0.02 age .gtoreq.50/age <50 0.46
(0.10-2.1) 0.32 male/female 3.1 (0.9-11.0) 0.07 AA/Caucasian 1.2
(0.5-2.7) 0.66 Stage III-IV/Stage I-II 4.4 (1.6-11.9) 0.004
Univariate analysis miR-181b High/Low 3.4 (1.6-7.5) 0.002 78
Multivariate analysis miR-181b High/Low 3.3 (1.3-8.2) 0.01 age
.gtoreq.50/age <50 0.39 (0.08-1.8) 0.23 Male/female 2.2
(0.7-7.2) 0.17 AA/Caucasian 1.1 (0.5-2.5) 0.82 Stage III-IV/Stage
I-II 3.1 (1.2-8.1) 0.02 Univariate analysis let-7g High/Low
2.7(1.3-5.9) 0.01 84 Multivariate analysis let-7g High/Low 2.5
(1.1-5.5) 0.03 age .gtoreq.50/age <50 0.5 (0.1-2.4) 0.39
Male/female 1.5 (0.5-4.4) 0.50 AA/Caucasian 1.3 (0.6-2.9) 0.50
Stage III-VI/Stage I-II 3.6 (1.4-9.2) 0.006. Univariate analysis
miR-103-2 High/Low 2.5 (1.1-5.6) 0.03 81 Multivariate analysis
miR-103-2 High/Low 3.1 (1.3-7.5) 0.01 age .gtoreq.50/age <50 0.5
(0.1-2.2) 0.36 male/female 1.6 (0.6-4.9) 0.38 AA/Caucasian 0.8
(0.4-1.9) 0.69 Stage III-IV/Stage I-II 4.4 (1.7-11.1) 0.002
Univariate analysis miR-16b High/Low 4.6 (1.7-12.5) 0.003 69
Multivariate analysis miR-16b High/Low 5.1 (1.8-15.9) 0.003 age
.gtoreq.50/age <50 0.4 (0.08-1.7) 0.20 male/female 3.2 (0.8-1.7)
0.12 AA/Caucasian 0.9 (1.9-22.4) 0.003 Stage III-IV/Stage I-II 6.5
(1.9-22.4 0.003 Univariate analysis miR-106a High/Low 2.6 (1.1-6.1)
0.01 82 Multivariate analysis miR-106a High/Low 2.4 (1.0-5.7) 0.05
age .gtoreq.50/age <50 0.54 (0.11--2.5) 0.44 male/female 1.8
(0.5-6.5) 0.34 AA/Caucasian 1.1 (0.5-2.5) 0.84 Stage III-IV/Stage
I-II 5.4 (1.8-16.0) 0.002 Univariate analysis miR-203 High/Low 3.8
(1.4-10.5) 0.01 57 Multivariate analysis miR-203 High/Low 3.2
(1.1-9.4) 0.03 age .gtoreq.50/age <50 1.0 (0.1--8.1) 0.97
male/female 1.4 (0.4-5.1) 0.61 AA/Caucasian 0.9 (0.4-2.3) 0.83
Stage III-IV/Stage I-II 3.9 (1.3-11.8) 0.02 Univariate analysis
miR-29a High/Low 3.1 (1.3-7.3) 0.01 77 Multivariate analysis
miR-29a High/Low 3.2 (1.3-7.9) 0.01 age .gtoreq.50/age <50 0.5
(0.1--2.2) 0.35 male/female 2.2 (0.6-7.4) 0.22 AA/Caucasian 0.9
(0.4-2.1) 0.76 Stage III-IV/Stage I-II 4.5 (1.7-12.2) 0.003
Univariate analysis miR-10a High/Low 2.7 (1.3-5.7) 0.01 84
Multivariate analysis miR-10a High/Low 3.5 (1.5-7.8) 0.003 age
.gtoreq.50/age <50 0.4 (0.1--1.9) 0.26 male/female 1.7 (0.6-5.0)
0.34 AA/Caucasian 1.0 (0.45-2.3) 0.98 Stage III-IV/Stage I-II 4.9
(1.9-12.2) 0.001
[0300] MiR-21 expression is elevated in tumors (Table 7). The
miR-21 TIN ratios are also associated with clinical staging and
survival prognosis for colon cancer patients as well (Table 9, FIG.
8a).
[0301] There was a trend that individuals with more advanced TNM
staging have higher TIN ratios (p=0.034). TIN ratios were
dichotomized based on median values for each of the 80 individuals
with data. Individuals with high miR-21 TIN expression ratios had a
worse survival prognosis based on Kaplan Meier analysis (p=0.004)
suggesting that tumors expressing high levels of miR-21 is
predictive of poor prognosis. These results were further analyzed
with Cox regression analysis.
[0302] Individuals with high TIN ratios of miR-21 were at higher
risk with both univariate (HR=3.0; p=0.01) and multivariate
(HR=2.8; p=0.02) analysis adjusting for age, gender, race and TNM
staging (Table 9).
[0303] This result suggested that miR-21 expression levels can be
useful as prognostic prediction methods and can provide more
predictive value for survival prognosis than TNM staging alone.
miR-21 has been found to be differentially expressed in many tumor
types.
[0304] Studies have also demonstrated that high levels of miR-21
can lead to an inhibition of apoptosis in glioblastoma cells while
inhibition of miR-21 can lead to increased cell proliferation in
HeLa cells.
[0305] The inventors herein discovered that miR-21 is now believed
to be contributing to colon carcinogenesis in a similar manner.
[0306] We found that miR-106a elevated in tumors (Table 7) and
miR-106a TIN ratios are associated with survival prognosis (Table
9, FIG. 8b).
[0307] MiR-106a is a member of a class of paralogous miRNAs
including miR-17, miR-20, miR-106a, and miR-106h. These miRNAs are
very similar to one another in that they differ by only 1-2
nucleotides. Due to their similarity, they are all likely to have
similar targets. Interestingly, all four of these miRNAs show
similar patterns of expression and associations with prognosis
(data not shown). We present herein associations for miR-106a, but
we do not formally rule out the possibility that any or all of the
other miRNA paralogs are contributing to this association. MiR-106a
TIN ratios were dichotomized based on median values for each of the
82 individuals with data. Individuals with high miR-106a TIN
expression ratios had a worse survival prognosis based on Kaplan
Meier analysis (p=0.013; FIG. 8b).
[0308] This suggests that tumors expressing high levels of miR-106a
are predictive of poor survival prognosis. Individuals with high
TIN ratios of miR-106a were at higher risk with both univariate
(FIR=2.6, p=0.01) and multivariate (HR=2.4; p=0.05) analysis
adjusting for age, gender, race and TNM staging (Table 7).
Therefore, miR-106a may be a useful prognostic predictor of colon
cancer prognosis independent of TNM staging, Interestingly, the
Retinoblastoma tumor suppressor gene has been shown to be a
functional target of miR-106a, supporting a mechanism of how
miR-106a may be mechanistically contributing to colon
carcinogenesis.
[0309] Overexpression of the miR-17-92 cluster, which contains
paralogs of miR-106a, resulted in accelerated tumor development in
mice. This experimentally shows that miRNAs of the miR-106a family
are capable of affecting carcinogenesis further strengthening the
hypothesis that miR-106a may be contributing to carcinogenesis and
tumor progression.
[0310] Expression patterns of seven additional miRNAs were
associated with clinical staging and poor survival prognosis (Table
9, FIGS. 8c-8i).
[0311] There is a trend that individuals diagnosed with more
advanced TNM staging had higher TIN ratios for let-7g (p=0.010),
miR-10a (p=0.008), miR-16h (p=0.048), miR-29a (p=0.005), miR-103-2
(p=0.033), miR-181b (p=0.016), and miR-203 (p=0.016) (FIG. 8).
[0312] TIN ratios were dichotomized based on median (miR-16h,
miR-29a, miR-103-2, miR-203) or highest quartile (let-7g, miR-10a,
miR-181b) and Kaplan Meier analysis revealed that high TIN ratios
for each were found to be predictors of poor survival prognosis
(FIG. 8c-8i).
[0313] Univariate and multivariate Cox regression analysis
confirmed that high TIN ratios of any one of these miRNAs were
predictive of poor colon cancer prognosis independent of TNM
staging (Table 9). Multivariate Cox regression models that adjusted
for age, gender, race and TNM staging showed that high TIN ratios
for miR-16b (HR=5.1; p=0.003), let-7g (HR=2.5; p=0.03), miR-10a
(HR=3.4; p=0.003), miR-29a (HR=3.2; p=0.01), miR-103-2 (HR=3.1;
p=0.01), miR-181b (HR=3.2; p=0.01), and miR-203 (HR=3.2; p=0.03)
were each predictive of poor survival prognosis. These results
suggested that patients with tumors expressing high levels of any
of these miRNAs are at an increased risk of dying from colon
cancer. Therefore, expression levels of any these miRNAs may be
useful biomarkers that can help predict survival risks for colon
cancer patients independent of staging.
[0314] MiRNA Expression Signature of 9 miRNAs Predicts Survival
Prognosis:
[0315] We used the TIN ratios for all 9 of the previously mentioned
miRNAs to develop a miRNA signature that could be used to predict
colon cancer prognosis. Individuals missing more than 2 of 9 of
these values were excluded from this analysis. Hierarchical
clustering of the TIN ratios of the 9 miRNAs resulted in grouping
the remaining 78 patients into two groups (FIG. 9a).
[0316] These groups had significantly different survival prognoses
(FIG. 9b; p=0.004). Univariate (HR=3.2, p=0.008) and multivariate
(HR=2.8; p=0.04) Cox regression analysis demonstrated that the
miRNA signature was associated with poor survival prognosis
independent of TNM staging (Table 10)
TABLE-US-00014 TABLE 10 Cox regression analysis of microRNA
signature Variable HR (95% Cl) p value Univariate Analysis 9 miR
Cluster B/A 3.2 (1.4-7.8) 0.008 Multivariate, adjusting for age,
gender and race 9 miR Cluster B/A 2.8 (1.0-7.4) 0.043 age
.gtoreq.50/age <50 0.4 (0.08--1.8) 0.23 male/female 1.9
(0.6-6.6) 0.29 AA/Caucasian 0.9 (1.4-10.7) 0.82 Stage III-IV/Stage
I-II 3.9 (1.4-10.7) 0.007
[0317] Univariate (above) and multivariate (adjusting for age,
gender race and staging; below) Cox regression analyses were
performed to show that individuals classified into group B using
the 9 miRNA signature were at higher risk of dying of colon cancer.
Neither age, gender nor race significantly contributed to survival
risk. This risk associated with cluster assignment is independent
of staging.
[0318] These results demonstrate that miRNA signatures may be used
as a biomarker to predict the survival prognosis of colon cancer
patients.
[0319] Discussion
[0320] Individual miRNAs are differentially expressed in colon
tumors suggesting that altered expression of these miRNAs may be
part of the cellular changes responsible for colon carcinogenesis.
In addition to these findings, we show herein that miRNA expression
profiles are associated with colon cancer staging and prognosis.
Therefore miRNAs, either analyzed individually or as part of a
miRNA signature, can be used as biomarkers that will enable
physicians to predict patient survival risk with more accuracy.
[0321] The strong associations with miRNA TIN ratios with survival
prognosis suggest that altered miRNA expression may be part of the
causal pathway in colon carcinogenesis and progression. If altered
expression of any of these mi RNAs is causal to carcinogenesis, it
may be possible to design antagomir-like pharmaceuticals that can
be used to treat cancer. Using miRNA profiling and miRNA based
therapeutics, it may be possible to design personalized drug
treatment strategies based on which of these nine miRNAs are
altered. Additionally, these strategies may be useful in preventing
colon cancer in people that are at high risk due to genetically
inherited risks or previous cancer history.
Example 3
Methods, Reagents and Kits for Diagnosing, Staging, Prognosing,
Monitoring and Treating Colon Cancer-Related Diseases
[0322] In one embodiment, there is provided a diagnostic method of
assessing whether a patient has a colon cancer-related disease or
has higher than normal risk for developing a colon cancer-related
disease, comprising the steps of comparing the level of expression
of a marker in a patient sample and the normal level of expression
of the marker in a control, e.g., a sample from a patient without a
colon cancer-related disease. A significantly higher level of
expression of the marker in the patient sample as compared to the
normal level is an indication that the patient is afflicted with a
colon cancer-related disease or has higher than normal risk for
developing a colon cancer-related disease.
[0323] The markers are selected such that the positive predictive
value of the methods is at least about 10%, and in certain
non-limiting embodiments, about 25%, about 50% or about 90%. Also
preferred for use in the methods are markers that are
differentially expressed, as compared to normal cells, by at least
two-fold in at least about 20%, and in certain non-limiting
embodiments, about 50% or about 75%.
[0324] In one diagnostic method of assessing whether a patient is
afflicted with a colon cancer-related disease (e.g., new detection
("screening"), detection of recurrence, reflex testing), the method
comprises comparing: a) the level of expression of a marker in a
patient sample, and b) the normal level of expression of the marker
in a control non-colon cancer-related disease sample. A
significantly higher level of expression of the marker in the
patient sample as compared to the normal level is an indication
that the patient is afflicted with a colon cancer-related
disease.
[0325] There is also provided diagnostic methods for assessing the
efficacy of a therapy for inhibiting a colon cancer-related disease
in a patient. Such methods comprise comparing: a) expression of a
marker in a first sample obtained from the patient prior to
providing at least a portion of the therapy to the patient, and b)
expression of the marker in a second sample obtained from the
patient following provision of the portion of the therapy. A
significantly lower level of expression of the marker in the second
sample relative to that in the first sample is an indication that
the therapy is efficacious for inhibiting a colon cancer-related
disease in the patient.
[0326] It will be appreciated that in these methods the "therapy"
may be any therapy for treating a colon cancer-related disease
including, but not limited to, pharmaceutical compositions, gene
therapy and biologic therapy such as the administering of
antibodies and chemokines. Thus, the methods described herein may
be used to evaluate a patient before, during and after therapy, for
example, to evaluate the reduction in disease state.
[0327] In certain aspects, the diagnostic methods are directed to
therapy using a chemical or biologic agent. These methods comprise
comparing: a) expression of a marker in a first sample obtained
from the patient and maintained in the presence of the chemical or
biologic agent, and b) expression of the marker in a second sample
obtained from the patient and maintained in the absence of the
agent. A significantly lower level of expression of the marker in
the second sample relative to that in the first sample is an
indication that the agent is efficacious for inhibiting a colon
cancer-related disease in the patient. In one embodiment, the first
and second samples can be portions of a single sample obtained from
the patient or portions of pooled samples obtained from the
patient.
[0328] There is also provided a monitoring method for assessing the
progression of a colon cancer-related disease in a patient, the
method comprising: a) detecting in a patient sample at a first time
point, the expression of a marker; b) repeating step a) at a
subsequent time point in time; and c) comparing the level of
expression detected in steps a) and b), and therefrom monitoring
the progression of a colon cancer-related disease in the patient. A
significantly higher level of expression of the marker in the
sample at the subsequent time point from that of the sample at the
first time point is an indication that the colon cancer-related
disease has progressed, whereas a significantly lower level of
expression is an indication that the colon cancer-related disease
has regressed.
[0329] There is further provided a diagnostic method for
determining whether a colon cancer-related disease has worsened or
is likely to worsen in the future, the method comprising comparing:
a) the level of expression of a marker in a patient sample, and b)
the normal level of expression of the marker in a control sample. A
significantly higher level of expression in the patient sample as
compared to the normal level is an indication that the colon
cancer-related disease has worsened or is likely to worsen in the
future.
[0330] There is also provided a test method for selecting a
composition for inhibiting a colon cancer-related disease in a
patient. This method comprises the steps of: a) obtaining a sample
comprising cells from the patient; b) separately maintaining
aliquots of the sample in the presence of a plurality of test
compositions; c) comparing expression of a marker in each of the
aliquots; and d) selecting one of the test compositions which
significantly reduces the level of expression of the marker in the
aliquot containing that test composition, relative to the levels of
expression of the marker in the presence of the other test
compositions.
[0331] There is additionally provided a test method of assessing
the harmful potential of a compound in causing a colon
cancer-related disease. This method comprises the steps of: a)
maintaining separate aliquots of cells in the presence and absence
of the compound; and b) comparing expression of a marker in each of
the aliquots. A significantly higher level of expression of the
marker in the aliquot maintained in the presence of the compound,
relative to that of the aliquot maintained in the absence of the
compound, is an indication that the compound possesses such harmful
potential.
[0332] In addition, there is further provided a method of
inhibiting a colon cancer-related disease in a patient. This method
comprises the steps of: a) obtaining a sample comprising cells from
the patient; b) separately maintaining aliquots of the sample in
the presence of a plurality of compositions; c) comparing
expression of a marker in each of the aliquots; and d)
administering to the patient at least one of the compositions which
significantly lowers the level of expression of the marker in the
aliquot containing that composition, relative to the levels of
expression of the marker in the presence of the other
compositions.
[0333] The level of expression of a marker in a sample can be
assessed, for example, by detecting the presence in the sample of:
the corresponding marker protein or a fragment of the protein (e.g.
by using a reagent, such as an antibody, an antibody derivative, an
antibody fragment or single-chain antibody, which binds
specifically with the protein or protein fragment) the
corresponding marker nucleic acid (e.g. a nucleotide transcript, or
a complement thereof), or a fragment of the nucleic acid (e.g. by
contacting transcribed polynucleotides obtained from the sample
with a substrate having affixed thereto one or more nucleic acids
having the entire or a segment of the nucleic acid sequence or a
complement thereof) a metabolite which is produced directly (i.e.,
catalyzed) or indirectly by the corresponding marker protein.
[0334] Any of the aforementioned methods may be performed using at
least one or a plurality (e.g., 2, 3, 5, or 10 or more) of colon
cancer-related disease markers, including colon cancer-related
disease markers.
[0335] In such methods, the level of expression in the sample of
each of a plurality of markers, at least one of which is a marker,
is compared with the normal level of expression of each of the
plurality of markers in samples of the same type obtained from
control humans not afflicted with a colon cancer-related disease. A
significantly altered (i.e., increased or decreased as specified in
the above-described methods using a single marker) level of
expression in the sample of one or more markers, or some
combination thereof, relative to that marker's corresponding normal
or control level, is an indication that the patient is afflicted
with a colon cancer-related disease. For all of the aforementioned
methods, the marker(s) are selected such that the positive
predictive value of the method is at least about 10%.
[0336] In another aspect, there is provided various diagnostic and
test kits. In one embodiment, a kit is useful for assessing whether
a patient is afflicted with a colon cancer-related disease. The kit
comprises a reagent for assessing expression of a marker. In
another embodiment, a kit is useful for assessing the suitability
of a chemical or biologic agent for inhibiting a colon
cancer-related disease in a patient. Such a kit comprises a reagent
for assessing expression of a marker, and may also comprise one or
more of such agents.
[0337] In a further embodiment, the kits are useful for assessing
the presence of colon cancer-related disease cells or treating
colon cancer-related diseases. Such kits comprise an antibody, an
antibody derivative or an antibody fragment, which binds
specifically with a marker protein or a fragment of the protein.
Such kits may also comprise a plurality of antibodies, antibody
derivatives or antibody fragments wherein the plurality of such
antibody agents binds specifically with a marker protein or a
fragment of the protein.
[0338] In an additional embodiment, the kits are useful for
assessing the presence of colon cancer-related disease cells,
wherein the kit comprises a nucleic acid probe that binds
specifically with a marker nucleic acid or a fragment of the
nucleic acid. The kit may also comprise a plurality of probes,
wherein each of the probes binds specifically with a marker nucleic
acid, or a fragment of the nucleic acid.
[0339] In a further aspect, there is provided methods for treating
a patient afflicted with a colon cancer-related disease or at risk
of developing a colon cancer-related disease. Such methods may
comprise reducing the expression and/or interfering with the
biological function of a marker. In one embodiment, the method
comprises providing to the patient an antisense oligonucleotide or
polynucleotide complementary to a marker nucleic acid, or a segment
thereof. For example, an antisense polynucleotide may be provided
to the patient through the delivery of a vector that expresses an
anti-sense polynucleotide of a marker nucleic acid or a fragment
thereof. In another embodiment, the method comprises providing to
the patient an antibody, an antibody derivative or antibody
fragment, which binds specifically with a marker protein, or a
fragment of the protein.
[0340] In a broad aspect, there is provided a method for producing
a non-human animal model for assessment of at least one colon
cancer-related disease. The method includes exposing the animal to
repeated doses of at least one chemical believed to cause colon
cancer. In certain aspects, the method further includes collecting
one or more selected samples from the animal; and comparing the
collected sample to one or more indicia of potential colon cancer
initiation or development.
[0341] In broad aspect, there is provides a method of producing the
animal model that includes: maintaining the animal in a specific
chemical-free environment and sensitizing the animal with at least
one chemical believed to cause colon cancer. In certain
embodiments, at least a part of the animal's colon is sensitized by
multiple sequential exposures.
[0342] In another broad aspect, there is provided a method of
screening for an agent for effectiveness against at least one colon
cancer-related disease. The method generally includes:
administering at least one agent to the animal, determining whether
the agent reduces or aggravates one or more symptoms of the colon
cancer-related disease; correlating a reduction in one or more
symptoms with effectiveness of the agent against the colon
cancer-related disease; or correlating a lack of reduction in one
or more symptoms with ineffectiveness of the agent.
[0343] The animal model is useful for assessing one or more
metabolic pathways that contribute to at least one of initiation,
progression, severity, pathology, aggressiveness, grade, activity,
disability, mortality, morbidity, disease sub-classification or
other underlying pathogenic or pathological feature of at least one
colon cancer-related disease. The analysis can be by one or more
of: hierarchical clustering, signature network construction, mass
spectroscopy proteomic analysis, surface plasmon resonance, linear
statistical modeling, partial least squares discriminant analysis,
and multiple linear regression analysis.
[0344] In a particular aspect, the animal model is assessed for at
least one colon cancer-related disease, by examining an expression
level of one or more markers, or a functional equivalent
thereto.
[0345] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example,
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989);
DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide
Synthesis (Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195;
Nucleic Acid Hybridization (Hames & Higgins eds., 1984);
Transcription And Translation (Hames & Higgins eds., 1984);
Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);
Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A
Practical Guide To Molecular Cloning (1984); the treatise, Methods
In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors
For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring
Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et
al. eds.), Immunochemical Methods In Cell And Molecular Biology
(Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds.,
1986); The Laboratory Rat, editor in chief: Mark A. Suckow;
authors: Sharp and LaRegina. CRC Press, Boston, 1988, which are
incorporated herein by reference) and chemical methods.
[0346] Described herein are newly discovered markers associated
with a colon cancer-induced state of various cells. It has been
discovered that the higher than normal level of expression of any
of these markers or combination of these markers correlates with
the presence of a colon cancer-related disease in a patient.
Methods are provided for detecting the presence of a colon
cancer-related disease in a sample; the absence of a in a sample;
the stage of a colon cancer-related disease; and, other
characteristics of a colon cancer-related disease that are relevant
to the assessment, prevention, diagnosis, characterization and
therapy of a colon cancer-related disease in a patient. Methods of
treating a colon cancer-related disease are also provided.
DEFINITIONS
[0347] As used herein, each of the following terms has the meaning
associated with it in this section.
[0348] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0349] A "marker" is a gene or protein whose altered level of
expression in a tissue or cell from its expression level in normal
or healthy tissue or cell is associated with a disease state.
[0350] The "normal" level of expression of a marker is the level of
expression of the marker in colon system cells of a human subject
or patient not afflicted with a colon cancer-related disease.
[0351] An "over-expression" or "significantly higher level of
expression" of a marker refers to an expression level in a test
sample that is greater than the standard error of the assay
employed to assess expression, and in certain embodiments, at least
twice, and in other embodiments, three, four, five or ten times the
expression level of the marker in a control sample (e.g., sample
from a healthy subject not having the marker associated disease)
and in certain embodiments, the average expression level of the
marker in several control samples.
[0352] A "significantly lower level of expression" of a marker
refers to an expression level in a test sample that is at least
twice, and in certain embodiments, three, four, five or ten times
lower than the expression level of the marker in a control sample
(e.g., sample from a healthy subject not having the marker
associated disease) and in certain embodiments, the average
expression level of the marker in several control samples.
[0353] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g., a probe, for specifically
detecting the expression of a marker. The kit may be promoted,
distributed or sold as a unit for performing the methods of the
present invention.
[0354] "Proteins" encompass marker proteins and their fragments;
variant marker proteins and their fragments; peptides and
polypeptides comprising an at least 15 amino acid segment of a
marker or variant marker protein; and fusion proteins comprising a
marker or variant marker protein, or an at least 15 amino acid
segment of a marker or variant marker protein.
[0355] The compositions, kits and methods described herein have the
following uses, among others: 1) assessing whether a patient is
afflicted with a colon cancer-related disease; 2) assessing the
stage of a colon cancer-related disease in a human patient; 3)
assessing the grade of a colon cancer-related disease in a patient;
4) assessing the nature of a colon cancer-related disease in a
patient; 5) assessing the potential to develop a colon
cancer-related disease in a patient; 6) assessing the histological
type of cells associated with a colon cancer-related disease in a
patient; 7) making antibodies, antibody fragments or antibody
derivatives that are useful for treating a colon cancer-related
disease and/or assessing whether a patient is afflicted with a
colon cancer-related disease; 8) assessing the presence of colon
cancer-related disease cells; 9) assessing the efficacy of one or
more test compounds for inhibiting a colon cancer-related disease
in a patient; 10) assessing the efficacy of a therapy for
inhibiting a colon cancer-related disease in a patient; 11)
monitoring the progression of a colon cancer-related disease in a
patient; 12) selecting a composition or therapy for inhibiting a
colon cancer-related disease in a patient; 13) treating a patient
afflicted with a colon cancer-related disease; 14) inhibiting a
colon cancer-related disease in a patient; 15) assessing the
harmful potential of a test compound; and 16) preventing the onset
of a colon cancer-related disease in a patient at risk for
developing a colon cancer-related disease.
[0356] Screening Methods
[0357] The animal models created by the methods described herein
will enable screening of therapeutic agents useful for treating or
preventing a colon cancer-related disease. Accordingly, the methods
are useful for identifying therapeutic agents for treating or
preventing a colon cancer-related disease. The methods comprise
administering a candidate agent to an animal model made by the
methods described herein, assessing at least one colon
cancer-related disease response in the animal model as compared to
a control animal model to which the candidate agent has not been
administered. If at least one colon cancer-related disease response
is reduced in symptoms or delayed in onset, the candidate agent is
an agent for treating or preventing the colon cancer-related
disease.
[0358] The candidate agents may be pharmacologic agents already
known in the art or may be agents previously unknown to have any
pharmacological activity. The agents may be naturally arising or
designed in the laboratory. They may be isolated from
microorganisms, animals or plants, or may be produced
recombinantly, or synthesized by any suitable chemical method. They
may be small molecules, nucleic acids, proteins, peptides or
peptidomimetics. In certain embodiments, candidate agents are small
organic compounds having a molecular weight of more than 50 and
less than about 2,500 daltons. Candidate agents comprise functional
groups necessary for structural interaction with proteins.
Candidate agents are also found among biomolecules including, but
not limited to: peptides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0359] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. There are,
for example, numerous means available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available
or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. In certain embodiments,
the candidate agents can be obtained using any of the numerous
approaches in combinatorial library methods art, including, by
non-limiting example: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection.
[0360] In certain further embodiments, certain pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs.
[0361] The same methods for identifying therapeutic agents for
treating a colon cancer-related disease can also be used to
validate lead compounds/agents generated from in vitro studies.
[0362] The candidate agent may be an agent that up- or
down-regulates one or more colon cancer-related disease response
pathways. In certain embodiments, the candidate agent may be an
antagonist that affects such pathway.
[0363] Methods for Treating a Colon Cancer-Related Disease
[0364] There is provided herein methods for treating, inhibiting,
relieving or reversing a colon cancer-related disease response. In
the methods described herein, an agent that interferes with a
signaling cascade is administered to an individual in need thereof,
such as, but not limited to, colon cancer-related disease patients
in whom such complications are not yet evident and those who
already have at least one colon cancer-related disease
response.
[0365] In the former instance, such treatment is useful to prevent
the occurrence of such colon cancer-related disease response and/or
reduce the extent to which they occur. In the latter instance, such
treatment is useful to reduce the extent to which such colon
cancer-related disease response occurs, prevent their further
development or reverse the colon cancer-related disease
response.
[0366] In certain embodiments, the agent that interferes with the
colon cancer-related disease response cascade may be an antibody
specific for such response.
[0367] Expression of a Marker
[0368] Expression of a marker can be inhibited in a number of ways,
including, by way of a non-limiting example, an antisense
oligonucleotide can be provided to the colon cancer-related disease
cells in order to inhibit transcription, translation, or both, of
the marker(s). Alternately, a polynucleotide encoding an antibody,
an antibody derivative, or an antibody fragment which specifically
binds a marker protein, and operably linked with an appropriate
promoter/regulator region, can be provided to the cell in order to
generate intracellular antibodies which will inhibit the function
or activity of the protein. The expression and/or function of a
marker may also be inhibited by treating the colon cancer-related
disease cell with an antibody, antibody derivative or antibody
fragment that specifically binds a marker protein. Using the
methods described herein, a variety of molecules, particularly
including molecules sufficiently small that they are able to cross
the cell membrane, can be screened in order to identify molecules
which inhibit expression of a marker or inhibit the function of a
marker protein. The compound so identified can be provided to the
patient in order to inhibit colon cancer-related disease cells of
the patient.
[0369] Any marker or combination of markers, as well as any certain
markers in combination with the markers, may be used in the
compositions, kits and methods described herein. In general, it is
desirable to use markers for which the difference between the level
of expression of the marker in colon cancer-related disease cells
and the level of expression of the same marker in normal colon
system cells is as great as possible. Although this difference can
be as small as the limit of detection of the method for assessing
expression of the marker, it is desirable that the difference be at
least greater than the standard error of the assessment method,
and, in certain embodiments, a difference of at least 2-, 3-, 4-,
5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 100-, 500-, 1000-fold or greater
than the level of expression of the same marker in normal
tissue.
[0370] It is recognized that certain marker proteins are secreted
to the extracellular space surrounding the cells. These markers are
used in certain embodiments of the compositions, kits and methods,
owing to the fact that such marker proteins can be detected in a
colon cancer-associated body fluid sample, which may be more easily
collected from a human patient than a tissue biopsy sample. In
addition, in vivo techniques for detection of a marker protein
include introducing into a subject a labeled antibody directed
against the protein. For example, the antibody can be labeled with
a radioactive marker whose presence and location in a subject can
be detected by standard imaging techniques.
[0371] In order to determine whether any particular marker protein
is a secreted protein, the marker protein is expressed in, for
example, a mammalian cell, such as a human colon line,
extracellular fluid is collected, and the presence or absence of
the protein in the extracellular fluid is assessed (e.g. using a
labeled antibody which binds specifically with the protein).
[0372] It will be appreciated that patient samples containing colon
cells may be used in the methods described herein. In these
embodiments, the level of expression of the marker can be assessed
by assessing the amount (e.g., absolute amount or concentration) of
the marker in a sample. The cell sample can, of course, be
subjected to a variety of post-collection preparative and storage
techniques (e.g., nucleic acid and/or protein extraction, fixation,
storage, freezing, ultrafiltration, concentration, evaporation,
centrifugation, etc.) prior to assessing the amount of the marker
in the sample.
[0373] It will also be appreciated that the markers may be shed
from the cells into the digestive system, the blood stream and/or
interstitial spaces. The shed markers can be tested, for example,
by examining the serum or plasma.
[0374] The compositions, kits and methods can be used to detect
expression of marker proteins having at least one portion which is
displayed on the surface of cells which express it. For example,
immunological methods may be used to detect such proteins on whole
cells, or computer-based sequence analysis methods may be used to
predict the presence of at least one extracellular domain (i.e.,
including both secreted proteins and proteins having at least one
cell-surface domain) Expression of a marker protein having at least
one portion which is displayed on the surface of a cell which
expresses it may be detected without necessarily lysing the cell
(e.g., using a labeled antibody which binds specifically with a
cell-surface domain of the protein).
[0375] Expression of a marker may be assessed by any of a wide
variety of methods for detecting expression of a transcribed
nucleic acid or protein. Non-limiting examples of such methods
include immunological methods for detection of secreted,
cell-surface, cytoplasmic or nuclear proteins, protein purification
methods, protein function or activity assays, nucleic acid
hybridization methods, nucleic acid reverse transcription methods
and nucleic acid amplification methods.
[0376] In a particular embodiment, expression of a marker is
assessed using an antibody (e.g., a radio-labeled,
chromophore-labeled, fluorophore-labeled or enzyme-labeled
antibody), an antibody derivative (e.g., an antibody conjugated
with a substrate or with the protein or ligand of a protein-ligand
pair), or an antibody fragment (e.g., a single-chain antibody, an
isolated antibody hypervariable domain, etc.) which binds
specifically with a marker protein or fragment thereof, including a
marker protein which has undergone all or a portion of its normal
post-translational modification.
[0377] In another particular embodiment, expression of a marker is
assessed by preparing mRNA/cDNA (i.e., a transcribed
polynucleotide) from cells in a patient sample, and by hybridizing
the mRNA/cDNA with a reference polynucleotide which is a complement
of a marker nucleic acid, or a fragment thereof. cDNA can,
optionally, be amplified using any of a variety of polymerase chain
reaction methods prior to hybridization with the reference
polynucleotide; preferably, it is not amplified. Expression of one
or more markers can likewise be detected using quantitative PCR to
assess the level of expression of the marker(s). Alternatively, any
of the many methods of detecting mutations or variants (e.g.,
single nucleotide polymorphisms, deletions, etc.) of a marker may
be used to detect occurrence of a marker in a patient.
[0378] In a related embodiment, a mixture of transcribed
polynucleotides obtained from the sample is contacted with a
substrate having fixed thereto a polynucleotide complementary to or
homologous with at least a portion (e.g., at least 7, 10, 15, 20,
25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker
nucleic acid. If polynucleotides complementary to or homologous
with are differentially detectable on the substrate (e.g.,
detectable using different chromophores or fluorophores, or fixed
to different selected positions), then the levels of expression of
a plurality of markers can be assessed simultaneously using a
single substrate (e.g., a "gene chip" microarray of polynucleotides
fixed at selected positions). When a method of assessing marker
expression is used which involves hybridization of one nucleic acid
with another, it is desired that the hybridization be performed
under stringent hybridization conditions.
[0379] In certain embodiments, the biomarker assays can be
performed using mass spectrometry or surface plasmon resonance. In
various embodiment, the method of identifying an agent active
against a colon cancer-related disease can include a) providing a
sample of cells containing one or more markers or derivative
thereof; b) preparing an extract from said cells; c) mixing said
extract with a labeled nucleic acid probe containing a marker
binding site; and, d) determining the formation of a complex
between the marker and the nucleic acid probe in the presence or
absence of the test agent. The determining step can include
subjecting said extract/nucleic acid probe mixture to an
electrophoretic mobility shift assay.
[0380] In certain embodiments, the determining step comprises an
assay selected from an enzyme linked immunoabsorption assay
(ELISA), fluorescence based assays and ultra high throughput
assays, for example surface plasmon resonance (SPR) or fluorescence
correlation spectroscopy (FCS) assays. In such embodiments, the SPR
sensor is useful for direct real-time observation of biomolecular
interactions since SPR is sensitive to minute refractive index
changes at a metal-dielectric surface. SPR is a surface technique
that is sensitive to changes of 10.sup.5 to 10.sup.-6 refractive
index (RI) units within approximately 200 nm of the SPR
sensor/sample interface. Thus, SPR spectroscopy is useful for
monitoring the growth of thin organic films deposited on the
sensing layer.
[0381] Because the compositions, kits, and methods rely on
detection of a difference in expression levels of one or more
markers, it is desired that the level of expression of the marker
is significantly greater than the minimum detection limit of the
method used to assess expression in at least one of normal cells
and colon cancer-affected cells.
[0382] It is understood that by routine screening of additional
patient samples using one or more of the markers, it will be
realized that certain of the markers are over-expressed in cells of
various types, including specific colon cancer-related
diseases.
[0383] In addition, as a greater number of patient samples are
assessed for expression of the markers and the outcomes of the
individual patients from whom the samples were obtained are
correlated, it will also be confirmed that altered expression of
certain of the markers are strongly correlated with a colon
cancer-related disease and that altered expression of other markers
are strongly correlated with other diseases. The compositions,
kits, and methods are thus useful for characterizing one or more of
the stage, grade, histological type, and nature of a colon
cancer-related disease in patients.
[0384] When the compositions, kits, and methods are used for
characterizing one or more of the stage, grade, histological type,
and nature of a colon cancer-related disease in a patient, it is
desired that the marker or panel of markers is selected such that a
positive result is obtained in at least about 20%, and in certain
embodiments, at least about 40%, 60%, or 80%, and in substantially
all patients afflicted with a colon cancer-related disease of the
corresponding stage, grade, histological type, or nature. The
marker or panel of markers invention can be selected such that a
positive predictive value of greater than about 10% is obtained for
the general population (in a non-limiting example, coupled with an
assay specificity greater than 80%).
[0385] When a plurality of markers are used in the compositions,
kits, and methods, the level of expression of each marker in a
patient sample can be compared with the normal level of expression
of each of the plurality of markers in non-colon cancer samples of
the same type, either in a single reaction mixture (i.e. using
reagents, such as different fluorescent probes, for each marker) or
in individual reaction mixtures corresponding to one or more of the
markers. In one embodiment, a significantly increased level of
expression of more than one of the plurality of markers in the
sample, relative to the corresponding normal levels, is an
indication that the patient is afflicted with a colon
cancer-related disease. When a plurality of markers is used, 2, 3,
4, 5, 8, 10, 12, 15, 20, 30, or 50 or more individual markers can
be used; in certain embodiments, the use of fewer markers may be
desired.
[0386] In order to maximize the sensitivity of the compositions,
kits, and methods (i.e. by interference attributable to cells of
non-colon system origin in a patient sample), it is desirable that
the marker used therein be a marker which has a restricted tissue
distribution, e.g., normally not expressed in a non-colon system
tissue.
[0387] It is recognized that the compositions, kits, and methods
will be of particular utility to patients having an enhanced risk
of developing a colon cancer-related disease and their medical
advisors. Patients recognized as having an enhanced risk of
developing a colon cancer-related disease include, for example,
patients having a familial history of a colon cancer-related
disease.
[0388] The level of expression of a marker in normal human colon
system tissue can be assessed in a variety of ways. In one
embodiment, this normal level of expression is assessed by
assessing the level of expression of the marker in a portion of
colon system cells which appear to be normal and by comparing this
normal level of expression with the level of expression in a
portion of the colon system cells which is suspected of being
abnormal. Alternately, and particularly as further information
becomes available as a result of routine performance of the methods
described herein, population-average values for normal expression
of the markers may be used. In other embodiments, the `normal`
level of expression of a marker may be determined by assessing
expression of the marker in a patient sample obtained from a
non-colon cancer-afflicted patient, from a patient sample obtained
from a patient before the suspected onset of a colon cancer-related
disease in the patient, from archived patient samples, and the
like.
[0389] There is also provided herein compositions, kits, and
methods for assessing the presence of colon cancer-related disease
cells in a sample (e.g. an archived tissue sample or a sample
obtained from a patient). These compositions, kits, and methods are
substantially the same as those described above, except that, where
necessary, the compositions, kits, and methods are adapted for use
with samples other than patient samples. For example, when the
sample to be used is a parafinized, archived human tissue sample,
it can be necessary to adjust the ratio of compounds in the
compositions, in the kits, or the methods used to assess levels of
marker expression in the sample.
[0390] Kits and Reagents
[0391] The kits are useful for assessing the presence of colon
cancer-related disease cells (e.g. in a sample such as a patient
sample). The kit comprises a plurality of reagents, each of which
is capable of binding specifically with a marker nucleic acid or
protein. Suitable reagents for binding with a marker protein
include antibodies, antibody derivatives, antibody fragments, and
the like. Suitable reagents for binding with a marker nucleic acid
(e.g. a genomic DNA, an MRNA, a spliced MRNA, a cDNA, or the like)
include complementary nucleic acids. For example, the nucleic acid
reagents may include oligonucleotides (labeled or non-labeled)
fixed to a substrate, labeled oligonucleotides not bound with a
substrate, pairs of PCR primers, molecular beacon probes, and the
like.
[0392] The kits may optionally comprise additional components
useful for performing the methods described herein. By way of
example, the kit may comprise fluids (e.g. SSC buffer) suitable for
annealing complementary nucleic acids or for binding an antibody
with a protein with which it specifically binds, one or more sample
compartments, an instructional material which describes performance
of the method, a sample of normal colon system cells, a sample of
colon cancer-related disease cells, and the like.
[0393] Method of Producing Antibodies
[0394] There is also provided herein a method of making an isolated
hybridoma which produces an antibody useful for assessing whether a
patient is afflicted with a colon cancer-related disease. In this
method, a protein or peptide comprising the entirety or a segment
of a marker protein is synthesized or isolated (e.g. by
purification from a cell in which it is expressed or by
transcription and translation of a nucleic acid encoding the
protein or peptide in vivo or in vitro). A vertebrate, for example,
a mammal such as a mouse, rat, rabbit, or sheep, is immunized using
the protein or peptide. The vertebrate may optionally (and
preferably) be immunized at least one additional time with the
protein or peptide, so that the vertebrate exhibits a robust immune
response to the protein or peptide. Splenocytes are isolated from
the immunized vertebrate and fused with an immortalized cell line
to form hybridomas, using any of a variety of methods. Hybridomas
formed in this manner are then screened using standard methods to
identify one or more hybridomas which produce an antibody which
specifically binds with the marker protein or a fragment thereof.
There is also provided herein hybridomas made by this method and
antibodies made using such hybridomas.
[0395] Method of Assessing Efficacy
[0396] There is also provided herein a method of assessing the
efficacy of a test compound for inhibiting colon cancer-related
disease cells. As described above, differences in the level of
expression of the markers correlate with the abnormal state of
colon system cells. Although it is recognized that changes in the
levels of expression of certain of the markers likely result from
the abnormal state of colon system cells, it is likewise recognized
that changes in the levels of expression of other of the markers
induce, maintain, and promote the abnormal state of those cells.
Thus, compounds which inhibit a colon cancer-related disease in a
patient will cause the level of expression of one or more of the
markers to change to a level nearer the normal level of expression
for that marker (i.e. the level of expression for the marker in
normal colon system cells).
[0397] This method thus comprises comparing expression of a marker
in a first colon cell sample and maintained in the presence of the
test compound and expression of the marker in a second colon cell
sample and maintained in the absence of the test compound. A
significantly reduced expression of a marker in the presence of the
test compound is an indication that the test compound inhibits a
colon cancer-related disease. The colon cell samples may, for
example, be aliquots of a single sample of normal colon cells
obtained from a patient, pooled samples of normal colon cells
obtained from a patient, cells of a normal colon cell line,
aliquots of a single sample of colon cancer-related disease cells
obtained from a patient, pooled samples of colon cancer-related
disease cells obtained from a patient, cells of a colon
cancer-related disease cell line, or the like.
[0398] In one embodiment, the samples are colon cancer-related
disease cells obtained from a patient and a plurality of compounds
believed to be effective for inhibiting various colon
cancer-related diseases are tested in order to identify the
compound which is likely to best inhibit the colon cancer-related
disease in the patient.
[0399] This method may likewise be used to assess the efficacy of a
therapy for inhibiting a colon cancer-related disease in a patient.
In this method, the level of expression of one or more markers in a
pair of samples (one subjected to the therapy, the other not
subjected to the therapy) is assessed. As with the method of
assessing the efficacy of test compounds, if the therapy induces a
significantly lower level of expression of a marker then the
therapy is efficacious for inhibiting a colon cancer-related
disease. As above, if samples from a selected patient are used in
this method, then alternative therapies can be assessed in vitro in
order to select a therapy most likely to be efficacious for
inhibiting a colon cancer-related disease in the patient.
[0400] As described herein, the abnormal state of human colon cells
is correlated with changes in the levels of expression of the
markers. There is also provided a method for assessing the harmful
potential of a test compound. This method comprises maintaining
separate aliquots of human colon cells in the presence and absence
of the test compound. Expression of a marker in each of the
aliquots is compared. A significantly higher level of expression of
a marker in the aliquot maintained in the presence of the test
compound (relative to the aliquot maintained in the absence of the
test compound) is an indication that the test compound possesses a
harmful potential. The relative harmful potential of various test
compounds can be assessed by comparing the degree of enhancement or
inhibition of the level of expression of the relevant markers, by
comparing the number of markers for which the level of expression
is enhanced or inhibited, or by comparing both.
[0401] Various aspects are described in further detail in the
following subsections.
[0402] Isolated Proteins and Antibodies
[0403] One aspect pertains to isolated marker proteins and
biologically active portions thereof, as well as polypeptide
fragments suitable for use as immunogens to raise antibodies
directed against a marker protein or a fragment thereof. In one
embodiment, the native marker protein can be isolated from cells or
tissue sources by an appropriate purification scheme using standard
protein purification techniques. In another embodiment, a protein
or peptide comprising the whole or a segment of the marker protein
is produced by recombinant DNA techniques. Alternative to
recombinant expression, such protein or peptide can be synthesized
chemically using standard peptide synthesis techniques.
[0404] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein").
[0405] When the protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When the
protein is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals, i.e.,
it is separated from chemical precursors or other chemicals which
are involved in the synthesis of the protein. Accordingly such
preparations of the protein have less than about 30%, 20%, 10%, 5%
(by dry weight) of chemical precursors or compounds other than the
polypeptide of interest.
[0406] Biologically active portions of a marker protein include
polypeptides comprising amino acid sequences sufficiently identical
to or derived from the amino acid sequence of the marker protein,
which include fewer amino acids than the full length protein, and
exhibit at least one activity of the corresponding full-length
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the corresponding
full-length protein. A biologically active portion of a marker
protein can be a polypeptide which is, for example, 10, 25, 50, 100
or more amino acids in length. Moreover, other biologically active
portions, in which other regions of the marker protein are deleted,
can be prepared by recombinant techniques and evaluated for one or
more of the functional activities of the native form of the marker
protein. In certain embodiments, useful proteins are substantially
identical (e.g., at least about 40%, and in certain embodiments,
50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and
retain the functional activity of the corresponding
naturally-occurring marker protein yet differ in amino acid
sequence due to natural allelic variation or mutagenesis.
[0407] In addition, libraries of segments of a marker protein can
be used to generate a variegated population of polypeptides for
screening and subsequent selection of variant marker proteins or
segments thereof.
[0408] Predictive Medicine
[0409] There is also provided herein uses of the animal models and
markers in the field of predictive medicine in which diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring
clinical trials are used for prognostic (predictive) purposes to
thereby treat an individual prophylactically. Accordingly, there is
also provided herein diagnostic assays for determining the level of
expression of one or more marker proteins or nucleic acids, in
order to determine whether an individual is at risk of developing a
colon cancer-related disease. Such assays can be used for
prognostic or predictive purposes to thereby prophylactically treat
an individual prior to the onset of the colon cancer-related
disease.
[0410] In another aspect, the methods are useful for at least
periodic screening of the same individual to see if that individual
has been exposed to chemicals or toxins that change his/her
expression patterns.
[0411] Yet another aspect pertains to monitoring the influence of
agents (e.g., drugs or other compounds administered either to
inhibit a colon cancer-related disease or to treat or prevent any
other disorder (e.g., in order to understand any system effects
that such treatment may have) on the expression or activity of a
marker in clinical trials.
[0412] Pharmacogenomics
[0413] The markers are also useful as pharmacogenomic markers. As
used herein, a "pharmacogenomic marker" is an objective biochemical
marker whose expression level correlates with a specific clinical
drug response or susceptibility in a patient. The presence or
quantity of the pharmacogenomic marker expression is related to the
predicted response of the patient and more particularly the
patient's tumor to therapy with a specific drug or class of drugs.
By assessing the presence or quantity of the expression of one or
more pharmacogenomic markers in a patient, a drug therapy which is
most appropriate for the patient, or which is predicted to have a
greater degree of success, may be selected.
[0414] Monitoring Clinical Trials
[0415] Monitoring the influence of agents (e.g., drug compounds) on
the level of expression of a marker can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent to affect marker expression can be
monitored in clinical trials of subjects receiving treatment for a
colon cancer-related disease.
[0416] In one non-limiting embodiment, the present invention
provides a method for monitoring the effectiveness of treatment of
a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug candidate) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of one or more
selected markers in the pre-administration sample; (iii) obtaining
one or more post-administration samples from the subject; (iv)
detecting the level of expression of the marker(s) in the
post-administration samples; (v) comparing the level of expression
of the marker(s) in the pre-administration sample with the level of
expression of the marker(s) in the post-administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly.
[0417] For example, increased expression of the marker gene(s)
during the course of treatment may indicate ineffective dosage and
the desirability of increasing the dosage. Conversely, decreased
expression of the marker gene(s) may indicate efficacious treatment
and no need to change dosage.
[0418] Electronic Apparatus Readable Media, Systems, Arrays and
Methods of Using Same
[0419] As used herein, "electronic apparatus readable media" refers
to any suitable medium for storing, holding or containing data or
information that can be read and accessed directly by an electronic
apparatus. Such media can include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as compact disc;
electronic storage media such as RAM, ROM, EPROM, EEPROM and the
like; and general hard disks and hybrids of these categories such
as magnetic/optical storage media. The medium is adapted or
configured for having recorded thereon a marker as described
herein.
[0420] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus or other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as
personal digital assistants (PDAs), cellular phone, pager and the
like; and local and distributed processing systems.
[0421] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any method for
recording information on media to generate materials comprising the
markers described herein.
[0422] A variety of software programs and formats can be used to
store the marker information of the present invention on the
electronic apparatus readable medium. Any number of data processor
structuring formats (e.g., text file or database) may be employed
in order to obtain or create a medium having recorded thereon the
markers. By providing the markers in readable form, one can
routinely access the marker sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences in readable form to compare a
target sequence or target structural motif with the sequence
information stored within the data storage means. Search means are
used to identify fragments or regions of the sequences which match
a particular target sequence or target motif.
[0423] Thus, there is also provided herein a medium for holding
instructions for performing a method for determining whether a
subject has a colon cancer-related disease or a pre-disposition to
a colon cancer-related disease, wherein the method comprises the
steps of determining the presence or absence of a marker and based
on the presence or absence of the marker, determining whether the
subject has a colon cancer-related disease or a pre-disposition to
a colon cancer-related disease and/or recommending a particular
treatment for a colon cancer-related disease or pre-colon
cancer-related disease condition.
[0424] There is also provided herein an electronic system and/or in
a network, a method for determining whether a subject has a colon
cancer-related disease or a pre-disposition to a colon
cancer-related disease associated with a marker wherein the method
comprises the steps of determining the presence or absence of the
marker, and based on the presence or absence of the marker,
determining whether the subject has a colon cancer-related disease
or a pre-disposition to a colon cancer-related disease, and/or
recommending a particular treatment for the colon cancer-related
disease or pre-colon cancer-related disease condition. The method
may further comprise the step of receiving phenotypic information
associated with the subject and/or acquiring from a network
phenotypic information associated with the subject.
[0425] Also provided herein is a network, a method for determining
whether a subject has a colon cancer-related disease or a
pre-disposition to a colon cancer-related disease associated with a
marker, the method comprising the steps of receiving information
associated with the marker, receiving phenotypic information
associated with the subject, acquiring information from the network
corresponding to the marker and/or a colon cancer-related disease,
and based on one or more of the phenotypic information, the marker,
and the acquired information, determining whether the subject has a
colon cancer-related disease or a pre-disposition to a colon
cancer-related disease. The method may further comprise the step of
recommending a particular treatment for the colon cancer-related
disease or pre-colon cancer-related disease condition.
[0426] There is also provided herein a business method for
determining whether a subject has a colon cancer-related disease or
a pre-disposition to a colon cancer-related disease, the method
comprising the steps of receiving information associated with the
marker, receiving phenotypic information associated with the
subject, acquiring information from the network corresponding to
the marker and/or a colon cancer-related disease, and based on one
or more of the phenotypic information, the marker, and the acquired
information, determining whether the subject has a colon
cancer-related disease or a pre-disposition to a colon
cancer-related disease. The method may further comprise the step of
recommending a particular treatment for the colon cancer-related
disease or pre-colon cancer-related disease condition.
[0427] There is also provided herein an array that can be used to
assay expression of one or more genes in the array. In one
embodiment, the array can be used to assay gene expression in a
tissue to ascertain tissue specificity of genes in the array. In
this manner, up to about 7000 or more genes can be simultaneously
assayed for expression. This allows a profile to be developed
showing a battery of genes specifically expressed in one or more
tissues.
[0428] In addition to such qualitative determination, there is
provided herein the quantitation of gene expression. Thus, not only
tissue specificity, but also the level of expression of a battery
of genes in the tissue is ascertainable. Thus, genes can be grouped
on the basis of their tissue expression per se and level of
expression in that tissue. This is useful, for example, in
ascertaining the relationship of gene expression between or among
tissues. Thus, one tissue can be perturbed and the effect on gene
expression in a second tissue can be determined. In this context,
the effect of one cell type on another cell type in response to a
biological stimulus can be determined.
[0429] Such a determination is useful, for example, to know the
effect of cell-cell interaction at the level of gene expression. If
an agent is administered therapeutically to treat one cell type but
has an undesirable effect on another cell type, the method provides
an assay to determine the molecular basis of the undesirable effect
and thus provides the opportunity to co-administer a counteracting
agent or otherwise treat the undesired effect. Similarly, even
within a single cell type, undesirable biological effects can be
determined at the molecular level. Thus, the effects of an agent on
expression of other than the target gene can be ascertained and
counteracted.
[0430] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of a colon cancer-related disease, progression
of a colon cancer-related disease, and processes, such as cellular
transformation associated with a colon cancer-related disease.
[0431] The array is also useful for ascertaining the effect of the
expression of a gene or the expression of other genes in the same
cell or in different cells. This provides, for example, for a
selection of alternate molecular targets for therapeutic
intervention if the ultimate or downstream target cannot be
regulated.
[0432] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes that could serve as a
molecular target for diagnosis or therapeutic intervention.
[0433] Surrogate Markers
[0434] The markers may serve as surrogate markers for one or more
disorders or disease states or for conditions leading up to a colon
cancer-related disease state. As used herein, a "surrogate marker"
is an objective biochemical marker which correlates with the
absence or presence of a disease or disorder, or with the
progression of a disease or disorder. The presence or quantity of
such markers is independent of the disease. Therefore, these
markers may serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies, or when an assessment of disease
progression is desired before a potentially dangerous clinical
endpoint is reached.
[0435] The markers are also useful as pharmacodynamic markers. As
used herein, a "pharmacodynamic marker" is an objective biochemical
marker which correlates specifically with drug effects. The
presence or quantity of a pharmacodynamic marker is not related to
the disease state or disorder for which the drug is being
administered; therefore, the presence or quantity of the marker is
indicative of the presence or activity of the drug in a subject.
For example, a pharmacodynamic marker may be indicative of the
concentration of the drug in a biological tissue, in that the
marker is either expressed or transcribed or not expressed or
transcribed in that tissue in relationship to the level of the
drug. In this fashion, the distribution or uptake of the drug may
be monitored by the pharmacodynamic marker. Similarly, the presence
or quantity of the pharmacodynamic marker may be related to the
presence or quantity of the metabolic product of a drug, such that
the presence or quantity of the marker is indicative of the
relative breakdown rate of the drug in vivo.
[0436] Pharmacodynamic markers are of particular use in increasing
the sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker
transcription or expression, the amplified marker may be in a
quantity which is more readily detectable than the drug itself.
Also, the marker may be more easily detected due to the nature of
the marker itself; for example, using the methods described herein,
antibodies may be employed in an immune-based detection system for
a protein marker, or marker-specific radiolabeled probes may be
used to detect a mRNA marker. Furthermore, the use of a
pharmacodynamic marker may offer mechanism-based prediction of risk
due to drug treatment beyond the range of possible direct
observations.
[0437] Protocols for Testing
[0438] The method of testing for colon cancer-related diseases
comprises, for example measuring the expression level of each
marker gene in a biological sample from a subject over time and
comparing the level with that of the marker gene in a control
biological sample.
[0439] When the marker gene is one of the genes described herein
and the expression level is differentially expressed (for examples,
higher or lower than that in the control), the subject is judged to
be affected with a colon cancer-related disease. When the
expression level of the marker gene falls within the permissible
range, the subject is unlikely to be affected with a colon
cancer-related disease.
[0440] The standard value for the control may be pre-determined by
measuring the expression level of the marker gene in the control,
in order to compare the expression levels. For example, the
standard value can be determined based on the expression level of
the above-mentioned marker gene in the control. For example, in
certain embodiments, the permissible range is taken as .+-.2 S.D.
based on the standard value. Once the standard value is determined,
the testing method may be performed by measuring only the
expression level in a biological sample from a subject and
comparing the value with the determined standard value for the
control.
[0441] Expression levels of marker genes include transcription of
the marker genes to mRNA, and translation into proteins. Therefore,
one method of testing for a colon cancer-related disease is
performed based on a comparison of the intensity of expression of
mRNA corresponding to the marker genes, or the expression level of
proteins encoded by the marker genes.
[0442] The measurement of the expression levels of marker genes in
the testing for a colon cancer-related disease can be carried out
according to various gene analysis methods. Specifically, one can
use, for example, a hybridization technique using nucleic acids
that hybridize to these genes as probes, or a gene amplification
technique using DNA that hybridize to the marker genes as
primers.
[0443] The probes or primers used for the testing can be designed
based on the nucleotide sequences of the marker genes. The
identification numbers for the nucleotide sequences of the
respective marker genes are describer herein.
[0444] Further, it is to be understood that genes of higher animals
generally accompany polymorphism in a high frequency. There are
also many molecules that produce isoforms comprising mutually
different amino acid sequences during the splicing process. Any
gene associated with a colon cancer-related disease that has an
activity similar to that of a marker gene is included in the marker
genes, even if it has nucleotide sequence differences due to
polymorphism or being an isoform.
[0445] It is also to be understood that the marker genes can
include homologs of other species in addition to humans. Thus,
unless otherwise specified, the expression "marker gene" refers to
a homolog of the marker gene unique to the species or a foreign
marker gene which has been introduced into an individual.
[0446] Also, it is to be understood that a "homolog of a marker
gene" refers to a gene derived from a species other than a human,
which can hybridize to the human marker gene as a probe under
stringent conditions. Such stringent conditions are known to one
skilled in the art who can select an appropriate condition to
produce an equal stringency experimentally or empirically.
[0447] A polynucleotide comprising the nucleotide sequence of a
marker gene or a nucleotide sequence that is complementary to the
complementary strand of the nucleotide sequence of a marker gene
and has at least 15 nucleotides, can be used as a primer or probe.
Thus, a "complementary strand" means one strand of a double
stranded DNA with respect to the other strand and which is composed
of A:T (U for RNA) and G:C base pairs.
[0448] In addition, "complementary" means not only those that are
completely complementary to a region of at least 15 continuous
nucleotides, but also those that have a nucleotide sequence
homology of at least 40% in certain instances, 50% in certain
instances, 60% in certain instances, 70% in certain instances, at
least 80%, 90%, and 95% or higher. The degree of homology between
nucleotide sequences can be determined by an algorithm, BLAST,
etc.
[0449] Such polynucleotides are useful as a probe to detect a
marker gene, or as a primer to amplify a marker gene. When used as
a primer, the polynucleotide comprises usually 15 bp to 100 bp, and
in certain embodiments 15 bp to 35 bp of nucleotides. When used as
a probe, a DNA comprises the whole nucleotide sequence of the
marker gene (or the complementary strand thereof), or a partial
sequence thereof that has at least 15 bp nucleotides. When used as
a primer, the 3' region must be complementary to the marker gene,
while the 5' region can be linked to a restriction
enzyme-recognition sequence or a tag.
[0450] "Polynucleotides" may be either DNA or RNA. These
polynucleotides may be either synthetic or naturally-occurring.
Also, DNA used as a probe for hybridization is usually labeled.
Those skilled in the art readily understand such labeling methods.
Herein, the term "oligonucleotide" means a polynucleotide with a
relatively low degree of polymerization. Oligonucleotides are
included in polynucleotides.
[0451] Tests for a colon cancer-related disease using hybridization
techniques can be performed using, for example, Northern
hybridization, dot blot hybridization, or the DNA microarray
technique. Furthermore, gene amplification techniques, such as the
RT-PCR method may be used. By using the PCR amplification
monitoring method during the gene amplification step in RT-PCR, one
can achieve a more quantitative analysis of the expression of a
marker gene.
[0452] In the PCR gene amplification monitoring method, the
detection target (DNA or reverse transcript of RNA) is hybridized
to probes that are labeled with a fluorescent dye and a quencher
which absorbs the fluorescence. When the PCR proceeds and Taq
polymerase degrades the probe with its 5'-3' exonuclease activity,
the fluorescent dye and the quencher draw away from each other and
the fluorescence is detected. The fluorescence is detected in real
time. By simultaneously measuring a standard sample in which the
copy number of a target is known, it is possible to determine the
copy number of the target in the subject sample with the cycle
number where PCR amplification is linear. Also, one skilled in the
art recognizes that the PCR amplification monitoring method can be
carried out using any suitable method.
[0453] The method of testing for a colon cancer-related disease can
be also carried out by detecting a protein encoded by a marker
gene. Hereinafter, a protein encoded by a marker gene is described
as a "marker protein." For such test methods, for example, the
Western blotting method, the immunoprecipitation method, and the
ELISA method may be employed using an antibody that binds to each
marker protein.
[0454] Antibodies used in the detection that bind to the marker
protein may be produced by any suitable technique. Also, in order
to detect a marker protein, such an antibody may be appropriately
labeled. Alternatively, instead of labeling the antibody, a
substance that specifically binds to the antibody, for example,
protein A or protein G, may be labeled to detect the marker protein
indirectly. More specifically, such a detection method can include
the ELISA method.
[0455] A protein or a partial peptide thereof used as an antigen
may be obtained, for example, by inserting a marker gene or a
portion thereof into an expression vector, introducing the
construct into an appropriate host cell to produce a transformant,
culturing the transformant to express the recombinant protein, and
purifying the expressed recombinant protein from the culture or the
culture supernatant. Alternatively, the amino acid sequence encoded
by a gene or an oligopeptide comprising a portion of the amino acid
sequence encoded by a full-length cDNA are chemically synthesized
to be used as an immunogen.
[0456] Furthermore, a test for a colon cancer-related disease can
be performed using as an index not only the expression level of a
marker gene but also the activity of a marker protein in a
biological sample. Activity of a marker protein means the
biological activity intrinsic to the protein. Various methods can
be used for measuring the activity of each protein.
[0457] Even if a patient is not diagnosed as being affected with a
colon cancer-related disease in a routine test in spite of symptoms
suggesting these diseases, whether or not such a patient is
suffering from a colon cancer-related disease can be easily
determined by performing a test according to the methods described
herein.
[0458] More specifically, in certain embodiments, when the marker
gene is one of the genes described herein, an increase or decrease
in the expression level of the marker gene in a patient whose
symptoms suggest at least a susceptibility to a colon
cancer-related disease indicates that the symptoms are primarily
caused by a colon cancer-related disease.
[0459] In addition, the tests are useful to determine whether a
colon cancer-related disease is improving in a patient. In other
words, the methods described herein can be used to judge the
therapeutic effect of a treatment for a colon cancer-related
disease. Furthermore, when the marker gene is one of the genes
described herein, an increase or decrease in the expression level
of the marker gene in a patient, who has been diagnosed as being
affected by a colon cancer-related disease, implies that the
disease has progressed more.
[0460] The severity and/or susceptibility to a colon cancer-related
disease may also be determined based on the difference in
expression levels. For example, when the marker gene is one of the
genes described herein, the degree of increase in the expression
level of the marker gene is correlated with the presence and/or
severity of a colon cancer-related disease.
[0461] Animal Models
[0462] In another aspect, there is provided herein animal models
for a colon cancer-related disease where the expression level of
one or more marker genes or a gene functionally equivalent to the
marker gene has been elevated in the animal model. A "functionally
equivalent gene" as used herein generally is a gene that encodes a
protein having an activity similar to a known activity of a protein
encoded by the marker gene. A representative example of a
functionally equivalent gene includes a counterpart of a marker
gene of a subject animal, which is intrinsic to the animal.
[0463] The animal model for a colon cancer-related disease is
useful for detecting physiological changes due to a colon
cancer-related disease. In certain embodiments, the animal model is
useful to reveal additional functions of marker genes and to
evaluate drugs whose targets are the marker genes.
[0464] In one embodiment, an animal model for a colon
cancer-related disease can be created by controlling the expression
level of a counterpart gene or administering a counterpart gene.
The method can include creating an animal model for a colon
cancer-related disease by controlling the expression level of a
gene selected from the group of genes described herein. In another
embodiment, the method can include creating an animal model for a
colon cancer-related disease by administering the protein encoded
by a gene described herein, or administering an antibody against
the protein. It is to be also understood, that in certain other
embodiments, the marker can be over-expressed such that the marker
can then be measured using appropriate methods.
[0465] In another embodiment, an animal model for a colon
cancer-related disease can be created by introducing a gene
selected from such groups of genes, or by administering a protein
encoded by such a gene.
[0466] In another embodiment, a colon cancer-related disease can be
induced by suppressing the expression of a gene selected from such
groups of genes or the activity of a protein encoded by such a
gene. An antisense nucleic acid, a ribozyme, or an RNAi can be used
to suppress the expression. The activity of a protein can be
controlled effectively by administering a substance that inhibits
the activity, such as an antibody.
[0467] The animal model is useful to elucidate the mechanism
underlying a colon cancer-related disease and also to test the
safety of compounds obtained by screening. For example, when an
animal model develops the symptoms of colon cancer-related disease,
or when a measured value involved in a certain a colon
cancer-related disease alters in the animal, a screening system can
be constructed to explore compounds having activity to alleviate
the disease.
[0468] As used herein, the expression "an increase in the
expression level" refers to any one of the following: where a
marker gene introduced as a foreign gene is expressed artificially;
where the transcription of a marker gene intrinsic to the subject
animal and the translation thereof into the protein are enhanced;
or where the hydrolysis of the protein, which is the translation
product, is suppressed. As used herein, the expression "a decrease
in the expression level" refers to either the state in which the
transcription of a marker gene of the subject animal and the
translation thereof into the protein are inhibited, or the state in
which the hydrolysis of the protein, which is the translation
product, is enhanced. The expression level of a gene can be
determined, for example, by a difference in signal intensity on a
DNA chip. Furthermore, the activity of the translation product--the
protein--can be determined by comparing with that in the normal
state.
[0469] It is also within the contemplated scope that the animal
model can include transgenic animals, including, for example
animals where a marker gene has been introduced and expressed
artificially; marker gene knockout animals; and knock-in animals in
which another gene has been substituted for a marker gene. A
transgenic animal, into which an antisense nucleic acid of a marker
gene, a ribozyme, a polynucleotide having an RNAi effect, or a DNA
functioning as a decoy nucleic acid or such has been introduced,
can be used as the transgenic animal. Such transgenic animals also
include, for example, animals in which the activity of a marker
protein has been enhanced or suppressed by introducing a
mutation(s) into the coding region of the gene, or the amino acid
sequence has been modified to become resistant or susceptible to
hydrolysis. Mutations in an amino acid sequence include
substitutions, deletions, insertions, and additions.
[0470] In addition, the expression itself of a marker gene can be
controlled by introducing a mutation(s) into the transcriptional
regulatory region of the gene. Those skilled in the art understand
such amino acid substitutions. Also, the number of amino acids that
are mutated is not particularly restricted, as long as the activity
is maintained. Normally, it is within 50 amino acids, in certain
non-limiting embodiments, within 30 amino acids, within 10 amino
acids, or within 3 amino acids. The site of mutation may be any
site, as long as the activity is maintained.
[0471] In yet another aspect, there is provided herein screening
methods for candidate compounds for therapeutic agents to treat a
colon cancer-related disease. One or more marker genes are selected
from the group of genes described herein. A therapeutic agent for a
colon cancer-related disease can be obtained by selecting a
compound capable of increasing or decreasing the expression level
of the marker gene(s).
[0472] It is to be understood that the expression "a compound that
increases the expression level of a gene" refers to a compound that
promotes any one of the steps of gene transcription, gene
translation, or expression of a protein activity. On the other
hand, the expression "a compound that decreases the expression
level of a gene", as used herein, refers to a compound that
inhibits any one of these steps.
[0473] In particular aspects, the method of screening for a
therapeutic agent for a colon cancer-related disease can be carried
out either in vivo or in vitro. This screening method can be
performed, for example, by (1) administering a candidate compound
to an animal subject; (2) measuring the expression level of a
marker gene(s) in a biological sample from the animal subject; or
(3) selecting a compound that increases or decreases the expression
level of a marker gene(s) as compared to that in a control with
which the candidate compound has not been contacted.
[0474] In still another aspect, there is provided herein a method
to assess the efficacy of a candidate compound for a pharmaceutical
agent on the expression level of a marker gene(s) by contacting an
animal subject with the candidate compound and monitoring the
effect of the compound on the expression level of the marker
gene(s) in a biological sample derived from the animal subject. The
variation in the expression level of the marker gene(s) in a
biological sample derived from the animal subject can be monitored
using the same technique as used in the testing method described
above. Furthermore, based on the evaluation, a candidate compound
for a pharmaceutical agent can be selected by screening.
[0475] All patents, patent applications and references cited herein
are incorporated in their entirety by reference. While the
invention has been described and exemplified in sufficient detail
for those skilled in this art to make and use it, various
alternatives, modifications and improvements should be apparent
without departing from the spirit and scope of the invention. One
skilled in the art readily appreciates that the present invention
is well adapted to carry out the objects and obtain the ends and
advantages mentioned, as well as those inherent therein.
[0476] The methods and reagents described herein are representative
of preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Modifications therein
and other uses will occur to those skilled in the art. These
modifications are encompassed within the spirit of the invention
and are defined by the scope of the claims. It will also be readily
apparent to a person skilled in the art that varying substitutions
and modifications may be made to the invention disclosed herein
without departing from the scope and spirit of the invention.
[0477] It should be understood that although the present invention
has been specifically disclosed by preferred embodiments and
optional features, modifications and variations of the concepts
herein disclosed may be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended
claims.
* * * * *