U.S. patent application number 16/696210 was filed with the patent office on 2020-03-19 for methods of treating dementia associated with alzheimer's disease with protective protein/cathepsin a (ppca).
This patent application is currently assigned to St. Jude Children's Research Hospital. The applicant listed for this patent is St. Jude Children's Research Hospital. Invention is credited to Ida Annunziata, Alessandra D'Azzo, Shai White-Gilbertson.
Application Number | 20200087704 16/696210 |
Document ID | / |
Family ID | 46881159 |
Filed Date | 2020-03-19 |
United States Patent
Application |
20200087704 |
Kind Code |
A1 |
Annunziata; Ida ; et
al. |
March 19, 2020 |
METHODS OF TREATING DEMENTIA ASSOCIATED WITH ALZHEIMER'S DISEASE
WITH PROTECTIVE PROTEIN/CATHEPSIN A (PPCA)
Abstract
Methods are provided for the prognosis, diagnosis and treatment
of various pathological states, including cancer, chemotherapy
resistance and dementia associated with Alzheimer's disease. The
methods provided herein are based on the discovery that various
proteins with a high level of sialylation are shown herein to be
associated with disease states, such as, cancer, chemotherapy
resistance and dementia associated with Alzheimer's disease. Such
methods provide a lysosomal exocytosis activity profile comprising
one or more values representing lysosomal exocytosis activity. Also
provided herein, is the discovery that low lysosomal sialidase
activity is associated with various pathological states. Thus, the
methods also provide a lysosomal sialidase activity profile,
comprising one or more values representing lysosomal sialidase
activity. A lysosomal sialidase activity profile is one example of
a lysosomal exocytosis activity profile.
Inventors: |
Annunziata; Ida; (Memphis,
TN) ; D'Azzo; Alessandra; (Memphis, TN) ;
White-Gilbertson; Shai; (North Charleston, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
St. Jude Children's Research Hospital |
Memphis |
TN |
US |
|
|
Assignee: |
St. Jude Children's Research
Hospital
Memphis
TN
|
Family ID: |
46881159 |
Appl. No.: |
16/696210 |
Filed: |
November 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15808075 |
Nov 9, 2017 |
10533208 |
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16696210 |
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15187349 |
Jun 20, 2016 |
9840727 |
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15808075 |
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14239728 |
Feb 19, 2014 |
9399791 |
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PCT/US2012/052629 |
Aug 28, 2012 |
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15187349 |
|
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61544855 |
Oct 7, 2011 |
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61529675 |
Aug 31, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/48 20130101;
A61K 38/4813 20130101; Y02A 90/10 20180101; G16H 20/00 20180101;
G01N 33/57484 20130101; G01N 33/573 20130101; G01N 33/57496
20130101; G16H 20/40 20180101; G01N 2333/924 20130101; G06F 19/34
20130101; C12Q 1/34 20130101; Y02A 90/26 20180101; G01N 2800/2821
20130101; A61K 38/47 20130101; C12Y 302/01018 20130101; A61P 25/28
20180101; G01N 2800/56 20130101; A61P 35/00 20180101 |
International
Class: |
C12Q 1/34 20060101
C12Q001/34; G01N 33/574 20060101 G01N033/574; G01N 33/573 20060101
G01N033/573; A61K 38/48 20060101 A61K038/48; A61K 38/47 20060101
A61K038/47 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Federal Government support
under GM060950 awarded by the National Institutes of Health. The
United States Government has certain rights in the invention. This
invention was also supported by the American Lebanese Syrian
Associated Charities (ALSAC) of St. Jude Children's Research
Hospital.
Claims
1. A method of determining the prognosis for a subject with cancer,
comprising the steps of a) providing a subject profile comprising a
lysosomal sialidase activity profile comprising two or more values
from different lysosomal sialidase activity markers, a NEU1
substrate sialylation activity profile or a NEU1 level activity
profile from a tumor sample from said subject; b) providing a
corresponding reference profile comprising a lysosomal sialidase
activity profile comprising two or more values from different
lysosomal sialidase activity markers, a NEU1 substrate sialylation
activity profile or a NEU1 level activity profile from a control
sample, wherein the subject profile and the reference profile
comprise one or more values representing lysosomal sialidase
activity, NEU1 substrate sialylation activity or NEU1 level
activity; and c) comparing said subject and said reference
lysosomal sialidase activity profiles to thereby determine the
prognosis for said subject with cancer, wherein a lower lysosomal
sialidase activity, a higher NEU1 substrate sialylation activity or
a higher NEU1 level activity of said subject as compared to the
lysosomal sialidase activity, NEU1 substrate sialylation activity
or NEU1 level activity of said reference results in a prediction of
an invasive cancer for said subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/805,075, filed Nov. 9, 2017, which is a
continuation of U.S. patent application Ser. No. 15/187,349, filed
Jun. 20, 2016, issued as U.S. Pat. No. 9,840,727 on Dec. 12, 2017,
which is a continuation of U.S. patent application Ser. No.
14/239,728, filed Feb. 19, 2014, issued as U.S. Pat. No. 9,399,791
on Jul. 26, 2016, which is a National Stage Application of
PCT/US2012/052629, filed Aug. 28, 2012, which claims the benefit of
U.S. Provisional Application Ser. No. 61/544,855, filed Oct. 7,
2011, and U.S. Provisional Application Ser. No. 61/529,675, filed
Aug. 31, 2011. Each of these priority applications is incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of molecular
biology, cancer and Alzheimer's disease therapeutics and
diagnostics.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0004] The official copy of the sequence listing is submitted
concurrently with the specification as a text file via EFS-Web, in
compliance with the American Standard Code for Information
Interchange (ASCII), with a file name of
S88435_1110US_C3_Seq_List.txt, a creation date of Nov. 26, 2019,
and a size of 12 KB. The sequence listing filed via EFS-Web is part
of the specification and is hereby incorporated in its entirety by
reference herein.
BACKGROUND OF THE INVENTION
[0005] The prognosis of a disease or pathological condition in a
subject can be greatly improved with an early diagnosis. However,
reliable prognostic and diagnostic methods are lacking for managing
disease states. For example, for Alzheimer's disease, the only
definitive diagnostic test is to determine whether amyloid plaques
are present in a subject's brain tissue, a determination that can
only be made after death. Thus, due to the lack of suitable
diagnostic methods only a tentative diagnosis can be provided. In
another example, diagnosis and prognosis of a cancer are important
for choosing the best treatment options in order to improve
outcome. There is also a need for diagnostic and prognostic tests
to predict the efficacy of a particular chemotherapy regime to
determine the best treatment options for a subject.
[0006] Therefore, there is a significant need in the art for more
accurate and reliable diagnostic and prognostic methods for cancer
and Alzheimer's disease.
BRIEF SUMMARY OF THE INVENTION
[0007] Methods are provided for the prognosis, diagnosis and
treatment of various pathological states, including cancer,
chemotherapy resistance and dementia associated with Alzheimer's
disease. The methods provided herein are based on the discovery
that various proteins with a high level of sialylation are shown
herein to be associated with disease states, such as, cancer,
chemotherapy resistance and dementia associated with Alzheimer's
disease. Such methods provide a lysosomal exocytosis activity
profile comprising one or more values representing lysosomal
exocytosis activity. Also provided herein, is the discovery that
low lysosomal sialidase activity is associated with various
pathological states. Thus, the methods also provide a lysosomal
sialidase activity profile, comprising one or more values
representing lysosomal sialidase activity. A lysosomal sialidase
activity profile is one example of a lysosomal exocytosis activity
profile. As such, the level of lysosomal exocytosis activity and/or
lysosomal sialidase activity is predictive of a diagnosis and/or
prognosis of cancer, chemotherapy resistance or dementia associated
with Alzheimer's disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 displays a summary model of the role of NEU1 in
cancer.
[0009] FIG. 2 depicts the presence of lysosomal proteins in the CSF
and the correlation of these proteins with Alzheimer's disease.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the inventions are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0011] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0012] I. Overview
[0013] Provided herein are methods for the diagnosis and prognosis
of various pathological states by looking at the lysosomal
exocytosis activity in a sample. The level of lysosomal exocytosis
activity can serve as a marker for the diagnosis and/or prognosis
of pathological conditions including, for example, cancer,
chemotherapy resistance and dementia associated with Alzheimer's
disease. Various activity profiles are provided herein for the
diagnosis and/or prognosis of cancer, chemotherapy resistance, and
dementia associated with Alzheimer's disease.
[0014] II. Types of Profiles
[0015] As used herein, a "profile" comprises one or more values
corresponding to a measurement of a marker(s) representing an
activity in a sample. Various profiles are disclosed herein which
can be used for the prognosis and/or diagnosis of a given
pathological state. Such profiles include: a lysosomal exocytosis
activity profile, a sialylation activity profile, a lysosomal
sialidase activity profile, a NEU1 substrate sialylation activity
profile and a NEU1 level activity profile. Each of these profiles
is explained in detail herein and summarized in Table 1
herewith.
[0016] By "lysosomal exocytosis activity profile" is meant a
profile of one or more values representing lysosomal exocytosis
activity. As used herein, "lysosomal exocytosis activity" is meant
a measure of the level of exocytosis in a sample. Various markers
can be used to determine the lysosomal exocytosis activity of a
sample. Such markers include one or more of the following: (1) the
level of NEU1 protein or direct enzymatic activity of NEU1; (2) the
protein level of one or more NEU1 substrates; (3) the protein level
of one or more lysosomal proteins; (4) the protein level of one or
more lysosomal proteases; (5) the protein level of LAMP-1; (6) the
protein level of hexosaminidase beta; (7) the protein level of
mannosidase alpha; or (8) the protein level of one or more
cathepsins; (9) any marker for a sialylation activity profile
provided herein; or (10) any marker for a lysosomal sialidase
activity profile provided herein. Once the level of each of a given
marker is determined, it becomes a value in the lysosomal
exocytosis activity profile. The lysosomal exocytosis activity
profile can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or more lysosomal exocytosis activity values of the various
lysosomal exocytosis activity markers provided herein.
[0017] In one embodiment, one type of lysosomal exocytosis activity
profile is a sialylation activity profile. By "sialylation activity
profile" is meant a profile of one or more values representing
sialylation activity. As used herein, "sialylation activity" is
meant a measure of the sialylation level of a population of
proteins in a sample or the sialylation level of one or more
proteins in a sample. Various markers can be used to determine
sialylation activity. Such markers include one or more of the
following: (1) the overall level of sialylation in a sample; (2)
the level of NEU1 protein or direct enzymatic activity of NEU1; (3)
the level of sialylation of one or more NEU1 substrates; (4) the
protein level of one or more NEU1 substrates; or (5) any marker for
a lysosomal sialidase activity profile, as discussed in further
detail elsewhere herein or outlined in Table 1. Once the level of
each of a given marker is determined, it becomes a value in the
sialylation activity profile. The sialylation activity profile can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more
sialylation activity values of the various sialylation activity
markers provided herein.
[0018] In one embodiment, one type of sialylation activity profile
is a lysosomal sialidase activity profile. By "lysosomal sialidase
activity profile" is meant a profile of one or more values
representing lysosomal sialidase activity. By "lysosomal sialidase
activity" is meant a direct or indirect measure of lysosomal
sialidase activity. Various markers can be used to determine
lysosomal sialidase activity in a sample. The various markers
representing the lysosomal sialidase activity in a sample include
any one or more of the following: (1) the level of NEU1 protein or
the level of direct enzymatic activity of NEU1; (2) the protein
level of one or more NEU1 substrate; (3) the level of sialylation
of one or more NEU1 substrate; or (4) the activity level of one or
more NEU1 substrate. Once the level or activity of a given marker
is determined, it becomes a value in the lysosomal sialidase
activity profile. Thus, the lysosomal sialidase activity profile
can comprise any combination of the lysosomal sialidase activity
markers provided herein. The lysosomal sialidase activity profile
can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or
more lysosomal sialidase activity values of the various lysosomal
sialidase activity markers provided herein.
[0019] In one embodiment, one type of lysosomal sialidase activity
profile is a NEU1 substrate sialylation activity profile. By "NEU1
substrate sialylation activity profile" is meant measuring
lysosomal sialidase activity in a sample by determining the level
of sialylation of one or more NEU1 substrates. The various markers
representing lysosomal sialidase activity that are encompassed in a
NEU1 substrate sialylation activity profile include: (1) the level
of sialylation of one or more NEU1 substrate; (2) the level of
sialylation of LAMP-1; (3) The level of sialylation of MUC-1; or
(4) the level of sialylation of NEU1 and MUC-1. The NEU1 substrate
sialylation activity profile can comprise 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15 or more marker values provided by the
sialylation level of the various NEU1 substrates.
[0020] In another embodiment, one type of lysosomal sialidase
activity profile is a NEU1 level activity profile. By "NEU1 level
activity profile" is meant measuring lysosomal sialidase activity
in a sample by determining the protein level of any non-MUC-1 NEU1
substrate or of NEU1 itself. The various markers representing
lysosomal sialidase activity that are encompassed in a NEU1 level
activity profile include: (1) the level of NEU1 protein; (2) the
protein level of any one or more non-MUC-1 NEU1 substrate; or (3)
the protein level of LAMP-1. The NEU1 level activity profile can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more
marker values provided by the protein level of the various NEU1
substrates.
[0021] If multiple markers are present in a given profile, not all
the markers must show an altered activity as compared to the marker
value in a corresponding control or reference profile in order to
produce the prognosis and/or diagnosis provided herein. In some
instances, the alteration of a single marker may be sufficient for
a diagnosis and/or prognosis. In other embodiments, an alteration
in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more marker values in a given
profile as compared to the values in corresponding control or
reference profile is sufficient for a diagnosis and/or
prognosis.
TABLE-US-00001 TABLE 1 Summary of various markers employed to
establish a specific type of activity profile. NEU1 Lysosomal
Lysosomal Substrate Exocytosis Sialylation Sialidase Sialylation
NEU1 Level Activity Activity Activity Activity Activity Marker
Profile Profile Profile Profile Profile the level of NEU1 + + + +
protein the level of direct + + + enzymatic activity of NEU1 the
protein level of one + + + + or more NEU1 At least one substrates
NEU1 substrate other than MUC-1 must be detected the protein level
of + + + + LAMP-1 the protein level of + + + + MUC-1 Only in
combination with another NEU1 substrate the protein level of + + +
+ LAMP-1 and MUC-1 the level of any one or + more lysosomal
proteins the protein level of one + or more lysosomal proteases the
protein level of one + or more cathepsins the protein level of +
Hexosaminidase beta the protein level of + mannosidase alpha the
activity level of + + + one or more NEU1 substrates the activity
level of + + + LAMP-1 the activity level of + + + MUC-1 the
activity level of + + + LAMP-1 and MUC-1 the overall level of + +
sialylation in a sample the sialylation level of + + + + a NEU1
substrate (including levels of a population of substrates and/or
the levels of a single substrate) the sialylation level of + + + +
LAMP-1 the sialylation level of + + + + MUC-1 the sialylation level
of + + + + LAMP-1 and MUC-1 the protein level and + + + the
sialylation level of one or more NEU1 substrates the level of NEU1
+ + + protein, the protein level of one or more NEU1 substrates and
the sialylation level of one or more NEU1 substrates the level of
NEU1 + + + protein, the level of NEU1 enzymatic activity, the
protein level of one or more NEU1 substrates and the sialylation
level of one or more NEU1 substrates
[0022] III. Assays for Markers of the Various Activity Profiles
[0023] The methods for diagnosis and/or prognosis provided herein
are based on analyzing a sample for lysosomal exocytosis activity,
sialylation activity, lysosomal sialidase activity, NEU1 substrate
sialylation activity and/or NEU1 level activity and comparing it to
a reference value for lysosomal exocytosis activity, sialylation
activity, lysosomal sialidase activity, NEU1 substrate sialylation
activity and/or NEU1 level activity from a control sample.
Measuring the "level" or "amount" of a protein, sialylation, or an
activity in a sample means quantifying the lysosomal exocytosis
activity, sialylation activity, lysosomal sialidase activity, NEU1
substrate sialylation activity or NEU1 level activity by
determining, for example, the relative or absolute amount of
protein and/or sialylation of a protein and/or the activity of a
protein. One aspect of the methods provided herein relates to
assays for detecting lysosomal exocytosis activity, sialylation
activity, lysosomal sialidase activity, NEU1 substrate sialylation
activity and NEU1 level activity in the context of a sample. These
assays determine the values that make up the lysosomal exocytosis
activity profile, sialylation activity profile, lysosomal sialidase
activity profile, NEU1 substrate sialylation activity profile or
NEU1 level activity profile of a sample.
[0024] A "sample" or "subject sample", as used herein, can comprise
any sample in which one desires to determine the lysosomal
exocytosis activity, sialylation activity, lysosomal sialidase
activity, NEU1 substrate sialylation activity and/or NEU1 level
activity. By "subject" is intended any animal (i.e. mammals) such
as, humans, primates, rodents, agricultural and domesticated
animals such as, but not limited to, dogs, cats, cattle, horses,
pigs, sheep, and the like, in which one desires to determine the
lysosomal exocytosis activity, sialylation activity and/or
lysosomal sialidase activity. The sample may be derived from any
cell, tissue, or biological fluid from the animal of interest. The
sample may comprise any clinically relevant tissue, such as, but
not limited to, bone marrow, cerebrospinal fluid, tumor biopsy,
fine needle aspirate, or a sample of body fluid, such as blood,
plasma, serum, lymph, ascetic fluid, cystic fluid or urine. The
sample used in the methods provided herein will vary based on the
assay format, nature of the detection method, and the tissues,
cells or extracts which are used as the sample.
[0025] A "reference" lysosomal exocytosis activity, sialylation
activity, lysosomal sialidase activity, NEU1 substrate sialylation
activity and/or NEU1 level activity as used herein is provided in a
control sample. A "control" or "control sample" provides a
reference point for measuring changes in lysosomal exocytosis
activity, sialylation activity, lysosomal sialidase activity, NEU1
substrate sialylation level activity and/or NEU1 level activity of
a subject sample. The control may be a predetermined value based on
a group of samples or it may be a single value based on an
individual sample. The control may be a sample tested in parallel
with the subject sample. A control sample may comprise, for
example: (a) any sample from healthy individual(s); (b) a normal
tissue sample taken from a location adjacent to a tumor from the
same subject; (b) a tissue sample from healthy individual(s) taken
from the same tissue type as a subject tumor; (c) a serum or plasma
sample taken from healthy individual(s); (d) a cerebrospinal fluid
sample taken from healthy individual(s); or (e) a urine sample from
healthy individual(s).
[0026] As used herein a "higher" or "increased" level for a given
marker (i.e. any of the various markers provided herein) is meant
any significant increase in the level of the marker in a sample as
compared to the level of the corresponding marker in a control
sample. An increased or higher level for a given marker can be any
statistically significant increase in the level of the marker of at
least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 400% or more as compared
to a reference level in a control sample. Alternatively, an
increase in the level for a given marker can be any fold increase
of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 20-fold
or more over the value for the level of the corresponding marker in
a control sample. In some embodiments, an increase in the level of
a given marker can result in an increase in a specific activity in
the sample (i.e. the lysosomal exocytosis activity, sialylation
activity, lysosomal sialidase activity, NEU1 substrate sialylation
activity or NEU1 level activity). In other embodiments, an increase
in the level of a given marker can result in a decrease in a
specific activity in a sample.
[0027] As used herein, a "decreased", "lower" or "reduced" level
for a given marker (i.e. any of the various markers provided
herein) is meant any significant decrease in the level of the
marker in a sample as compared to the level of the corresponding
marker in a control sample. By lower or reduced level of a marker
is meant a statistically significant reduction in the level of a
marker in a subject sample of at least 5%, 10%, 15%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
as compared to a reference level in a control sample.
Alternatively, a decrease in the level for a given marker can be
any fold decrease of at least 1.5-fold, 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold,
16-fold, 20-fold or more as compared to the level of the
corresponding marker in a control sample. In some embodiments, a
decrease in the level of a given marker can result in a decrease in
a specific activity in the sample (i.e. the lysosomal exocytosis
activity, sialylation activity, lysosomal sialidase activity, NEU1
substrate sialylation activity or NEU1 level activity). In other
embodiments, a decrease in the level of a given marker can result
in an increase in a specific activity in the sample.
[0028] Table 2 provides non-limiting examples of markers for the
various activity profiles provided herein and denotes if an
increase or a decrease in the marker is reflective of an increase
or a decrease in the activity in a sample (i.e. the lysosomal
exocytosis activity, sialylation activity, lysosomal sialidase
activity, NEU1 substrate sialylation activity or NEU1 level
activity).
[0029] A. Lysosomal Exocytosis Activity
[0030] In one embodiment, the lysosomal exocytosis activity of one
or more lysosomal exocytosis activity markers in a sample is
provided. As used herein, "exocytosis" is a process of cellular
secretion in which substances contained in vesicles are discharged
from the cell by fusion of the vesicular membrane with the outer
cell membrane. There are two types of exocytosis, constitutive and
regulated. Constitutive exocytosis is not regulated by calcium,
while regulated exocytosis is dependent on calcium. Exocytosis
involves vesicle recruitment, tethering and docking of the vesicle
to the plasma membrane and fusion of the vesicle membrane with the
plasma membrane thereby releasing the contents of the vesicle into
the extracellular space. During exocytosis, the vesicles release
various components into the extracellular environment. Some
examples of components of secretory vesicles include, but are not
limited to, enzymes, proteases, extracellular matrix components,
hormones, neurotransmitters and cytotoxic compounds.
[0031] Lysosomal exocytosis is one type of exocytosis. By
"lysosomal exocytosis" is meant the process by which lysosomes
release their contents to the extracellular space. Lysosomal
exocytosis is a calcium dependent process that involves the
recruitment and docking of lysosomes to the plasma membrane, fusion
of the lysosomal membrane with the plasma membrane and the release
of lysosomal luminal content into the extracellular environment.
Some examples of lysosomal contents include, but are not limited
to, enzymes, such as lipases, proteases, nucleases and amylase, and
other proteins related to lysosomal function, such as sialidases
and proteins involved in lysosomal exocytosis.
[0032] In one embodiment, a subject sample has a higher or
increased lysosomal exocytosis activity as compared to a control
sample. By "higher lysosomal exocytosis activity" or "increased
lysosomal exocytosis activity" is meant a statistically significant
alteration in the level of one or more markers in the lysosomal
exocytosis activity profile. Table 2 provides non-limiting examples
of markers for the lysosomal exocytosis activity profile and
denotes if an increase or a decrease in the marker is reflective of
a higher lysosomal exocytosis activity.
[0033] In one embodiment, an increase in lysosomal exocytosis
activity is denoted in a given profile by an alteration in at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the
lysosomal exocytosis activity markers as compared to a control
sample. In some cases, an alteration in lysosomal exocytosis
activity of one marker is sufficient for a diagnosis and/or
prognosis. In other cases an alteration in two or more lysosomal
exocytosis activity markers is sufficient for a diagnosis and/or
prognosis.
[0034] Assays to measure lysosomal exocytosis activity for an
exocytosis marker are provided herein. One measure of lysosomal
exocytosis activity is the level of a protein in a sample (i.e.
NEU1, NEU1 substrates or lysosomal proteins). A variety of assays
for detecting protein in a sample are known in the art and include
direct and indirect assays for protein. An exemplary method for
detecting the presence or absence or the quantity of a protein in a
sample involves obtaining a sample and contacting the sample with a
compound or agent capable of specifically binding and detecting the
protein, such that the presence of the protein is detected in the
sample. Results obtained with a sample from a subject may be
compared to results obtained with a biological sample from a
control subject.
[0035] In one embodiment, an agent for detecting a protein is an
antibody capable of specifically binding to that protein.
Antibodies can be polyclonal or monoclonal. The term "labeled",
with regard to the antibody is intended to encompass direct
labeling of the antibody by coupling (i.e. physically linking) a
detectable substance to the antibody as well as indirect labeling
of the antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary
antibody.
[0036] The level of a protein in a sample can be quantitatively
measured by a variety of assays utilizing antibodies for a specific
protein. These include, for example, immunoassays,
radioimmunoassays, enzyme-linked immunosorbant assays and
two-antibody sandwich assays. Quantitative western blotting can
also be used to determine the level of protein. Western blots can
be quantitated by well-known methods such as scanning densitometry.
In addition, antibodies can be used to detect and quantitate the
level of protein in a sample of a tissue by fluorescence or
confocal microscopy by using a fluorescently labeled antibody or
secondary reagent.
[0037] In another embodiment, a marker is the level of sialylation
of a sample. Assays for measuring the level of sialylation in a
sample are provided elsewhere herein, for example, in the section
on sialylation activity.
[0038] In yet another embodiment, a marker is the level of protein
activity in a sample. The protein activity for any protein provided
herein can be measured by assaying for the activity of the specific
protein in a sample. For example, if the protein is an enzyme, the
activity of the enzyme can be measured in an enzyme activity assay.
Various assays are known in the art for measuring enzymatic
activity. For example, NEU1 enzyme activity in a sample can be
measured by incubating the sample with a sialylated NEU1 substrate
and detecting the amount of free sialic acid present in the sample
after incubation. As such, the units of enzyme activity can be
calculated (i.e. the amount of activity per milligram of
protein).
[0039] B. Sialylation Activity
[0040] In one embodiment, the sialylation activity of one or more
sialylation activity markers in a sample is provided. As used
herein, a protein or lipid is "sialylated" if a sialic acid is
present on the terminal portion of a glycoprotein or glycolipid. By
"sialylation" is meant the transfer of sialic acid to the terminal
portions of the sialylated glycolipids or to the N- or O-linked
sugar chains of glycoproteins. Sialylation can be catalyzed by a
number of different sialyltransferases, each with specificity for a
particular sugar substrate. Non-limiting examples of
sialyltransferases known in the art include, for example,
sialyltransferase, beta-galactosamide alpha-2,6-sialyltransferase,
alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase,
beta-galactoside alpha-2,3-sialyltransferase, N-acetyllactosaminide
alpha-2,3-sialyltransferase, alpha-N-acetyl-neuraminide
alpha-2,8-sialyltransferase and lactosylceramide
alpha-2,3-sialyltransferase.
[0041] Sialyltransferases can transfer sialic acid to a substrate
by various linkages. For example, some sialyltransferases add
sialic acid with an alpha-2,3 linkage to galactose, while other
sialyltransferases add sialic acid with an alpha-2,6 linkage to
galactose or N-acetylgalactosamine. Another group of
sialyltransferases can add sialic acid to other sialic acids by an
alpha-2,8 linkage. In one embodiment, the sialic acid is added with
an alpha-2,6 linkage to a glycoprotein. In another embodiment, the
sialic acid is added with an alpha 2,3 linkage to a
glycoprotein.
[0042] In one embodiment, a subject sample has a higher or
increased sialylation activity as compared to a control sample. By
"higher sialylation activity" or "increased sialylation activity"
is meant a statistically significant alteration in the level of one
or more markers in the sialylation activity profile. Table 2
provides non-limiting examples of markers for the sialylation
activity profile and denotes if an increase or a decrease in the
marker is reflective of a higher sialylation activity.
[0043] In one embodiment, an increase in sialylation activity is
denoted in a given profile by an alteration in at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the sialylation
activity markers as compared to a control sample. In some cases, an
alteration in sialylation activity of one marker is sufficient for
a diagnosis and/or prognosis. In other cases an alteration in two
or more sialylation activity markers is sufficient for a diagnosis
and/or prognosis.
[0044] Various assays are known to measure sialylation levels in a
sample. For example, a sample can be incubated with sambuscus nigra
lectin (SNA) that binds preferentially to sialic acid attached to a
terminal galactose in position alpha-2,6. Other assays for
sialylation are known in the art and include the use of Machia
amurentis lectin that binds sialic acids attached with an alpha-2,3
linkage.
[0045] In one embodiment, sialylation activity can be measured for
a population of proteins to determine global sialylation of
proteins in a sample. For example, sialylation in this context can
be assayed for in a sample by lectin binding assays. The lectin
binding assays can be ELISA based or can be gel based. In another
embodiment, the sialylation activity of a single protein can be
measured. In this instance, an ELISA based or gel based lectin
assay can be coupled with a specific antibody to a protein of
interest. The level of sialylation in a sample can be quantitated
by using samples of known different sialylation levels as standards
in the assay.
[0046] C. Lysosomal Sialidase Activity
[0047] In one embodiment, the sialylation activity comprises the
level of lysosomal sialidase activity. "Sialidases" are enzymes
that remove the terminal sialic acid from glycoproteins by a
process called desialylation. In mammals, there are at least four
types of sialidases including, for example, Neuraminidase 1 (NEU1),
NEU2, NEU3 and NEU4 which differ in substrate specificity and
subcellular localization. NEU1, for example, is localized to the
lysosome and cleaves terminal sialic acid residues from substrates
such as glycoproteins. As such, NEU1 is an enzyme that contributes
to the overall sialylation activity of a sample.
[0048] In the lysosome, NEU1 is part of a heterotrimeric complex
together with beta-galactosidase and protective protein/cathepsin A
(PPCA). The presence of PPCA in the NEU1 complex stabilizes NEU1 in
the lysosome. NEU1 has various substrates. As used herein, a "NEU1
substrate" is any protein that is desialylated by NEU1. Some
non-limiting examples of NEU1 substrates include LAMP-1, Cathepsin
A, mucins (i.e. MUC1), cathepsin D, cathepsin B and Amyloid
Precursor Protein. NEU1 can catalyze the hydrolysis of alpha 2-3
and alpha 2-6 sialyl linkages of terminal sialic acid residues in
oligosaccharides, glycoproteins and glycolipids. Desialylation of a
glycoprotein, for example, leads to the destabilization and
degradation of the protein. Thus, the sialidase, NEU1, contributes
to the turnover of glycoproteins.
[0049] In addition to its role as a sialidase, NEU1 has a related
effect on the constitutive process of lysosomal exocytosis. As
described elsewhere herein, lysosomal exocytosis involves the
recruitment and docking of lysosomes to the plasma membrane, fusion
of the lysosomal membrane with the plasma membrane and the release
of lysosomal luminal content into the extracellular environment.
The recruitment and docking step is facilitated by the lysosomal
associated protein-1 (LAMP-1). LAMP-1 is a NEU1 substrate, and thus
the stability and turnover rate of LAMP-1 can be influenced by
lysosomal sialidase activity.
[0050] In one embodiment, a subject sample has a lower or decreased
lysosomal sialidase activity as compared to a control sample. By
"lower lysosomal sialidase activity" or "decreased lysosomal
sialidase activity" is meant a statistically significant alteration
in the level of one or more markers in the lysosomal sialidase
activity profile. Table 2 provides non-limiting examples of markers
for the lysosomal sialidase activity profile and denotes if an
increase or a decrease in the marker is reflective of a lower
lysosomal sialidase activity.
[0051] In one embodiment, a decrease in lysosomal sialidase
activity is denoted in a given profile by an alteration in at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the
lysosomal sialidase activity markers as compared to a control
sample. In some cases, an alteration in lysosomal sialidase
activity of one marker is sufficient for a diagnosis and/or
prognosis. In other cases, an alteration in two or more lysosomal
sialidase activity markers is sufficient for a diagnosis and/or
prognosis.
[0052] In one embodiment, lysosomal sialidase activity is measured
by the level of NEU1 protein or enzymatic activity of NEU1. Assays
to measure NEU1 protein level are well known in the art and include
contacting a sample with an antibody to NEU1. In addition, NEU1
enzymatic activity can be measured directly in a sample by assaying
for NEU1 enzyme activity of a sample in the presence of a
sialylated NEU1 substrate. Thus, when NEU1 protein and/or enzyme
activity levels in a sample are low or absent, NEU1 substrates will
not be desialylated or will be desialylated at a lower rate
resulting in an increase in sialylation of the substrate and/or an
increase in the stability of the substrate and thus an increase in
the protein level of the NEU1 substrate in a sample. For example,
under conditions where NEU1 protein is not present in a sample
(i.e. in a NEU1 knockout), LAMP-1 is over-sialylated, accumulates
in the lysosome, recruits the lysosome to the plasma membrane and
facilitates docking of the lysosome to the plasma membrane. In such
cases, the loss of NEU1 protein/activity results in an increase in
lysosomal exocytosis.
[0053] As used herein, an increase in sialylation of any one or
more NEU1 substrates results in a lower lysosomal sialidase
activity in a sample. Further, an increase in the protein level of
any one or more of the NEU1 substrates provided herein also results
in a lower lysosomal sialidase activity. As such, these values are
markers for lysosomal sialidase activity and indicative of low
protein and activity levels of NEU1 in a sample. Assays to measure
for sialylation levels in a sample or the sialylation level of a
specific protein in a sample are discussed elsewhere herein. Assays
to measure the protein level of any of the various lysosomal
sialidase activity markers are known in the art and are described
in detail elsewhere herein.
[0054] In another embodiment, the enzymatic activity level of NEU1
or the activity level of any of the various NEU1 substrates are
markers for lysosomal sialidase activity. Assays to measure the
protein activity for various proteins is known in the art and
described elsewhere herein.
[0055] D. NEU1 Substrate Sialylation Activity
[0056] In one embodiment, one type of lysosomal sialidase profile
is a NEU1 substrate sialylation activity profile. Non-limiting
examples of the various NEU1 substrate sialylation activity markers
are summarized in Table 1.
[0057] In one embodiment, a subject sample has a higher or
increased NEU1 substrate sialylation activity as compared to a
control sample. By "higher NEU1 substrate sialylation activity" or
"increased NEU1 substrate sialylation activity" is meant a
statistically significant alteration in the level of one or more
markers in the NEU1 substrate sialylation activity profile. Table 2
provides non-limiting examples of markers for the NEU1 substrate
sialylation activity profile and denotes if an increase or a
decrease in the marker is reflective of a higher NEU1 substrate
sialylation activity.
[0058] In one embodiment, an increase in NEU1 substrate sialylation
activity is denoted in a given profile by an alteration in at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the
NEU1 substrate sialylation activity markers as compared to a
control sample. In some cases, an alteration in NEU1 substrate
sialylation activity of one marker is sufficient for a diagnosis
and/or prognosis. In other cases an alteration in two or more NEU1
substrate sialylation activity markers is sufficient for a
diagnosis and/or prognosis.
[0059] Assays to measure the NEU1 substrate sialylation activity of
the various NEU1 substrate sialylation activity markers are known
in the art and include measuring the level of sialylation of any of
the various NEU1 substrates provided herein. Such assays are
described elsewhere herein.
[0060] E. NEU1 Level Activity
[0061] In another embodiment, one type of lysosomal sialidase
activity profile is a NEU1 level activity profile. Non-limiting
examples of the various NEU1 level activity markers are summarized
in Table 1.
[0062] In one embodiment, a subject sample has a higher or
increased NEU1 level activity as compared to a control sample. By
"higher NEU1 level activity" or "increased NEU1 level activity" is
meant a statistically significant alteration in the level of two or
more markers in the NEU1 level activity profile. Table 2 provides
non-limiting examples of markers for the NEU1 level activity
profile and denotes if an increase or a decrease in the marker is
reflective of a higher NEU1 level activity.
[0063] In one embodiment, an increase in NEU1 level activity is
denoted in a given profile by an alteration in at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the NEU1 level
activity markers as compared to a control sample. In some cases, an
alteration in NEU1 level activity of two or more markers is
sufficient for a diagnosis and/or prognosis.
[0064] Assays to measure NEU1 level activity of the various NEU1
level activity markers are known in the art and include, for
example, immuno-blotting using an antibody specific for a NEU1
level activity marker or ELISA assay using an antibody specific for
a NEU1 level activity marker. These assays are discussed in detail
elsewhere herein.
TABLE-US-00002 TABLE 2 Summary of alterations in various markers
employed to establish an increase or decrease in a specific type of
activity. Marker Level of Marker Compared to Control Increased
Lysosomal exocytosis activity the level of NEU1 protein Decreased
the level of NEU1 enzymatic Decreased activity the protein level of
one or more Increased NEU1 substrates the protein level of LAMP-1
Increased the protein level of MUC-1 Increased the protein level of
any one or Increased more lysosomal proteins the protein level of
one or more Increased lysosomal proteases the activity level of one
or more Increased NEU1 substrates the activity level of LAMP-1
Increased the activity level of MUC-1 Increased The overall level
of sialylation Increased in a sample the sialylation level of one
or Increased more NEU1 substrates the sialylation level of LAMP-1
Increased the sialylation level of MUC-1 Increased the protein
level of one or more Increased cathepsins the protein level of
Increased Hexosaminidase beta the protein level of mannosidase
Increased alpha Increased Sialylation Activity the level of NEU1
protein Decreased The level of NEU1 enzymatic Decreased activity
the protein level of one or more Increased NEU1 substrates the
protein level of LAMP-1 Increased the protein level of MUC-1
Increased the activity level of one or more Increased NEU1
substrates the activity level of LAMP-1 Increased the activity
level of MUC-1 Increased the overall level of sialylation in
Increased a sample the sialylation level of one or Increased more
NEU1 substrates the sialylation level of LAMP-1 Increased the
sialylation level of MUC-1 Increased Decreased Lysosomal Sialidase
Activity the level of NEU1 protein Decreased the level of NEU1
enzymatic Decreased activity the protein level of one or more
Increased NEU1 substrates the protein level of LAMP-1 Increased the
protein level of MUC-1 Increased The activity level of one or more
Increased NEU1 substrates the activity level of LAMP-1 Increased
the activity level of MUC-1 Increased the sialylation level of one
or Increased more NEU1 substrates the sialylation level of LAMP-1
Increased the sialylation level of MUC-1 Increased Increased NEU1
Substrate Sialylation Activity the sialylation level of one or
Increased more NEU1 substrates the sialylation level of LAMP-1
Increased the sialylation level of MUC-1 Increased Increased NEU1
Level Activity the protein level of one or more Increased non-MUC-1
NEU1 substrates the protein level of MUC-1-only Increased in
combination with another NEU1 substrate the protein level of LAMP-1
Increased the protein level of MUC-1 Increased
[0065] IV. Cancer
[0066] The various profiles provided herein can be used in methods
of prognosis of a chemotherapy regime, diagnosis of cancer and
prognosis of cancer in a subject. As provided herein "prognosis" is
the likely outcome of a pathological condition or disease (i.e. the
expected morbidity or mortality, the expected outcome of a therapy,
or the risk of metastasis). "Diagnosis" refers to determining
whether a subject is likely to have a disease or condition.
[0067] As mentioned in the previous section, NEU1 is a regulator of
lysosomal exocytosis. Moreover, NEU1 is the only known regulator of
lysosomal exocytosis. Defects in lysosomal exocytosis have been
associated with various diseases. For example, NEU1 deficiency
results in the lysosomal storage disease sialidosis.
[0068] Under conditions where NEU1 levels or enzyme activity are
low, the NEU1 substrate, LAMP-1, accumulates in an over-sialylated
state in the lysosome. As discussed, LAMP-1 enhances lysosomal
exocytosis. As such, a lower lysosomal sialidase activity results
in an increase in lysosomal exocytosis and release of lysosomal
contents into the extracellular space.
[0069] Described herein is the discovery that cancer cells and
tumors from various types of cancers have a low lysosomal sialidase
activity (i.e. as measured using any of the lysosomal sialidase
activity markers provided herein). See, for example, Example 1
described elsewhere herein. In such cases, the down-regulation of
NEU1 leads to a deregulation of lysosomal exocytosis in the cancer
cells, thus increasing lysosomal exocytosis.
[0070] Excessive lysosomal exocytosis can have profound effects on
cancer diagnosis, prognosis and chemotherapy as discussed herein.
The methods of determining the prognosis of a lysosomotropic
chemotherapeutic agent, and the diagnosis and prognosis of cancer
provided herein encompass any type of cancer in a subject.
Non-limiting examples of types of cancer encompassed by the methods
herein include, sarcomas, leukemia, lymphoma, breast cancer, colon
cancer, rhabdomyosarcoma, Ewing's sarcoma, lung cancer, bladder
cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain
tumors, acute lymphoblastic leukemia, and bone cancer. In specific
embodiments, the cancer comprises rhabdomyosarcoma, breast cancer,
colon cancer, pancreatic cancer or Ewing's sarcoma.
[0071] A. Methods of Prognosis of a Chemotherapy Regime
[0072] Provided herein are methods of determining the prognosis for
a lysosomotropic chemotherapeutic agent regime in a subject with
cancer. As used herein, a "lysosomotropic chemotherapeutic agent"
is meant any chemotherapeutic agent that accumulates preferentially
in the lysosomes of cells. Many commonly used chemotherapeutic
agents accumulate in the acidic lysosome due to their weakly basic
nature. Some non-limiting examples of lysosomotropic
chemotherapeutic agents include doxorubicin, cisplatin and
docetaxel.
[0073] In cases where NEU1 is down-regulated in a cancer, this
leads to a deregulation of lysosomal exocytosis in the cancer
cells, thus increasing lysosomal exocytosis. As such,
chemotherapeutic agents which accumulate in the lysosomes are
released from the cancer cells into the extracellular space thereby
preventing the chemotherapy from having an effect on the cell.
Thus, a low lysosomal sialidase activity is predictive of
chemotherapy resistance to lysosomotropic chemotherapeutic agents.
By "resistant" to chemotherapy is meant the ability of a cell or
tumor to withstand the effects of a chemotherapeutic agent(s).
[0074] The prognosis for a lysosomotropic chemotherapeutic agent
regime in a subject with cancer can be determined by obtaining a
lysosomal sialidase activity profile of a sample from the subject
with cancer. In such cases, an alteration in the lysosomal
sialidase activity of any one or more lysosomal sialidase activity
markers as compared to a control sample, as depicted, for example,
in Table 2, results in a lower or decreased lysosomal sialidase
activity for the sample. In the case where the lysosomal sialidase
activity is lower in the subject sample as compared to a control
sample, it is predicted that the cancer will be resistant to the
lysosomotropic chemotherapy.
[0075] In one embodiment, the method of determining the prognosis
for a lysosomotropic chemotherapeutic agent regime in a subject
with cancer comprises the steps of: (a) providing a subject
lysosomal sialidase activity profile from a tumor sample from the
subject; (b) providing a reference lysosomal sialidase activity
profile from a control sample, wherein the subject lysosomal
sialidase activity profile and the reference lysosomal sialidase
activity profile comprise one or more values representing lysosomal
sialidase activity; and (c) comparing the subject and the reference
lysosomal sialidase activity profiles to thereby determine the
prognosis for a lysosomotropic chemotherapeutic agent regime in the
subject, wherein a lower lysosomal sialidase activity of the
subject as compared to the lysosomal sialidase activity of the
reference results in a prediction that the cancer will be resistant
to the lysosomotropic chemotherapeutic agent.
[0076] In one embodiment, the lysosomal sialidase activity profile
comprises any number and combination of lysosomal sialidase
activity values for any of the various lysosomal sialidase activity
markers provided herein. Non-limiting examples of the lysosomal
sialidase activity profile of a sample are provided in Table 1.
[0077] In a specific embodiment, the lysosomal sialidase activity
comprises the level of LAMP-1 protein. In another embodiment, the
lysosomal sialidase activity comprises the level of MUC-1 protein.
In yet another embodiment, the lysosomal sialidase activity
comprises the level of the NEU1 substrates LAMP-1 and MUC-1. In a
further embodiment, the lysosomal sialidase activity comprises the
level of LAMP-1 sialylation. In yet another embodiment, the
lysosomal sialidase activity comprises the level of MUC-1
sialylation. In another specific embodiment, the level of lysosomal
sialidase activity comprises the level of LAMP-land MUC-1
sialylation. In still further embodiments, the lysosomal sialidase
activity comprises the level of LAMP-1, the level of MUC-1, the
level of sialylation of LAMP-1 and the level of MUC-1
sialylation.
[0078] Knowledge of the lysosomal sialidase activity status of a
tumor from a subject will allow the physician to predict the most
appropriate therapy for a subject having a cancer with a low
lysosomal sialidase activity profile. For example, lysosomotropic
chemotherapeutic agents would not be chosen for treating a tumor
with low lysosomal sialidase activity profile since this is
predictive that the tumor will be resistant to these agents. Thus,
a treatment regime with chemotherapeutic drugs that do not
accumulate in the lysosome would be a better treatment option.
[0079] B. Methods of Diagnosis and Prognosis of Cancer
[0080] The methods herein also provide a method of determining the
prognosis and diagnosis for a subject with cancer. Information
obtained from the diagnosis and prognosis can be useful in
selecting an appropriate treatment.
[0081] As described elsewhere herein, NEU1 is a negative regulator
of lysosomal exocytosis and low lysosomal sialidase activity
results in an increase in lysosomal exocytosis. Excess lysosomal
exocytosis can have profound effects on the extracellular
environment of a cell. For example, the lysosome contains proteases
which breakdown the extracellular matrix resulting in a remodeling
of the extracellular environment. The breakdown of the
extracellular matrix increases the vulnerability of tissue to
invasion. As such, a high concentration of proteases in the
extracellular matrix surrounding a cancer cell can enhance the
invasive potential and metastasis of a cancer cell.
[0082] A cancer that is "invasive" has the ability to spread to the
surrounding tissue. "Metastasis", as used herein, refers to the
process by which a cancer spreads or transfers from the site of
origin to other regions of the body. As depicted elsewhere herein,
cancers that have low lysosomal sialidase activity have an
increased invasive potential. Assays that measure the invasiveness
of a cancer are known in the art and an example invasion assay is
described in detail in Example 1 provided elsewhere herein.
[0083] Invasive cancers are more likely to metastasize and thus
have an unfavorable prognosis, whereas non-invasive cancers are
less likely to metastasize and therefore have a favorable
prognosis. The term "unfavorable prognosis" in regards to tumors or
subjects diagnosed with cancer refers to a tumor or subject with a
high probability of metastasis and/or a high probability of causing
death or dying. A "favorable prognosis" in regards to a subject
diagnosed with cancer refers to a tumor or subject with a low
probability of metastasis and/or a low probability of causing death
or dying.
[0084] The lysosomal sialidase activity of a sample, for the
purpose of diagnosis and prognosis of cancer, can be determined by
measuring the values for any two or more of the various lysosomal
sialidase activity markers provided herein. Thus, lower levels of
lysosomal sialidase activity in a subject sample as compared to a
reference lysosomal sialidase activity of a control sample are
indicative that a tumor has increased invasive potential (i.e. an
unfavorable prognosis), while higher or normal levels of lysosomal
sialidase activity in a subject sample as compared to a reference
lysosomal sialidase activity of a control sample are predictive of
a less invasive potential (i.e. a favorable prognosis).
[0085] In some embodiments the diagnosis and/or prognosis of cancer
can be determined by measuring the NEU1 substrate sialylation
activity or the NEU1 level activity of a sample. These activities
can be determined by measuring the values for any of the various
markers provided in Tables 1 and 2. For a NEU1 substrate
sialylation activity, a higher level of any one or more NEU1
substrate sialylation activity markers results in a higher NEU1
substrate sialylation activity and is indicative of cancer and an
unfavorable prognosis. For a NEU1 level activity, a higher level of
any two or more NEU1 substrate activity markers results in a higher
NEU1 level activity and is indicative of cancer and an unfavorable
prognosis.
[0086] In one embodiment, a method of determining the prognosis for
a subject with a cancer is provided and comprises the steps of: (a)
providing a subject lysosomal sialidase activity profile comprising
two or more values from different lysosomal sialidase activity
markers, a NEU1 substrate sialylation activity profile or a NEU1
level activity profile from a tumor sample from the subject; (b)
providing a corresponding reference lysosomal sialidase activity
profile comprising two or more values from different lysosomal
sialidase activity markers, a NEU1 substrate sialylation activity
profile or a NEU1 level activity profile from a control sample,
wherein the subject profile and the reference profile comprise one
or more values representing lysosomal sialidase activity, NEU1
substrate sialylation activity or NEU1 level activity; and (c)
comparing the subject and the reference lysosomal sialidase
activity profiles, NEU1 substrate sialylation activity profiles or
NEU1 level activity profiles to thereby determine the prognosis for
the subject with cancer, wherein a lower lysosomal sialidase
activity, a higher NEU1 substrate sialylation activity or a higher
NEU1 level activity of the subject as compared to the lysosomal
sialidase activity, NEU1 substrate sialylation activity or NEU1
level activity of the reference results in a prediction of an
invasive cancer for the subject.
[0087] In another embodiment, a method of diagnosing cancer in a
subject is provided, the method comprising: (a) providing a subject
profile comprising a lysosomal sialidase activity profile
comprising two or more values from different lysosomal sialidase
activity markers, a NEU1 substrate sialylation activity profile or
a NEU1 level activity profile from a tumor sample from the subject;
(b) providing a corresponding reference profile comprising a
lysosomal sialidase activity profile comprising two or more values
from different lysosomal sialidase activity markers, a NEU1
substrate sialylation activity profile or a NEU1 level activity
profile from a control sample, wherein the subject profile and the
reference profile comprise one or more values representing
lysosomal sialidase activity, NEU1 substrate sialylation activity
or NEU1 level activity; and (c) comparing the subject and the
reference lysosomal sialidase activity profiles, NEU1 substrate
sialylation profiles or NEU1 level profiles to thereby determine
the diagnosis for the subject, wherein the subject is diagnosed
with cancer if the lysosomal sialidase activity of the subject is
lower, the NEU1 substrate sialylation activity is higher or the
NEU1 level activity is higher than the lysosomal sialidase
activity, the NEU1 substrate sialidase activity or the NEU1 level
activity of the reference.
[0088] Knowledge of the level of lysosomal sialidase activity, NEU1
substrate sialylation activity or NEU1 level activity in a subject
sample allows a practitioner to diagnose a subject as having
cancer, predict the aggressiveness of a cancer and thereby select
the appropriate therapy for the subject with cancer.
[0089] V. Methods of Diagnosis of Dementia Associated With
Alzheimer's Disease
[0090] Also provided herein are methods for the diagnosis of
dementia associated with Alzheimer's disease. Provided herein, is a
demonstration that the lysosomal exocytosis activity profile of a
sample from a subject is predictive of dementia associated with
Alzheimer's disease.
[0091] As used herein, "dementia associated with Alzheimer's
disease" is characterized by the standard criteria for dementia as
reported in the Recommendations from the National Institute on
Aging-Alzheimer's Association workgroups on diagnostic guidelines
for Alzheimer's disease. The standard criteria for dementia include
(1) cognitive or behavioral symptoms that interfere with the
ability to function at usual activities or work, denote a decline
from previous functioning and performing levels and cannot be
explained by a major psychiatric disorder or delirium; (2)
detection and diagnosis of cognitive impairment through a
combination of history-taking from the patient and a knowledgeable
informant and an objective cognitive assessment; and (3) the
cognitive or behavioral impairment involves two or more of the
following: (a) impaired ability to acquire and remember new
information; (b) impaired reasoning and handling of complex tasks,
poor judgment; (c) impaired visuospatial abilities; (d) impaired
language functions; and (e) changes in personality, behavior or
comportment. Dementia associated with Alzheimer's can further have
one or more of the following characteristics: (1) meets all
criteria for dementia as described above; (2) insidious onset; (3)
a history of worsening or cognition by report or observation; (4)
amnestic presentation; and (5) nonamnestic presentations, such as,
language presentation, visuospatial presentation or executive
dysfunction. The Alzheimer's disease dementia guidelines are
described in detail in McKhann et al. (2011) Alzheimer's &
Dementia 7:263-69, herein incorporated by reference in its
entirety.
[0092] As described elsewhere herein, NEU1 is a negative regulator
of lysosomal exocytosis. In such instances when NEU1 protein levels
or enzymatic activity are low, lysosomal exocytosis is enhanced. As
shown herein, under conditions where the NEU1 protein level is low,
highly sialylated proteins can be detected in the cerebrospinal
fluid (CSF). In such cases, the composition of the CSF is changed
and many of the highly sialylated proteins also have increased
levels in the CSF. These proteins that are changed in the CSF under
conditions of low NEU1 protein and activity levels correlate with
biomarkers for predicting dementia associated with Alzheimer's
disease. For example, amyloid precursor protein (APP) is shown
herein to be a NEU1 substrate and accumulates in the brain and CSF
in a highly sialylated form under low NEU1 conditions. Lysosomes
also comprise proteases that can process APP to form toxic A.beta.
peptides. Thus, excessive lysosomal exocytosis (i.e. when NEU1
protein or enzymatic activity levels are low) enhances the plaque
formation that is characteristic of Alzheimer's disease. See, for
example, Example 3, provided elsewhere herein. Thus, in one
embodiment, an increased lysosomal exocytosis activity, as
described in detail elsewhere herein, in the CSF can be predictive
of dementia associated with Alzheimer's disease.
[0093] A variety of proteins can have an increased sialylation
level and/or have increased levels in the CSF. In some embodiments,
the proteins having an increased sialylation level and/or protein
level are NEU1 substrates. In other embodiments, the proteins
having an increased sialylation level and/or protein level are
lysosomal proteins. Non-limiting examples of proteins with
increased sialylation and/or protein level include LAMP-1, MUC-1,
Cathepsin B, Cathepsin D, Complement system proteins, Fibrinogen,
Hexosaminidase beta, Mannosidase alpha, Transthyretin, beta-2
microglobulin and Amyloid Precursor Protein. Any one or more of
these proteins can be a marker for lysosomal exocytosis
activity.
[0094] Provided herein is a method of diagnosing dementia
associated with Alzheimer's disease in a subject, the method
comprising: (a) providing a subject lysosomal exocytosis activity
profile of a sample of cerebrospinal fluid from the subject; (b)
providing a reference lysosomal exocytosis activity profile of a
control sample of cerebrospinal fluid, wherein the subject
lysosomal exocytosis activity profile and the corresponding
reference lysosomal exocytosis activity profile comprise one or
more values representing lysosomal exocytosis activity; and (c)
comparing the subject and the reference lysosomal exocytosis
activity profiles, wherein the subject is diagnosed with dementia
associated with Alzheimer's disease if the subject has a higher
lysosomal exocytosis activity as compared to the reference
lysosomal exocytosis activity.
[0095] In one embodiment the lysosomal exocytosis activity profile
comprises a lysosomal sialidase activity profile. The lysosomal
sialidase activity profile can comprise any combination of any of
the various lysosomal sialidase activity markers provided herein.
In such cases, a low lysosomal sialidase activity in a subject
sample as compared to a reference lysosomal sialidase activity in a
control sample results in a subject being diagnosed with dementia
associated with Alzheimer's disease.
[0096] In another embodiment, the lysosomal exocytosis activity
profile comprises a sialylation activity profile. The sialylation
activity profile can comprise any combination of any of the various
sialylation activity markers provided herein. In such cases, a high
sialylation activity in a subject sample as compared to a reference
sialylation activity in a control sample results in a subject being
diagnosed with dementia associated with Alzheimer's disease.
[0097] For the diagnosis of dementia associated with Alzheimer's
disease, the lysosomal exocytosis activity profiles, the lysosomal
sialidase activity profiles or the sialylation activity profiles
can comprise any one or more of the various markers provided herein
(i.e. see Tables 1 and 2).
[0098] Knowledge of the sialylation activity profile of a subject
sample will allow the physician to make a diagnosis of dementia
associated with Alzheimer's disease in a subject. Thus, an early
diagnosis can be made and the appropriate treatment options can be
considered for the subject.
[0099] VI. Methods of Generating a Lysosomal Sialidase Activity
Profile and an Lysosomal Exocytosis Activity Profile
[0100] Methods of generating a lysosomal sialidase activity profile
and/or a lysosomal exocytosis activity profile for a sample are
also provided. As presented herein, the lysosomal sialidase
activity profile of a sample can comprise one or more lysosomal
sialidase activity markers representing lysosomal sialidase
activity (i.e. any of the various markers of lysosomal sialidase
activity provided herein, see Table 1). Also herein, the lysosomal
exocytosis activity profile of a sample can comprise one or more
lysosomal exocytosis activity markers representing lysosomal
exocytosis activity (i.e. any or the various markers of lysosomal
exocytosis activity provided herein, see Table 1).
[0101] In one embodiment, a method of generating a lysosomal
sialidase activity profile comprises: (a) obtaining a sample from a
tumor from a subject; and (b) assaying for the level of LAMP-1
protein or the level of LAMP-1 sialylation. In a further
embodiment, the method comprises assaying for one or more
additional lysosomal sialidase activity markers. In yet another
embodiment of the method, the one or more additional lysosomal
sialidase activity markers comprise a NEU1 substrate. Assays for
measuring the various lysosomal sialidase activity markers are
provided elsewhere herein.
[0102] In another embodiment, a method of generating a lysosomal
exocytosis activity profile from cerebrospinal fluid comprises: (a)
obtaining a sample of cerebrospinal fluid from a subject; and (b)
assaying for lysosomal exocytosis activity. In a specific
embodiment, assaying for lysosomal exocytosis activity comprises
assaying for the level of LAMP-1 protein or the level of LAMP-1
sialylation.
[0103] In a non-limiting embodiment, assaying for lysosomal
exocytosis activity comprises assaying for the level of one or more
proteins comprising LAMP-1, MUC-1, amyloid precursor protein,
Cathepsin B, Cathepsin D, Fibrinogen, Hexosaminidase beta,
Mannosidase alpha, Transthyretin, beta-2 microglobulin or
Immunoglobulin heavy chain.
[0104] VII. Methods of Treatment
[0105] Further provided are methods of treating a subject having a
cancer or having dementia associated with Alzheimer's disease. By
"treating" a subject with cancer or dementia associated with
Alzheimer's disease is intended administration of a therapeutically
effective amount of NEU1 or an active variant or fragment thereof,
administration of a therapeutically effective amount of protective
protein/cathepsin A (PPCA) or an active variant or fragment thereof
or administration of a therapeutically effective amount of a
combination of NEU1 and PPCA to a subject that has cancer or
dementia associated with Alzheimer's disease, where the purpose is
to cure, heal, alleviate, relieve, alter, remedy, ameliorate,
improve, or affect the condition or the symptoms of the cancer or
dementia associated with Alzheimer's disease.
[0106] Also provided herein are methods of preventing a cancer or
dementia associated with Alzheimer's disease in a subject. By
"preventing" a cancer or dementia associated with Alzheimer's
disease in a subject is intended administration of a
therapeutically effective amount of NEU1 or an active variant or
fragment thereof, administration of a therapeutically effective
amount of protective protein/cathepsin A (PPCA) or an active
variant or fragment thereof or administration of a therapeutically
effective amount of a combination of NEU1 and PPCA to a subject,
where the purpose is to protect the subject from development of a
cancer or dementia associated with Alzheimer's disease. In some
embodiments, a therapeutically effective amount of NEU1 or an
active variant or fragment thereof, protective protein/cathepsin A
(PPCA) or an active variant or fragment thereof or a combination of
NEU1 and PPCA is administered to a subject, such as a human, that
is at risk for developing a cancer or dementia associated with
Alzheimer's disease.
[0107] A "therapeutically effective amount" as used herein refers
to that amount which provides a therapeutic effect for a given
condition and administration regimen. Thus, the phrase
"therapeutically effective amount" is used herein to mean an amount
sufficient to cause an improvement in a clinically significant
condition in the host. In particular aspects, a "therapeutically
effective amount" refers to an amount of NEU1, PPCA, or a
combination of NEU1 and PPCA provided herein that when administered
to a subject brings about a positive therapeutic response with
respect to the treatment of a subject for a cancer or dementia
associated with Alzheimer's disease. A positive therapeutic
response in regard to treating a cancer includes curing or
ameliorating the symptoms of the disease. In the present context, a
deficit in the response of the host can be evidenced by continuing
or spreading of the cancer. An improvement in a clinically
significant condition in the host includes a decrease in the size
of a tumor, increased necrosis of a tumor, clearance of the tumor
from the host tissue, reduction or amelioration of metastasis, or a
reduction in any symptom associated with the cancer. A positive
therapeutic response in regard to treating a subject with dementia
associated with Alzheimer's disease includes curing or ameliorating
the symptoms of the disease. In this context, a deficit in the
response of the host can be evidenced by continuing or worsening of
the dementia associated with Alzheimer's disease. An improvement in
a clinically significant condition in the host includes a decrease
in dementia (i.e. an improvement in memory, judgment, visuospatial
abilities, language functions, behavior or any of the other
symptoms of dementia provided elsewhere herein) in the subject.
[0108] In particular aspects, a "therapeutically effective amount"
refers to an amount of NEU1, PPCA, or a combination of NEU1 and
PPCA provided herein that when administered to a subject brings
about a positive therapeutic response with respect to the
prevention of a cancer or dementia associated with Alzheimer's
disease in a subject. A positive therapeutic response with respect
to preventing a cancer or dementia associated with Alzheimer's
disease in a subject, for example, is the prevention of development
of the disease in a subject.
[0109] In one embodiment, a method of treating a subject having a
cancer comprises administering to a subject in need thereof a
therapeutically effective amount of Neuraminidase 1 (NEU1) having
an amino acid sequence with at least 85% sequence identity to SEQ
ID NO: 2 or an active variant or fragment thereof.
[0110] In another embodiment, a method of treating a subject with
dementia associated with Alzheimer's disease comprises
administering to a subject in need thereof a therapeutically
effective amount of Neuraminidase 1 (NEU1) having an amino acid
sequence with at least 85% sequence identity to SEQ ID NO: 2 or an
active variant or fragment thereof.
[0111] In some embodiments, the methods can further comprise
administration of Protective Protein/Cathepsin A (PPCA) having an
amino acid sequence with at least 85% sequence identity to SEQ ID
NO: 4.
[0112] In other embodiments, the administration of NEU1 and PPCA
can be separate or NEU1 and PPCA can be administered to a subject
simultaneously. The administration can be by any known method of
administration as described elsewhere herein. In one embodiment,
the administration of NEU1 and/or PPCA comprises administration of
a viral vector comprising a nucleotide sequence having at least 85%
sequence identity to SEQ ID NO: 1 and/or a nucleotide sequence
having at least 85% sequence identity to SEQ ID NO: 3.
[0113] Active variants and fragments of NEU1 can be used in the
methods provided herein. Such active variants can comprise at least
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 2, wherein the
active variants retain biological activity and hence have sialidase
activity. Sialidase activity is described in detail elsewhere
herein. Active variants of NEU1 are known in the art. There are
over 130 types of neuraminidases known from various species ranging
from viruses to humans. See, for example, Monti et al. (2010) Adv.
Carbohydr. Chem. Biochem. 64:403-79, herein incorporated by
reference in its entirety.
[0114] Active variants and fragments of PPCA can be used in the
methods provided herein. Such active variants can comprise at least
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO:4, wherein the
active variants retain biological activity and hence enhances NEU1
enzymatic activity. Assays to measure for NEU1 enzymatic activity
are described elsewhere herein. Active variants of PPCA are known
in the art. See, for example, Galjart et al. (1988) Cell
54(6):755-64, herein incorporated by reference in its entirety.
[0115] VIII. Methods of Administration
[0116] The methods of treatment for cancer and dementia associated
with Alzheimer's disease provided herein can encompass
administration of treatment via any parenteral route, including,
but not limited, to intramuscular, intraperitoneal, intravenous,
and the like.
[0117] Further, as used herein "pharmaceutically acceptable
carriers" are well known to those skilled in the art and include,
but are not limited to, 0.01-0.1 M, or 0.05M phosphate buffer or
0.8% saline. Additionally, such pharmaceutically acceptable
carriers may be aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers such as those based on Ringer's dextrose,
and the like. Preservatives and other additives may also be
present, such as, for example, antimicrobials, antioxidants,
collating agents, inert gases and the like.
[0118] Controlled or sustained release compositions include
formulation in lipophilic depots (e.g. fatty acids, waxes, oils).
Also comprehended herein are particulate compositions coated with
polymers (e.g. poloxamers or poloxamines) and the compound coupled
to antibodies directed against tissue-specific receptors, ligands
or antigens or coupled to ligands of tissue-specific receptors.
Other embodiments of the compositions presented herein incorporate
particulate forms protective coatings, protease inhibitors or
permeation enhancers for various routes of administration,
including parenteral, pulmonary, nasal and oral.
[0119] When administered, compounds are often cleared rapidly from
mucosal surfaces or the circulation and may therefore elicit
relatively short-lived pharmacological activity. Consequently,
frequent administrations of relatively large doses of bioactive
compounds may be required to sustain therapeutic efficacy.
Compounds modified by the covalent attachment of water-soluble
polymers such as polyethylene glycol, copolymers of polyethylene
glycol and polypropylene glycol, carboxymethyl cellulose, dextran,
polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to
exhibit substantially longer half-lives in blood following
intravenous injection than do the corresponding unmodified
compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre
et al., 1987). Such modifications may also increase the compound's
solubility in aqueous solution, eliminate aggregation, enhance the
physical and chemical stability of the compound, and greatly reduce
the immunogenicity and reactivity of the compound. As a result, the
desired in vivo biological activity may be achieved by the
administration of such polymer-compound abducts less frequently or
in lower doses than with the unmodified compound.
[0120] Dosages.
[0121] The sufficient amount may include but is not limited to from
about 1 .mu.g/kg to about 100 .mu.g/kg, from about 100 .mu.g/kg to
about 1 mg/kg, from about 1 mg/kg to about 10 mg/kg, about 10 mg/kg
to about 100 mg/kg, from about 100 mg/kg to about 500 mg/kg or from
about 500 mg/kg to about 1000 mg/kg. The amount may be 10 mg/kg.
The pharmaceutically acceptable form of the composition includes a
pharmaceutically acceptable carrier.
[0122] The preparation of therapeutic compositions which contain an
active component is well understood in the art. Typically, such
compositions are prepared as an aerosol of the polypeptide
delivered to the nasopharynx or as injectables, either as liquid
solutions or suspensions, however, solid forms suitable for
solution in, or suspension in, liquid prior to injection can also
be prepared. The preparation can also be emulsified. The active
therapeutic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the composition can contain minor amounts
of auxiliary substances such as wetting or emulsifying agents, pH
buffering agents which enhance the effectiveness of the active
ingredient.
[0123] An active component can be formulated into the therapeutic
composition as neutralized pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the polypeptide) and which
are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed from the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0124] The component or components of a therapeutic composition
provided herein may be introduced parenterally, transmucosally,
e.g., orally, nasally, pulmonarily, or rectally, or transdermally.
Preferably, administration is parenteral, e.g., via intravenous
injection, and also including, but is not limited to,
intra-arteriole, intramuscular, intradermal, subcutaneous,
intraperitoneal, intraventricular, and intracranial administration.
The term "unit dose" when used in reference to a therapeutic
composition provided herein refers to physically discrete units
suitable as unitary dosage for humans, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
diluent; i.e., carrier, or vehicle.
[0125] In another embodiment, the active compound can be delivered
in a vesicle, in particular a liposome (see Langer (1990) Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid).
[0126] In yet another embodiment, the therapeutic compound can be
delivered in a controlled release system. For example, the protein
may be administered using intravenous infusion, an implantable
osmotic pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump may be used (see Langer,
supra; Sefton (1987) CRC Crit. Ref Biomed. Eng. 14:201; Buchwald et
al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med.
321:574). In another embodiment, polymeric materials can be used
(see Medical Applications of Controlled Release, Langer and Wise
(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983) J.
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.
(1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351;
Howard et al. (1989) J. Neurosurg. 71:105). In yet another
embodiment, a controlled release system can be placed in proximity
of the therapeutic target, i.e., the brain or a tumor, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)). Other controlled release systems are discussed in
the review by Langer (1990) Science 249:1527-1533.
[0127] A subject in whom administration of an active component as
set forth above is an effective therapeutic regimen for a cancer or
dementia associated with Alzheimer's disease is preferably a human,
but can be any animal. Thus, as can be readily appreciated by one
of ordinary skill in the art, the methods and pharmaceutical
compositions provided herein are particularly suited to
administration to any animal, particularly a mammal, and including,
but by no means limited to, domestic animals, such as feline or
canine subjects, farm animals, such as but not limited to bovine,
equine, caprine, ovine, and porcine subjects, wild animals (whether
in the wild or in a zoological garden), research animals, such as
mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e.,
for veterinary medical use.
[0128] In the therapeutic methods and compositions provided herein,
a therapeutically effective dosage of the active component is
provided. A therapeutically effective dosage can be determined by
the ordinary skilled medical worker based on patient
characteristics (age, weight, sex, condition, complications, other
diseases, etc.), as is well known in the art. Furthermore, as
further routine studies are conducted, more specific information
will emerge regarding appropriate dosage levels for treatment of
various conditions in various patients, and the ordinary skilled
worker, considering the therapeutic context, age and general health
of the recipient, is able to ascertain proper dosing. Generally,
for intravenous injection or infusion, dosage may be lower than for
intraperitoneal, intramuscular, or other route of administration.
The dosing schedule may vary, depending on the circulation
half-life, and the formulation used. The compositions are
administered in a manner compatible with the dosage formulation in
the therapeutically effective amount. Precise amounts of active
ingredient required to be administered depend on the judgment of
the practitioner and are peculiar to each individual. However,
suitable dosages may range from about 0.1 to 20, preferably about
0.5 to about 10, and more preferably one to several, milligrams of
active ingredient per kilogram body weight of individual per day
and depend on the route of administration. Suitable regimes for
initial administration and booster shots are also variable, but are
typified by an initial administration followed by repeated doses at
one or more hour intervals by a subsequent injection or other
administration. Alternatively, continuous intravenous infusion
sufficient to maintain concentrations of ten nanomolar to ten
micromolar in the blood are contemplated.
[0129] Administration with Other Compounds.
[0130] For treatment of cancer or dementia associated with
Alzheimer's disease, one may administer the present active
component in conjunction with one or more pharmaceutical
compositions used for treating cancer or dementia associated with
Alzheimer's disease, including but not limited to (1)
chemotherapeutic agents; or (2) other drugs for treating symptoms
of Alzheimer's including domepezil, galantamine, memantine,
rivastigmine or tacrine. Administration may be simultaneous (for
example, administration of a mixture of the present active
component and a chemotherapeutic agent), or may be in seriatim.
[0131] Also contemplated are dry powder formulations comprising at
least one protein provided herein and another therapeutically
effective drug, such as a chemotherapeutic agent or a drug for
treating Alzheimer's disease.
[0132] Contemplated for use herein are oral solid dosage forms,
which are described generally in Remington's Pharmaceutical
Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at
Chapter 89, which is herein incorporated by reference. Solid dosage
forms include tablets, capsules, pills, troches or lozenges,
cachets or pellets. Also, liposomal or proteinoid encapsulation may
be used to formulate the present compositions (as, for example,
proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
Liposomal encapsulation may be used and the liposomes may be
derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).
A description of possible solid dosage forms for the therapeutic is
given by Marshall, K. In: Modern Pharmaceutics Edited by G. S.
Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by
reference. In general, the formulation will include the component
or components (or chemically modified forms thereof) and inert
ingredients which allow for protection against the stomach
environment, and release of the biologically active material in the
intestine.
[0133] Also specifically contemplated are oral dosage forms of the
above derivatized component or components. The component or
components may be chemically modified so that oral delivery of the
derivative is efficacious. Generally, the chemical modification
contemplated is the attachment of at least one moiety to the
component molecule itself, where the moiety permits (a) inhibition
of proteolysis; and (b) uptake into the blood stream from the
stomach or intestine. Also desired is the increase in overall
stability of the component or components and increase in
circulation time in the body. Examples of such moieties include:
polyethylene glycol, copolymers of ethylene glycol and propylene
glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone and polyproline. Abuchowski and Davis (1981)
"Soluble Polymer-Enzyme Abducts" In: Enzymes as Drugs, Hocenberg
and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383;
Newmark, et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers
that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.
Preferred for pharmaceutical usage, as indicated above, are
polyethylene glycol moieties.
[0134] For the component (or derivative) the location of release
may be the stomach, the small intestine (the duodenum, the jejunum,
or the ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
protein (or derivative) or by release of the biologically active
material beyond the stomach environment, such as in the
intestine.
[0135] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), EUDRAGIT
L30D coating, AQUATERIC coating, cellulose acetate phthalate (CAP),
EUDRAGIT L coating, EUDRAGIT S coating, and Shellac. These coatings
may be used as mixed films.
[0136] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0137] The peptide therapeutic can be included in the formulation
as fine multiparticulates in the form of granules or pellets of
particle size about 1 mm. The formulation of the material for
capsule administration could also be as a powder, lightly
compressed plugs or even as tablets. The therapeutic could be
prepared by compression.
[0138] Colorants and flavoring agents may all be included. For
example, the protein (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0139] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, a-lactose, anhydrous lactose, cellulose,
sucrose, modified dextran and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are FAST-FLO diluent, EMDEX diluent, STA-RX 1500
diluent, EMCOMPRESS diluent and AVICEL diluent.
[0140] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, EXPLOTAB disintegrant.
Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants. Binders may be used to hold the therapeutic agent
together to form a hard tablet and include materials from natural
products such as acacia, tragacanth, starch and gelatin. Others
include methyl cellulose (MC), ethyl cellulose (EC) and
carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0141] An antifrictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, CARBOWAX4000 lubricant, and CARBOWAX 6000
lubricant.
[0142] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0143] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0144] Additives which potentially enhance uptake of the protein
(or derivative) are for instance the fatty acids oleic acid,
linoleic acid and linolenic acid.
[0145] In one embodiment, the method comprises the use of viruses
for administering NEU1 and/or PPCA to a subject. Administration can
be by the use of viruses that express NEU1 and/or PPCA, such as
recombinant retroviruses, recombinant adeno-associated viruses,
recombinant adenoviruses, and recombinant Herpes simplex viruses
(see, for example, Mulligan, Science 260:926 (1993), Rosenberg et
al., Science 242:1575 (1988), LaSalle et al., Science 259:988
(1993), Wolff et al., Science 247:1465 (1990), Breakfield and
Deluca, The New Biologist 3:203 (1991)).
[0146] A NEU1 and/or PPCA gene can be delivered using recombinant
viral vectors, including for example, adenoviral vectors (e.g.,
Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA 90:11498 (1993),
Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215 (1994), Li et al.,
Hum. Gene Ther. 4:403 (1993), Vincent et al., Nat. Genet. 5:130
(1993), and Zabner et al., Cell 75:207 (1993)),
adenovirus-associated viral vectors (Flotte et al., Proc. Nat'l
Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki
Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,
Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat.
Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus
vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993),
Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)),
pox viruses, such as canary pox virus or vaccinia virus
(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), and
Flexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and
retroviruses (e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et
al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res
33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and
Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Pat.
No. 5,399,346). Within various embodiments, either the viral vector
itself, or a viral particle, which contains the viral vector may be
utilized in the methods described below.
[0147] As an illustration of one system, adenovirus, a
double-stranded DNA virus, is a well-characterized gene transfer
vector for delivery of a heterologous nucleic acid molecule (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994); Douglas
and Curiel, Science & Medicine 4:44 (1997)). The adenovirus
system offers several advantages including: (i) the ability to
accommodate relatively large DNA inserts, (ii) the ability to be
grown to high-titer, (iii) the ability to infect a broad range of
mammalian cell types, and (iv) the ability to be used with many
different promoters including ubiquitous, tissue specific, and
regulatable promoters. In addition, adenoviruses can be
administered by intravenous injection, because the viruses are
stable in the bloodstream.
[0148] Using adenovirus vectors where portions of the adenovirus
genome are deleted, inserts are incorporated into the viral DNA by
direct ligation or by homologous recombination with a
co-transfected plasmid. In an exemplary system, the essential E1
gene is deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. Although an adenoviral delivery system with an
E1 gene deletion cannot replicate in the host cells, the host's
tissue will express and process an encoded heterologous protein.
Host cells will also secrete the heterologous protein if the
corresponding gene includes a secretory signal sequence. Secreted
proteins will enter the circulation from tissue that expresses the
heterologous gene (e.g., the highly vascularized liver).
[0149] Moreover, adenoviral vectors containing various deletions of
viral genes can be used to reduce or eliminate immune responses to
the vector. Such adenoviruses are E1-deleted, and in addition,
contain deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022
(1998); Raper et al., Human Gene Therapy 9:671 (1998)). The
deletion of E2b has also been reported to reduce immune responses
(Amalfitano et al., J. Virol. 72:926 (1998)). By deleting the
entire adenovirus genome, very large inserts of heterologous DNA
can be accommodated. Generation of so called "gutless"
adenoviruses, where all viral genes are deleted, are particularly
advantageous for insertion of large inserts of heterologous DNA
(for a review, see Yeh. and Perricaudet, FASEB J. 11:615
(1997)).
[0150] High titer stocks of recombinant viruses capable of
expressing a therapeutic gene can be obtained from infected
mammalian cells using standard methods. For example, recombinant
herpes simplex virus can be prepared in Vero cells, as described by
Brandt et al., J. Gen. Virol. 72:2043 (1991), Herold et al., J.
Gen. Virol. 75:1211 (1994), Visalli and Brandt, Virology 185:419
(1991), Grau et al., Invest. Ophthalmol. Vis. Sci. 30:2474 (1989),
Brandt et al., J. Virol. Meth. 36:209 (1992), and by Brown and
MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
[0151] When the subject treated with a recombinant virus is a
human, then the therapy is preferably somatic cell gene therapy.
That is, the preferred treatment of a human with a recombinant
virus does not entail introducing into cells a nucleic acid
molecule that can form part of a human germ line and be passed onto
successive generations (i.e., human germ line gene therapy).
[0152] IX. Variants and Fragments
[0153] Fragments and variants of the polynucleotides encoding the
NEU1 and PPCA polypeptides can be employed in the various methods
and compositions of the invention. By "fragment" is intended a
portion of the polynucleotide and hence the protein encoded thereby
or a portion of the polypeptide. Fragments of a polynucleotide may
encode protein fragments that retain the biological activity of the
native protein. Thus, fragments of a polynucleotide may range from
at least about 20 nucleotides, about 50 nucleotides, about 100
nucleotides, about 150, about 200, about 250, about 300, about 350,
about 400, about 450, about 500, about 550, about 600 and up to the
full-length polynucleotide encoding the NEU1 or PPCA
polypeptide.
[0154] A fragment of a polynucleotide that encodes a biologically
active portion of a NEU1 or PPCA polypeptide will encode at least
15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up
to the total number of amino acids present in a full-length NEU1 or
PPCA polypeptide.
[0155] A biologically active portion of a NEU1 or PPCA polypeptide
can be prepared by isolating a portion of one of the
polynucleotides encoding the portion of the NEU1 or PPCA
polypeptide and expressing the encoded portion of the polypeptide
(e.g., by recombinant expression in vitro), and assessing the
activity of the portion of the NEU1 or PPCA polypeptide.
Polynucleotides that encode fragments of a NEU1 or PPCA polypeptide
can comprise nucleotide sequence comprising at least 16, 20, 50,
75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 nucleotides, or
up to the number of nucleotides present in a full-length NEU1 or
PPCA nucleotide sequence disclosed herein.
[0156] "Variant" sequences have a high degree of sequence
similarity. For polynucleotides, conservative variants include
those sequences that, because of the degeneracy of the genetic
code, encode the amino acid sequence of one of the NEU1 or PPCA
polypeptides. Variants such as these can be identified with the use
of well-known molecular biology techniques, as, for example,
polymerase chain reaction (PCR) and hybridization techniques.
Variant polynucleotides also include synthetically derived
nucleotide sequences, such as those generated, for example, by
using site-directed mutagenesis but which still encode a NEU1 or
PPCA polypeptide. Generally, variants of a particular
polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to that particular
polynucleotide as determined by sequence alignment programs and
parameters described elsewhere herein.
[0157] Variants of a particular polynucleotide can also be
evaluated by comparison of the percent sequence identity between
the polypeptide encoded by a variant polynucleotide and the
polypeptide encoded by the reference polynucleotide. Thus, for
example, isolated polynucleotides that encode a polypeptide with a
given percent sequence identity to the NEU1 or PPCA polypeptides
set forth herein. Percent sequence identity between any two
polypeptides can be calculated using sequence alignment programs
and parameters described. Where any given pair of polynucleotides
is evaluated by comparison of the percent sequence identity shared
by the two polypeptides they encode, the percent sequence identity
between the two encoded polypeptides is at least about 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more sequence identity.
[0158] By "variant" protein is intended a protein derived from the
native protein by deletion (so-called truncation) or addition of
one or more amino acids to the N-terminal and/or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein; or substitution of one
or more amino acids at one or more sites in the native protein.
Variant proteins are biologically active, that is they continue to
possess the desired biological activity of the native protein. Such
variants may result from, for example, genetic polymorphism or from
human manipulation. Biologically active variants of a NEU1 or PPCA
polypeptides will have at least about 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to the amino acid sequence for the
native protein as determined by sequence alignment programs and
parameters described elsewhere herein. A biologically active
variant of a protein may differ from that protein by as few as 1-15
amino acid residues, as few as 1-10, such as 6-10, as few as 5, as
few as 4, 3, 2, or even 1 amino acid residue.
[0159] Proteins may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known in the art. For example,
amino acid sequence variants of the NEU1 or PPCA proteins can be
prepared by mutations in the DNA. Methods for mutagenesis and
nucleotide sequence alterations are well known in the art. See, for
example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.
4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York) and the references
cited therein. Guidance as to appropriate amino acid substitutions
that do not affect biological activity of the protein of interest
may be found in the model of Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington,
D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be preferable.
[0160] Thus, the polynucleotides used in the invention can include
the naturally occurring sequences, the "native" sequences, as well
as mutant forms. Likewise, the proteins used in the methods of the
invention encompass naturally occurring proteins as well as
variations and modified forms thereof. Such variants will continue
to possess the ability to implement a recombination event.
Generally, the mutations made in the polynucleotide encoding the
variant polypeptide should not place the sequence out of reading
frame, and/or create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication
No. 75,444.
[0161] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays.
[0162] Variant polynucleotides and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different NEU1 or PPCA coding sequences can be manipulated to
create new NEU1 or PPCA polypeptides possessing the desired
properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. Strategies for such DNA shuffling are known in the art. See,
for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.
(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0163] X. Sequence Identity
[0164] As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0165] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0166] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0167] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. It is further to be
understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides are approximate, and are provided for
description.
[0168] The subject matter of the present disclosure is further
illustrated by the following non-limiting examples.
TABLE-US-00003 TABLE 3 Summary of SEQ ID NOS. SEQ ID NO NA/AA
Description 1 NA Neuraminidase 1 nucleic acid sequence. 2 AA
Neuraminidase 1 amino acid sequence. 3 NA PPCA nucleic acid
sequence. 4 AA PPCA amino acid sequence.
[0169] Non-limiting examples of methods disclosed herein are as
follows:
1. A method of determining the prognosis for a subject with cancer,
comprising the steps of
[0170] a) providing a subject profile comprising a lysosomal
sialidase activity profile comprising two or more values from
different lysosomal sialidase activity markers, a NEU1 substrate
sialylation activity profile or a NEU1 level activity profile from
a tumor sample from said subject;
[0171] b) providing a corresponding reference profile comprising a
lysosomal sialidase activity profile comprising two or more values
from different lysosomal sialidase activity markers, a NEU1
substrate sialylation activity profile or a NEU1 level activity
profile from a control sample, wherein the subject profile and the
reference profile comprise one or more values representing
lysosomal sialidase activity, NEU1 substrate sialylation activity
or NEU1 level activity; and
[0172] c) comparing said subject and said reference lysosomal
sialidase activity profiles to thereby determine the prognosis for
said subject with cancer, wherein a lower lysosomal sialidase
activity, a higher NEU1 substrate sialylation activity or a higher
NEU1 level activity of said subject as compared to the lysosomal
sialidase activity, NEU1 substrate sialylation activity or NEU1
level activity of said reference results in a prediction of an
invasive cancer for said subject.
2. A method of diagnosing cancer in a subject, the method
comprising:
[0173] a) providing a subject profile comprising a lysosomal
sialidase activity profile comprising two or more values from
different lysosomal sialidase activity markers, a NEU1 substrate
sialylation activity profile or a NEU1 level activity profile from
a tumor sample from said subject;
[0174] b) providing a corresponding reference profile comprising a
NEU1 activity profile comprising two or more values from different
lysosomal sialidase activity markers, a NEU1 substrate sialylation
activity profile or a NEU1 level activity profile from a control
sample, wherein the subject profile and the reference profile
comprise one or more values representing lysosomal sialidase
activity, NEU1 substrate sialylation activity or NEU1 level
activity; and
[0175] c) comparing said subject and said reference lysosomal
sialidase activity profiles to thereby determine the diagnosis for
said subject, wherein said subject is diagnosed with cancer if said
lysosomal sialidase activity of said subject is lower, the NEU1
substrate sialylation activity is higher or the NEU1 level activity
is higher than the lysosomal sialidase activity, NEU1 substrate
sialylation activity or NEU1 level activity of said reference.
3. A method of determining the prognosis for a lysosomotropic
chemotherapeutic agent regime in a subject with cancer, comprising
the steps of
[0176] a) providing a subject lysosomal sialidase activity profile
from a tumor sample from said subject;
[0177] b) providing a reference lysosomal sialidase activity
profile from a control sample, wherein the subject lysosomal
sialidase activity profile and the reference lysosomal sialidase
activity profile comprise one or more values representing lysosomal
sialidase activity; and
[0178] c) comparing said subject and said reference lysosomal
sialidase activity profiles to thereby determine the prognosis for
a lysosomotropic chemotherapy agent regime in the subject, wherein
a lower lysosomal sialidase activity of said subject as compared to
the lysosomal sialidase activity of said reference results in a
prediction that said cancer will be resistant to said
lysosomotropic chemotherapeutic agent.
4. The method of any one of embodiments 1, 2 or 3, wherein the
control sample is from normal tissue adjacent to said tumor from
said subject. 5. The method of any one of embodiments 1, 2 or 3,
wherein the one or more values representing lysosomal sialidase
activity comprise the level of LAMP-1 protein. 6. The method of any
one of embodiments 1, 2 or 3, wherein the one or more values
representing lysosomal sialidase activity comprise the level of
LAMP-1 and MUC-1 protein. 7. The method of any one of embodiments
1, 2 or 3, wherein the one or more values representing lysosomal
sialidase activity comprise the level of LAMP-1 sialylation. 8. The
method of any one of embodiments 1, 2 or 3, wherein the one or more
values representing lysosomal sialidase activity comprise the level
of MUC-1 sialylation. 9. The method of any one of embodiments 1, 2
or 3, wherein the one or more values representing lysosomal
sialidase activity comprise the level of LAMP-1 and MUC-1
sialylation. 10. The method of any one of embodiments 1, 2 or 3,
wherein the one or more values representing lysosomal sialidase
activity comprise the level of LAMP-1, the level of MUC-1 protein,
the level of LAMP-1 sialylation and the level of MUC-1 sialylation.
11. The method of embodiment 1, wherein the one or more values
representing lysosomal sialidase activity comprise the level of
MUC-1 protein. 12. The method of any one of embodiments 1-11,
wherein the cancer comprises rhabdomyosarcoma, breast cancer, colon
cancer, pancreatic cancer, acute lymphoblastic leukemia or Ewing's
sarcoma. 13. A method of diagnosing dementia associated with
Alzheimer's disease in a subject, the method comprising:
[0179] a) providing a subject lysosomal exocytosis activity profile
of a sample of cerebrospinal fluid from said subject;
[0180] b) providing a reference lysosomal exocytosis activity
profile of a control sample of cerebrospinal fluid, wherein the
subject lysosomal exocytosis activity profile and the corresponding
reference lysosomal exocytosis activity profile comprise one or
more values representing lysosomal exocytosis activity; and,
[0181] c) comparing said subject and said reference lysosomal
exocytosis activity profiles, wherein said subject is diagnosed
with dementia associated with Alzheimer's disease if the subject
has a higher lysosomal exocytosis activity as compared to the
reference lysosomal exocytosis activity.
14. The method of embodiment 13, wherein said subject lysosomal
exocytosis activity profile and said reference lysosomal exocytosis
activity profile comprise
[0182] (a) a lysosomal sialidase activity profile, wherein the
subject lysosomal sialidase activity profile and the corresponding
reference lysosomal sialidase activity profile comprise one or more
values representing lysosomal sialidase activity; or
[0183] (b) a sialylation activity profile, wherein the subject
sialylation activity profile and the corresponding reference
sialylation activity profile comprise one or more values
representing sialylation activity.
15. The method of embodiment 13, wherein the one or more values
representing lysosomal exocytosis activity comprise the level of
LAMP-1 sialylation. 16. The method of embodiment 13, wherein the
one or more values representing lysosomal exocytosis activity
comprise the level of MUC-1 sialylation. 17. The method of
embodiment 13, wherein the one or more values representing
lysosomal exocytosis activity comprise the level of LAMP-1 and
MUC-1 sialylation. 18. The method of embodiment 13, wherein the one
or more values representing lysosomal exocytosis activity comprise
the level of amyloid precursor protein sialylation. 19. The method
of embodiment 13, wherein the one or more values representing
lysosomal exocytosis activity comprises the level of sialylation of
one or more proteins comprising LAMP-1, MUC-1, amyloid precursor
protein, Cathepsin B, Cathepsin D, Fibrinogen, Transthyretin,
beta-2 microglobulin or Immunoglobulin heavy chain. 20. The method
of embodiment 13, wherein the one or more values representing
lysosomal exocytosis activity comprise the level of LAMP-1 protein.
21. The method of embodiment 13, wherein the one or more values
representing lysosomal exocytosis activity comprise the level of
MUC-1 protein. 22. The method of embodiment 13, wherein the one or
more values representing lysosomal exocytosis activity comprise the
level of amyloid precursor protein. 23. The method of embodiment
13, wherein the one or more values representing lysosomal
exocytosis activity comprise the level of LAMP-1 and MUC-1 protein.
24. The method of embodiment 13, wherein the one or more values
representing lysosomal exocytosis activity comprise the level of
LAMP-1 protein, the level of MUC-1 protein, the level of LAMP-1
sialylation and the level of MUC-1 sialylation. 25. The method of
embodiment 13, wherein the one or more values representing
lysosomal exocytosis activity comprises the level of protein of one
or more proteins comprising LAMP-1, MUC-1, amyloid precursor
protein, Cathepsin B, Cathepsin D, Fibrinogen, Hexosaminidase beta,
Mannosidase alpha, Transthyretin, beta-2 microglobulin or
Immunoglobulin heavy chain. 26. A method of treating a subject
having a cancer comprising administering to a subject in need
thereof a therapeutically effective amount of Neuraminidase 1
(NEU1) polypeptide having an amino acid sequence with at least 85%
sequence identity to SEQ ID NO: 2, wherein said polypeptide has
sialidase activity. 27. A method of treating a subject with
dementia associated with Alzheimer's disease comprising
administering to a subject in need thereof a therapeutically
effective amount of Neuraminidase 1 (NEU1) polypeptide having an
amino acid sequence with at least 85% sequence identity to SEQ ID
NO: 2, wherein said polypeptide has sialidase activity. 28. The
method of any one of embodiments 26 or 27, further comprising the
administration of Protective Protein/Cathepsin A (PPCA) polypeptide
having an amino acid sequence with at least 85% sequence identity
to SEQ ID NO: 4, wherein said PPCA polypeptide enhances NEU1
enzymatic activity. 29. The method of embodiment 28, wherein the
NEU1 polypeptide and PPCA polypeptide are administered separately
or simultaneously. 30. The method of embodiment 29, wherein
administration of the NEU1 polypeptide comprises administration of
a viral vector comprising a nucleotide sequence having at least 85%
sequence identity to SEQ ID NO: 1. 31. A method of generating a
lysosomal sialidase activity profile comprising:
[0184] (a) obtaining a sample from a tumor from a subject; and,
[0185] (b) assaying for the level of LAMP-1 protein or the level of
LAMP-1 sialylation.
32. The method of embodiment 31, comprising assaying for one or
more additional lysosomal sialidase activity markers. 33. The
method of embodiment 32, wherein the one or more additional
lysosomal sialidase activity markers comprise a NEU1 substrate. 34.
A method of generating a lysosomal exocytosis activity profile from
cerebrospinal fluid comprising:
[0186] (a) obtaining a sample of cerebrospinal fluid from a
subject; and,
[0187] (b) assaying for lysosomal exocytosis activity.
35. The method of embodiment 34, wherein assaying for lysosomal
exocytosis activity comprises assaying for the level of LAMP-1
protein or the level of LAMP-1 sialylation. 36. The method of
embodiment 34, wherein assaying for lysosomal exocytosis activity
comprises assaying for the level of one or more proteins comprising
LAMP-1, MUC-1, amyloid precursor protein, Cathepsin B, Cathepsin D,
Fibrinogen, Hexosaminidase beta, Mannosidase alpha, Transthyretin,
beta-2 microglobulin or Immunoglobulin heavy chain. 37. The method
of any one of embodiments 31-33, comprising assembling a lysosomal
sialidase activity profile in view of the activity values obtained.
38. The method of any one of embodiments 34-36, comprising
assembling a lysosomal exocytosis activity profile in view of the
activity values obtained. 39. The method of any one of embodiments
1-38, wherein the subject is a human.
[0188] The subject matter of the present disclosure is further
illustrated by the following non-limiting examples.
EXPERIMENTAL
Overview
[0189] We have discovered a novel association between lysosomal
sialidase NEU1-regulated lysosomal exocytosis and two pathological
states: (1) cancer and (2) Alzheimer's disease. The loss of NEU1
results in accumulation of its substrate LAMP-1, which, in turn,
facilitates the exocytosis of lysosomal contents. The physiological
consequences of this depend on the affected tissue. For instance,
release of active proteases into the extracellular environment may
cause remodeling of tissue surrounding a tumor. In the brain, this
release may result in processing of amyloidogenic proteins and
formation of plaques. In addition, xenobiotics, which accumulate in
the lysosome may undergo efflux through this mechanism, altering
drug metabolism. This application is of particular importance as a
possible predictor of chemotherapy resistance in cancer cells and
as prognostic marker of dementia related to Alzheimer's
disease.
[0190] We have identified two read-outs for the loss of NEU1 which
may be used to visualize NEU1 deficiency. These are substrates of
NEU1, mucins and the aforementioned LAMP-1. We suggest that this is
actually a possible proxy marker for NEU1 deficiency/downregulation
and, therefore, increased lysosomal exocytosis. Thus, this marker,
when combined with other NEU1 substrates, could be indicative of
deregulated lysosomal exocytosis of cancer, which predicts both
invasiveness and chemotherapy resistance.
[0191] Measuring NEU1 expression or catalytic activity in a cancer
biopsy may have two somewhat related prognostic applications: 1)
determine the state of the cancer: higher NEU1 activity=less
aggressive/better prognosis; lower NEU1 activity=more
aggressive/poorer prognosis; and 2) predict responsiveness to
chemotherapy: higher NEU1 activity=less lysosomal exocytosis/less
drug efflux extracellularly/more responsive; lower NEU1
activity=increased lysosomal exocytosis/more drug efflux/less
responsive.
[0192] Alternatively, this information can be gleaned via a panel
of NEU1 substrates. Accumulation of substrate glycoproteins in
their oversialylated state as well as of active lysosomal enzymes
indicate a global change in processing rather than a discrete
upregulation in expression, which is currently assumed. This global
change could then predict outcomes and could be used as fingerprint
of invasive-low NEU1 tumors
[0193] This discovery also has therapeutic application. By
restoring the negative regulation of lysosomal exocytosis, cancer
cells can become more treatable with chemotherapeutic drugs and
less aggressive at the same time. This may be possible by
administering NEU1 itself or by administering its stabilizing
partner, protective protein/cathepsin A (PPCA), or by other means,
i.e. LAMP1 downregulation.
[0194] Likewise, the downstream effects of lysosomal exocytosis can
be used as a molecular fingerprint for Alzheimer's pathology. For
instance, high levels of active lysosomal enzymes and other
potential substrates of NEU1 in cerebral spinal fluid allow
diagnosis of dementia related to late onset Alzheimer's disease in
patients, filling a gap in patient care which currently exists.
[0195] This is a new approach for distinguishing more aggressive
from less aggressive cancers that can help guide therapeutic
decision-making. This invention also can lead to new cancer
treatments and neurodegeneration therapies that may complement
existing techniques or provide completely novel approaches.
Example 1: NEU1 Deficiency in Cancer Development, Progression, and
Chemotherapy Resistance
[0196] Deficiency of the lysosomal sialidase NEU1 results in the
lysosomal storage disease sialidosis. Type I sialidosis is a
catastrophic pediatric disease while Type II, or adult onset
sialidosis, is a relatively mild condition caused by gene mutations
which preserve residual activity of NEU1. Our own research into
NEU1 deficiency, performed in the mouse model of sialidosis, has
revealed a novel function of NEU1 as an inhibitor of lysosomal
exocytosis. In the absence of NEU1, its substrate LAMP-1
accumulates, increasing the number of lysosomes docked at the PM
and ready to engage in lysosomal exocytosis. As a result, lysosomal
contents, including active proteases such as cathepsins, are
aberrantly released extracellularly, most likely impacting the
extracellular matrix structure and composition. We hypothesized
that this phenotype could be advantageous for cancer cells, which
extensively modify their extracellular matrix. We have therefore
examined the expression of NEU1 in a variety of cancer cell lines
from four cancer types: breast carcinoma, colon carcinoma, Ewing's
sarcoma, and alveolar rhabdomyosarcoma. For each type of cancer
examined, lower levels of NEU1 activity correlated with increased
expression of over-sialylated LAMP-1. Here we report on a
correlation between a low-NEU1, highly exocytic phenotype and the
invasive capacity of cells. In some cases, the invasiveness of
tested cell lines was known. For instance, the syngeneic system of
SW480 and SW620 colon cancer lines is composed of cells derived
from a primary or metastatic tumor, respectively, from the same
patient. In other cases, such as for Ewings sarcoma and
rhabdomyosarcoma, invasive potential of the tested cell lines was
determined in our hands using an ex vivo model of peritoneal
invasion. These studies establish a new paradigm for understanding
the spread of cancer: invasive potential is enhanced by degradation
of extracellular matrix via lysosomal exocytosis of active
proteases.
[0197] Lysosomal exocytosis is part of constitutive cellular
physiology which has particular importance for cancer cells.
Translocation of lysosomal contents to the extracellular matrix
(ECM) results in ECM remodeling and increased vulnerability of
healthy tissue to invasion. In addition, many commonly used
chemotherapeutics accumulate in the acidic lysosome due to their
weakly basic nature. Lysosomal exocytosis therefore constitutes a
method of xenobiotic efflux, relieving cancer cells of toxic
burden. We have characterized the lysosomal enzyme Neuraminidase 1
as a negative regulator of lysosome exocytosis and here demonstrate
the downregulation of NEU1 in several cancer types as well as the
advantageous physiological consequences for cancer cells associated
with the loss of NEU1.
[0198] In addition to its canonical role as a sialidase, NEU1 has a
related and profound effect on the constitutive process of
lysosomal exocytosis. This functionality is mediated by the NEU1
substrate Lysosomal Associated Membrane Protein 1 (LAMP1) which is
left hyper-glycosylated in the absence of NEU1. This
hyper-glycosylated state of LAMP1 appears to facilitate lysosomal
docking at the plasma membrane (PM) and subsequent exocytosis
causing a range of significant physiological changes to both the
affected cell and its environment. Here we present data to
demonstrate that loss of NEU1 and exacerbation of LEX result in two
major physiological shifts in cancer cells: enhanced invasive
potential and increased resistance to chemotherapy.
[0199] To first establish a general role for NEU1 in human cancer,
we probed multiple tumor arrays for both NEU1 and two of its
natural substrates, LAMP-1 and mucins. We found that downregulation
of NEU1 in tumors compared to healthy tissue was occurred across
cancer types and that using either LAMP-1 or mucin staining
functioned as proxy markers for this change (data not shown). The
finding is significant in part because the mucin MUC-1 has long
been used as a trusted cancer marker and this research suggests
that it may be downstream of another change with many other
predictable, functional consequences.
[0200] In order to test functional consequences of changes to NEU1
levels in cancer, we evaluated several cell line systems (data not
shown). In each, we assessed the NEU1 activity in lysates and the
corresponding abundance of over-decorated LAMP-1. For each set
evaluated, the relative invasive potential was determined either
from literature or from matrigel invasion assays. The more invasive
cells consistently demonstrated reduced NEU1 activity and increased
LAMP1 levels compared to less invasive cells of the same cancer
type (data not shown). We chose the RH41 and RH30 cell lines for
further study because these alveolar rhabdomyosarcoma lines arise
from skeletal muscle, a long-standing interest of our lab. The
relatively high levels of NEU1 in RH41 cells compared to RH30 cells
were further confirmed by archived microarray data, real time PCR,
Western blot, and immunofluorescence. In addition to reduced LAMP-1
in RH41 cells, the high level of NEU1 correlates with a reduction
of lysosomal exocytosis as measured by media activity assay and
TIRF imaging (data not shown). To test the importance of NEU1
expression on these physiological markers, we generated stable
clones of each line, with upregulation of NEU1 in RH30 cells and
downregulation in RH41 cells, along with empty vector controls for
each. Once the expression level of each line was recapitulated in
the other, we tested for exocytosis changes via LAMP-1 and TIRF
imaging (as well as activity assays). As predicted, we robustly
demonstrated that NEU1 is a negative regulator of lysosomal
exocytosis in the cancer cell context, marked by an accumulation of
LAMP-1 (data not shown).
[0201] There are several immediate implications for identifying a
regulator of lysosomal exocytosis in cancer cells. This process has
been shown to contribute to both invasive potential and
chemotherapy resistance, although a specific target for altering
this process has not been proposed until NEU1. Lysosomal exocytosis
is the most likely method for the efflux of active lysosomal
enzymes into the extracellular matrix, and the presence of enzymes
such as cathepsin B in the ECM correlates with metastasis across
cancer types. Active proteases participate in ECM remodeling and
inhibit the microenvironment's ability to contain tumor spread.
Therefore, we tested the stable lines for their ability to invade a
matrigel substrate, primarily composed of laminin and collagen IV,
both susceptible to digestion by lysosomal enzymes such as
cathepsins.
[0202] The stable clone lines for RH41 and RH30 were each plated
onto matrigel plugs for two days. The plugs were then fixed,
embedded, sectioned, and stained with H&E to visualize the
ingress of cells into the substrate (data not shown). Regardless of
parental line, those clones with low NEU1 successfully invaded the
matrigel after two days whereas those cells with high NEU1 were
excluded from the gel. This experiment established that the NEU1
status of cancer cells can determine the potential of cells to
invade ECM.
[0203] However, we further wished to examine the longer term impact
of lysosomal exocytosis on non-malignant tissue. We hypothesized
that at a tumor border, excessive lysosomal exocytosis from the
cancer would condition surrounding tissue for invasion, making
healthy tissue more vulnerable to a metastatic event. We therefore
tested the ability of the parental RH30 and RH41 cells to invade in
an ex-vivo setting using peritoneum harvested from wild-type or
Neu1-knockout mice. Tissues collected from Neu1 knockout mice have
undergone constitutive excessive lysosomal exocytosis and can
therefore represent the healthy tissue at the border of cancer
undergoing excessive lysosomal exocytosis due to NEU1
downregulation. In fact, a the more invasive RH30 cells were able
to cross into the peritoneum of wild type animals while the RH41
cells were largely excluded, recapitulating the results from the
matrigel assay. Importantly, the less invasive RH41 cells were able
to invade the knock out peritoneum as successfully as RH30 cells
invaded the wild type. This result suggests that the damage done by
long term lysosomal exocytosis sensitizes tissue to invasion,
independent of the aggressiveness of the cancer. Furthermore, RH30
cells placed on knockout peritoneum resulted in the most aggressive
rates on invasion. Therefore, we conclude that lysosomal exocytosis
does significant damage to ECM, regardless of the source of the
exocytosis. In the case of cancer cells, those with higher rates of
exocytosis more successfully invade a standard substrate.
[0204] The second prediction for functional changes downstream of
NEU1 also proved to be relevant to rhabdomyosarcoma. The RH30 line
has a baseline resistance to doxorubicin which can be weakened by
the addition of NEU1. Conversely, RH41 cells are highly susceptible
to doxorubicin and can acquire resistance upon upregulation of
NEU1. This result points specifically to the efflux of the drug
through the lysosome for a number of reasons. First, doxorubicin,
like many chemotherapeutics, is a weak base which accumulates in
the acid lysosomal compartment. Secondly, neither of these cells
lines expresses p-glycoprotein, the traditionally studied method of
drug efflux. Instead, we were able to image the trafficking of
doxorubicin over a 12 hour period and observed that (1) lysosomes
condense around the nucleus in RH41 cells prior to collapse of the
cell (2) the lysosomal enzyme cathepsin B translocates to the
nucleus prior to apoptosis, (3) the doxorubicin load in these cells
is virtually entirely held within the nucleus and (4) resistant
cells maintain a mobile fraction of doxorubicin in lysosomes (data
not shown).
[0205] Alteration of NEU1 status reversed these trends; although it
did not completely sensitize the RH30 cells to apoptosis, PARD
cleavage could be observed at previously harmless doses of
doxorubicin (data not shown). We decided to chemically inhibit
lysosomal exocytosis to see if complete inhibition could fully
eliminate doxorubicin resistance. To do this we used verapamil, a
calcium channel blocker which has previously been considered an
inhibitor of p-glycoprotein. However, recent work has shown that
this drug can sensitize cells to drug regardless of p-glycoprotein
status, suggesting that another mechanism may be the real target of
the drug. Because lysosomal exocytosis is dependent on Ca++ influx,
chelation of calcium would chemically halt the process and provide
a testable change. Rh30 cells co-incubated with verapamil and
doxorubicin are fully sensitized to the drug, recapitulating the
phenotype of RH41 cells (data not shown). Doxorubicin can be
visualized almost exclusively in the nucleus of the
verapamil-sensitized RH30 cells, and the elimination of the mobile
fraction of the drug is demonstrated (data not shown).
[0206] In conclusion, we have presented a model whereby lysosomal
exocytosis, as regulated by NEU1, is a critical determinant in
cancer cell phenotype (see FIG. 1). The loss of NEU1 results in
accumulation of its substrates and alterations to baseline
physiology. Two consequences of translocation of lysosomal contents
to the extracellular space are degradation of the ECM and efflux of
lysosomally-accumulating chemotherapy drugs. Thus, downregulation
of NEU1 may be an important predictor for resistance to a class of
chemotherapy drugs, including the commonly used doxorubicin,
cisplatin, and docetaxel, all of which known to localize at least
in part to the lysosome. In addition, NEU1 substrates may represent
a rationally designed panel of cancer markers, adding sensitivity
to the growing field.
[0207] In addition, when taken together, these data predict that
genetic deficiency of NEU would render people more vulnerable to
acquiring cancers and that those cancers would tend toward
aggressiveness.
Example 2: The Role of NEU1 in Chemotherapy Resistance
Background
[0208] Rhabdomyosarcoma (RMS) is the most common soft tissue
malignancy in children. For children diagnosed with metastatic
disease, 3-year survival rates are only about 30%. Systemic
chemotherapy is currently the predominant treatment for these
patients--and several combination protocols are being used on site
here at St. Jude--but drug resistance often blunts response. We
have recently developed a novel hypothesis for how drug resistance
arises and here propose work to clarify the mechanism. In brief,
chemotherapy drugs often accumulate in lysosomes, which are
multi-functional acidic organelles. Lysosomes can then be
transported to the cell periphery, fuse their membrane with the
plasma membrane and release their contents into the extracellular
space. This process, called lysosomal exocytosis (LEX), could
effectively limit intracellular exposure to drug. Recent work in
our lab has identified the lysosomal sialidase NEU1 as a negative
regulator of LEX. Without NEU1, its substrate LAMP1 (for Lysosome
Associated Membrane Protein) accumulates in lysosomes and aids in
their translocation to the plasma membrane. Our preliminary work on
RMS cell lines has shown that stable knockdown of NEU1 results in
high levels of LAMP1, more LEX, and more drug resistance.
Conversely, upregulation of NEU1 results in less LAMP1, less LEX,
and reduced drug resistance. Thus, NEU1 downregulation may be
advantageous for cancer cells and we have observed such a loss in
pediatric RMS tumor samples. Simply administering NEU1 to patients
may not be feasible. The NEU1 protein requires complexing with its
chaperone, Protective Protein Cathepsin A (PPCA), which may prove
to be a rate-limiting step. However, enhancing PPCA is known to
significantly boost NEU1 residual activity and this may prove to be
a more tractable entry point into clinical control of LEX.
[0209] Hypotheses and Specific Aims:
[0210] Downregulation of lysosomal exocytosis will enhance RMS
response to chemotherapy. We intend to leverage our understanding
of LEX to identify opportunities for treatment enhancement in the
following two aims. (1) Determine if PPCA upregulation enhances
outcome via increasing NEU1 activity. Our lab has developed an
AAV-based delivery method for PPCA, which is entering clinical
trials as an enzyme replacement approach for children with
galactosialidosis. This work suggests that the vector may be
relevant to cancer treatment, as well. (2) Determine if targeting
LAMP1 results in improved outcome. A small portion of LAMP1 is
available to bind to trafficking machinery and facilitate
peripheral movement of lysosomes. We will use intracellular
delivery of antibody against this sequence to competitively bind
and limit movement of the organelles. Success with this methodology
will validate the LAMP1 site as a possible target for small
molecule development.
[0211] Design:
[0212] These experiments, as proof-of-principle in vitro work, will
occur in well-characterized alveolar RMS cell lines. For Specific
Aim 1, dose curves of AAV-PPCA and a panel of chemotherapy drugs
(doxorubicin, cisplatin, vincristine) will be tested for induction
of apoptosis. The same panel will be used in Specific Aim 2, along
with two concentrations of LAMP1 antibody according to established
protocols for intracellular delivery. For each intervention, LEX
will be measured by assaying levels of lysosomal enzymes in culture
media.
[0213] Potential Impact:
[0214] This work will establish LEX as a determinant of
chemotherapy outcome. The suite of proteins we examine, PPCA, NEU1,
and LAMP1, may then all be used as markers to characterize a given
patient's tumor for LEX capacity. Secondly, the research is
expected to indicate possible targeted methods for inhibiting LEX.
Not only could this have an impact on cancer treatment generally,
it directly addresses the main hurdle in treating pediatric
alveolar RMS, particularly once metastasized.
[0215] Background:
[0216] Chemotherapy resistance is the key problem facing children
with metastatic rhabdomyosarcoma. Cancer cells can evade
chemotherapy by "pumping" the drug out. For instance, drugs can
accumulate in organelles called lysosomes, which can then move to
the cell surface, fuse with the cell membrane and release their
contents to the outside in a process known as lysosomal exocytosis.
Here we examine the main players in this process and study how to
inhibit it so that chemotherapeutic drugs remain inside targeted
cells and provoke their demise. First, PPCA is a lysosomal protein
that guides the enzyme NEU1 into the lysosome and enhances its
activity. NEU1 then helps to degrade LAMP1, one of its target
substrates. This latter step is important to avoid LAMP1
accumulation in lysosomes, which in turn causes excessive
exocytosis. Here we propose testing methods to affect the upstream
(PPCA) and downstream (LAMP1) players in order to inhibit
exocytosis and thereby allow cancer cells to retain the tested
drugs.
[0217] Hypotheses and Specific Aims:
[0218] Lysosomal exocytosis of drugs decreases effectiveness of
chemotherapy but this can be reversed by upregulating PPCA or
downregulating LAMP1. Specific Aim1 will test upregulation of PPCA
using a virus delivery method. Specific Aim 2 will test inhibition
of LAMP1 through use of an antibody against its binding site so
that it cannot facilitate lysosomal movement.
[0219] Potential Impact:
[0220] This research is expected to establish lysosomal exocytosis
as a major determinant of chemotherapy responsiveness.
Understanding this mechanism will allow clinicians to predict tumor
exocytic capacity and tailor drug combinations/doses accordingly.
In addition, we hope to establish specific, potentially druggable
targets to inhibit lysosomal exocytosis and enhance patient
response.
Example 3: Early Stage Alzheimer's Disease-Phenotype Linked to
Deficiency of the Lysosomal Sialidase Neu1
[0221] Lysosomal sialidase NEU1 catalyses the hydrolysis of
sialo-glycoconjugates by removing their terminal sialic acid
residues. In humans, primary or secondary deficiency of this enzyme
leads to two clinically similar neurodegenerative lysosomal storage
disorders: sialidosis and galactosialidosis. Mice deficient in Neu1
recapitulate the early-onset severe form of sialidosis. We have
discovered that loss of Neu1 activity exacerbates the process of
lysosomal exocytosis in various cell types by influencing the
sialic acid content of Lamp-1. This increases the ability of a pool
of lysosomes to dock at the PM and engage in lysosomal exocytosis.
In this study we have investigated whether excessive lysosomal
exocytosis underlies some of the neurological aspects seen in the
brain of Neu1.sup.-/- mice. Histopathological examination of the
brain of these mice revealed a progressive and time dependent
deposition of inclusions/deposits containing APP/A.beta. peptide,
particularly in the CA3 region of the hippocampus and the adjacent
fimbria. The affected regions coincide with sites of high Neu1
expression in wild-type brain. This abnormality was paralleled by
abnormal expression of oversialylated Lamp-1 and activated
proteases, both features linked to excessive lysosomal exocytosis.
These findings represent an example of a spontaneously occurring
AD-like phenotype in a mouse model of a neurodegenerative disease
and could contribute to the understanding of some of the
pathological mechanisms of Alzheimer's disease.
[0222] Lysosomal storage diseases (LSDs) comprise a group of more
than 50 genetic disorders of lysosomal function, mostly caused by
defects in one of the glycan-cleaving lysosomal hydrolases. Enzyme
deficiency usually leads to impaired substrates' degradation and to
their accumulation in cells of multiple systemic organs and the
nervous system. Here we present evidence that mice lacking the
lysosomal sialidase Neu1 besides recapitulating the
neurodegenerative LSD sialidosis, develop pathological and
molecular changes in the brain, which are reminiscent of
early-stage Alzheimer's disease (AD). Consequent to Neu1
loss-of-function the combined occurrence of excessive lysosomal
exocytosis of neural cells and accumulation of oversialylated Neu1
substrates, including the amyloid precursor protein (APP),
underlies this pathogenic cascade. These findings uncover
previously unknown molecular mechanisms that could contribute
and/or predispose to AD.
[0223] The fundamental role of the mammalian lysosomal sialidase
NEU1 is to initiate the hydrolysis of sialoglyconjugates by
removing their terminal sialic acids. This activity is crucial to
cell homeostasis because genetic defects that alter NEU1 activity
disrupt lysosomal metabolism and result in the LSD sialidosis. We
have recently identified Neu1 as the only negative regulator of the
physiological process known as lysosomal exocytosis (LEX). The
latter is a Ca.sup.2+-dependent, regulated mechanism that involves
recruitment/docking of lysosomes to the plasma membrane (PM), a
step which is facilitated by the lysosomal associated protein-1
(LAMP-1) and is followed by the fusion of the lysosomal membrane
with the PM, and the release of lysosomal luminal content into the
extracellular space. We have shown that loss of Neu1 in mouse BM
macrophages increases the pool of lysosomes, decorated by
oversialylated LAMP-1 on their LM, which are poised to become
exocytic. The ensuing exacerbation of this process leads to
disease.
[0224] Here we tested if Neu1-dependent increase in LEX is the
underlying molecular mechanism responsible for neurodegeneration in
the mouse model of sialidosis (Neu1.sup.-/-). We found that Neu1
was present throughout the brain parenchyma but was predominantly
expressed in two regions of the wild-type brain: the hippocampus
and the choroid plexus (CP) (data not shown). The CP is the
exocytic structure of the brain, producing and secreting the
cerebrospinal fluid (CSF), and functions as a barrier interface
between the blood and the CSF. In the KO mice the CP underwent
overt morphologic changes associated with extensive vacuolization
and expansion of the lysosomal system (data not shown). This
phenotype was accompanied by increased expression of a long-lived
oversialylated Lamp-1 (data not shown), a target substrate of Neu1.
We have shown this feature in other cells and tissues of the
Neu1.sup.-/- mice and demonstrated it can be used as read-out of
excessive LEX. This was confirmed by measuring the activity of the
lysosomal enzymes .alpha.-mannosidase and .beta.-hexosaminidase
that were both increased in the KO CSF (data not shown).
[0225] We reasoned that excessive exocytosis of lysosomal content
into the CSF would dramatically alter its composition. We
investigated this possibility by comparing the total protein
content of the Neu1.sup.-/- and Neu1.sup.+/+ CSF samples using high
throughput proteomic analysis. We found many lysosomal enzymes,
including cathepsin D and cathepsin B, as well as other proteins to
be present in abnormal amounts in the KO CSF (FIG. 2). Increased
levels of several of these proteins were also observed in the CP
cells of KO animals (data not shown). We postulated that many of
the proteins increased in the CSF of KO mice represent undigested
substrates of NEU1, which are secreted extracellularly via
exacerbated LEX. Notably, multiple proteins differentially
regulated in the Neu1.sup.-/- CSF have also been identified as
possible dementia-predicting biomarkers associated with Alzheimer's
disease (FIG. 2). Thus, profound alterations of cellular physiology
in one normally Neu1-rich brain region may cause subsequent
downstream changes that are highly suggestive of an
Alzheimer's-like status when Neu1 is lost. For this reason, we
hypothesized that the observed changes in composition of the
Neu1.sup.-/- CSF would be paralleled by altered characteristics of
neural cells in the brain parenchyma. We were intrigued by the
observation that the other area of the WT brain expressing Neu1 at
high levels is the hippocampus (data not shown), one of the most
intensely studied structures of the brain in the AD field. In
agreement with our defining paradigm of Neu1 reduction resulting in
Lamp-1 accumulation and subsequent excessive LEX, we first looked
at Lamp1 and observed a marked increase of this protein throughout
the KO brain (data not shown). This was confirmed by immunoblot
analysis of brain hippocampal protein extracts that identified
increased amount of an oversialylated Lamp-1 (data not shown).
Based on these results, we hypothesized that cells in the brain
parenchyma of Neu1.sup.-/- mice could also exert excessive LEX.
Remarkably, Lamp1 was highly expressed in the microglia population
(F4/80 staining, data not shown) suggesting that this cell
population might be the most exocytic in the brain parenchyma, as
previously demonstrated for BM macrophages. We investigated this by
culturing WT and KO microglia. We tested their exocytic activity by
measuring the levels of active lysosomal hydrolases present in the
medium, and found a marked increase of active lysosomal
.beta.-hexosaminidase in the medium from KO microglia (data not
shown).
[0226] We next examined the histopathological characteristics of
the KO hippocampus and noticed numerous, abnormal eosinophilic
bodies (data not shown). They were variable in size and mostly
contained granular proteinaceous material (data not shown). At the
EM level, these bodies were identified as swollen dystrophic
neurites containing numerous vacuoles of abnormal morphology
resembling autophagic vacuoles (data not shown). These features
were highly reminiscent of the distended dystrophic neurites
associated with AD. Therefore, we began a full characterization of
these structures, starting with antibodies reactive to the
N-terminal portion of APP and found a time-dependent, progressive
accumulation of this protein in the pyramidal neurons of the third
subregion of the Cornus ammonis (CA3) of the hippocampus of
Neu1.sup.-/- brain (data not shown). To test if this phenotype was
directly linked to the Neu1 deficiency, we analyzed the sialylation
status of APP in the Neu1.sup.-/- brain. Evidence for
oversialylation of APP came from immunoprecipitation studies; equal
amount of hippocampal protein extracts from wild-type and
Neu1.sup.-/- brain samples were immunoprecipitated with an APP
C-terminal antibody and were examined with sambucus nigra lectin
(SNA) that binds preferentially to sialic acid attached to terminal
galactose with (.alpha.-2,6) linkages (data not shown).
[0227] It is well established that a slight overexpression of APP
is a risk factor for the development of AD and duplication of the
APP locus in familial AD and Down syndrome patients is the basis of
early onset AD. We therefore tested a number of canonical
histological markers commonly applied for the diagnosis of AD in
the brain of Neu1.sup.-/- mice. Swollen dystrophic neurites were
readily detected with thioflavin S fluorescence suggesting they
were structurally close to amyloid deposits (data not shown).
Modified Bielschowsky silver stain also highlighted scattered
silver-positive neuritic structures (data not shown), not found in
aged matched WT mice. The APP accumulating neurites were also
immunoreactive with antibodies recognizing APP/A.beta. (data not
shown), and were positive when stained with an antibody against the
.beta.-amyloid isoform ending at the 42nd amino acid (A1342) (data
not shown). Most importantly, almost all the APP+ dystrophic
neurites were immunostained with ubiquitin, neurofilaments and tau
antibodies indicating the presence of protein aggregates and
extensive cytoskeletal abnormalities in these structures (data not
shown). We believe that APP accumulation in these dystrophic
neurites contributes to the formation of toxic amyloid peptides
(A.beta.) because quantitative determination of A.beta.40 and
A.beta.42(43) showed elevated A.beta. peptides in the Neu1.sup.-/-
brain (data not shown). Based on these data, we conclude that Neu1
deficiency is directly linked to early pathogenic events observed
in AD.
[0228] The APP+ neurites may represent early events in the
pathogenesis of the neuropil threads characterized by true amyloid
and plaque deposition. APP overexpression in neurons or neurites
may be toxic and cause degeneration with release of this protein.
Exocytic microglia could then contribute to the pathogenic process
by releasing into the extracellular space activated lysosomal
enzymes that progressively process the APP producing toxic A.beta.
peptides.
[0229] Here we have identified a novel mechanism orchestrated by
deficiency of lysosomal Neu1 that promotes deposition of
oversialylated APP which in turn may constitute a risk factor for
the development of late-onset non-familiar AD.
The discovery of novel putative Neu1 substrates, including APP, and
the occurrence of deregulated LEX in the brain/CSF could provide a
novel set of biomarkers for the diagnosis of AD and set the stage
for innovative therapeutic approaches to prevent/modulate
APP/A.beta. formation.
Example 4: Metabolic Control of Chemotherapy Resistance and
Metastasis
Summary
[0230] The dual dangers of cancer progression are chemotherapy
resistance and metastatic growth, both of which depend on the
metabolic status of tumor cells. Here, we demonstrate that the
lysosomal sialidase NEU1 plays a defining role in the development
of both phenotypes by negatively regulating the physiological
process of lysosomal exocytosis. Cancer cells use this mechanism to
sequester and purge lysosomotropic chemotherapeutics, thereby
developing drug resistance. Moreover, exocytosed active lysosomal
enzymes from tumors degrade the extracellular matrix of surrounding
tissue, compromising its ability to contain tumor spread.
Tumor-prone mice haploinsufficient for Neu1 develop highly
aggressive rare forms of cancer, confirming a role for NEU1 in
controlling malignancy. In addition, downregulation of NEU1 is
common in multiple human cancers. We propose that NEU1 functions as
a bona fide tumor suppressor by restraining lysosomal exocytosis in
cancer cells, precluding the development of a drug resistant and
invasive phenotype.
Highlights
[0231] Lysosomal sialidase NEU1 negatively regulates lysosomal
exocytosis in cancer cells [0232] Increased lysosomal exocytosis
confers chemotherapy resistance and invasiveness [0233] Neu1
haploinsufficiency potentiates tumor growth and spread in
Arf.sup.-/- mice [0234] Downregulation of NEU1 is observed in
multiple human cancers
INTRODUCTION
[0235] The lysosomal glycosidase N-acetyl-.alpha.-neuraminidase 1
(NEU1) is the most abundant and widely expressed mammalian
sialidase. Its canonical function is to remove .alpha.2,6- or
.alpha.2,3-linked terminal sialic acids from the saccharide chains
of glycoproteins, glycolipids (gangliosides), oligosaccharides, and
polysaccharides (Monti et al., 2010). Genetic deficiency of NEU1
results in impaired catabolism of sialic acids on its target
substrates, which in turn, affects countless cellular functions and
leads to the loss of cell and tissue homeostasis. The pathogenic
effects of NEU1 loss of function are obvious in the lysosomal
storage disease sialidosis, a severe neurosomatic condition in
children and adolescents that affects most of the systemic organs
and the nervous system (d'Azzo, 2009; Thomas, 2001).
[0236] In cancer, altered sialylation of glycoconjugates at surface
membranes is considered a central determinant of the neoplastic
process, though it is often unclear how changes in glycan
composition result in aberrant biological outcome (Varki et al.,
2009; Wang, 2005). This dynamic posttranslational modification
involving a charged sugar moiety can greatly modify the biochemical
and functional properties of proteins and lipids, thereby affecting
cell-cell and cell-extracellular matrix (ECM) interactions, cell
migration and adhesion patterns, intracellular signaling and
metastatic potential (Varki et al., 2009; Wang, 2005; Hedlund et
al., 2008; Uemura et al., 2009). Excessive sialic acid content can
result from two opposing processes, upregulation of the synthetic
enzymes sialyltransferases that control the regulated transfer of
sialic acids to nascent oligosaccharide moieties; and loss of
activity of the sialidases that affect the same sugar nucleotide
linkages. For example, a compelling recent study has implicated the
overexpression of the sialyltransferase ST6GalNAc-V in the enhanced
metastatic potential of breast cancer cells, most likely through
abnormal sialylation of as yet unidentified proteins (Bos et al.,
2009). One could argue that loss or downregulation of NEU1 would
necessarily result in abnormal processing of sialylated substrates,
making the catabolic arm of this post-translational modification as
relevant for cancer progression and growth. In fact, changes in the
expression levels of sialidases have been associated with cell
migration and metastasis (Kato et al., 2001; Miyagi et al., 1994;
Sawada et al., 2002; Uemura et al., 2009).
[0237] A previously unknown function for the sialidase NEU1 with
great relevance to cancer has recently been discovered: that of
negative regulator of lysosomal exocytosis (LEX) (Yogalingam et
al., 2008). This ubiquitous, calcium-regulated physiological
process entails the recruitment to the cytoskeletal network of a
selective pool of lysosomes that dock at the plasma membrane (PM);
their limiting membrane then fuses with the PM in response to
calcium influx and their luminal content is released
extracellularly (Bossi and Griffiths, 2005; Andrews, 2000;
Rodriguez et al., 1997). NEU1's function in this process is
mediated via the lysosomal associated membrane protein 1 (LAMP1), a
natural substrate of NEU1 (Yogalingam et al., 2008), which was
previously implicated in the peripheral movement of lysosomes
(Reddy et al., 2001). LAMP1 is a heavily glycosylated and
sialylated structural component of the lysosomal membrane whose
regulated turnover is considerably delayed when the protein is
oversialylated due to loss of NEU1 activity (Yogalingam et al.,
2008). Long-lived oversialylated LAMP1 changes the lysosomal
membrane topology and trafficking, thereby increasing the number of
exocytic lysosomes docked at the PM ready to fuse and secrete their
contents (Yogalingam et al., 2008).
[0238] Recent reports have made explicit calls for more attention
to the area of regulated exocytosis and cancer (Chan and Weber,
2002; Hendrix et al., 2010; Palmer et al., 2002). The involvement
of this process as a mediator of malignant growth and invasiveness
has been postulated in view of the deregulated activities of
vesicular trafficking effectors observed in some cancers (Hendrix
et al., 2010; Palmer et al., 2002). Exocytosis can impact at least
two critical areas of malignant progression: abnormal remodeling of
the ECM and promoting the efflux of chemotherapeutic agents, many
of which are lysosomotropic. Anthracyclines, cisplatin, and
sunitinib are examples of such drugs that have been directly
visualized in lysosomes, whose trafficking could greatly influence
the intracellular exposure to the drug (Gotink et al., 2011;
Hurwitz et al., 1997; Safaei et al., 2005). The degradation and
remodeling of the ECM has been attributed to the presence of
lysosomal proteases in the extracellular environment and has been
strongly correlated with tumor invasiveness and metastasis (Khan et
al., 1998a; Khan et al., 1998b; Matarrese et al.).
[0239] In the present study we demonstrate that downregulation of
NEU1 activity levels in invasive cancer results in the accumulation
of an oversialylated LAMP1 and in enhanced lysosomal exocytosis.
The combination of these processes initiates a cascade of events
that favor tumor progression, invasiveness and resistance to
chemotherapeutic drugs. These results establish that NEU1 functions
as a tumor suppressor by negatively regulating lysosomal
exocytosis.
Results
NEU1 Expression is Inversely Related to Lysosomal Exocytosis in
Rhabdomyosarcoma Cell Lines
[0240] Rhabdomyosarcoma (RMS), which arises in skeletal muscle, is
the most common soft-tissue malignancy in children and adolescents
(Ognjanovic et al., 2009). This cancer is classified into two
subtypes, embryonal RMS, which typically has a favorable prognosis,
and alveolar RMS, which is associated with poor prognosis
(Ognjanovic et al., 2009). RMS is treated primarily with surgical
resection and chemotherapy; common complications are metastases and
chemotherapy resistance, both of which could result from excessive
LEX.
[0241] To investigate the role of NEU1 in regulating LEX in cancer
cells, we chose two human RMS cell lines that express different
amounts of NEU1. RH41 and RH30 cells were both derived from
alveolar RMS tumors (Houghton et al., 2007). Affymetrix mRNA
microarray analysis of NEU1 expression showed that RH41 cells
express a relatively high level of NEU1 mRNA compared with that
expressed by RH30 cells. We confirmed this finding using
semiquantitative and real-time PCR (data not shown). The mRNA
results correlated well with the levels of NEU1 activity (data not
shown). Immunofluorescent labeling also revealed a typical
lysosomal distribution of NEU1 in both cell lines, but expression
was markedly higher in the RH41 cells (data not shown).
[0242] On the basis of the different patterns of NEU1 expression in
the RMS cell lines, we characterized their LEX profiles. Western
blot analysis of LAMP1 confirmed that the protein was more abundant
in the low-NEU1 RH30 cells and had a higher molecular weight, which
was indicative of increased sialic acid content (data not shown).
In addition, using confocal immunofluorescence, we observed a
LAMP1.sup.+ signal on the cell surface of nonpermeabilized RH30
cells but not on RH41 cells. This suggests an enhanced tendency of
a LAMP1-marked pool of lysosomes in the low-NEU1 cells to dock at
and fuse with the PM, leading to accumulation of LAMP1 at that site
(data not shown). We further monitored lysosomal trafficking in
real time by capturing confocal images of lysotracker red-tagged
puncta. In RH30 cells, lysosomes were preferentially captured at
the cell periphery and continuously dispatched from the cell center
outward; in contrast, RH41 cells had virtually no lysosomes
residing outside the perinuclear space (data not shown). Total
internal reflection (TIRF) microscopic analysis confirmed that the
peripheral lysosomes in RH30 cells were in close proximity to the
PM. Live RH30 cells stained with lysotracker green showed evidence
of signal localization within the range of TIRF-sensing, but RH41
cells did not (data not shown). Finally, we measured the
extracellular activity of the lysosomal enzyme
.beta.-hexosaminidase (.beta.-Hex) in culture medium from each cell
line as a measure of released lysosomal content. RH30-conditioned
medium contained considerably more .beta.-Hex than did
RH41-conditioned medium, in inverse relation with their respective
levels of NEU1 (data not shown).
NEU1 Expression Levels Influence the Extent of Lysosomal
Exocytosis
[0243] To pinpoint the primary role of NEU1 in the exocytic
phenotype, we engineered stable NEU1-modified clones of the 2 RMS
cell lines by using retroviral vectors. Modified RH30 cells
overexpressing NEU1 (RH30.sup.NEU1) and RH41 cells with silenced
NEU1 (RH41.sup.shNEU1) were first characterized for their NEU1
protein levels and activity to confirm the successful reversion of
their NEU1 expression patterns with respect to the corresponding
empty-vector controls (data not shown). LEX was then measured in
both modified cell lines and corresponding controls using 3
parameters: LAMP1 levels, TIRF analysis, and enzyme activities in
conditioned media. LAMP1 levels were inversely proportional to the
levels of NEU1 activity in the modified lines (data not shown). In
addition, LAMP1.sup.+ immunofluorescence was notably localized to
the cell periphery of RH41.sup.shNEU1 cells and control
RH30.sup.empty cells compared to their high-NEU1 counterparts
(RH30.sup.NEU1; RH41.sup.empty) (data not shown). TIRF imaging
confirmed the peripheral trafficking of lysosomes and their
tendency to cluster at the termini of cell extensions in the
RH41.sup.shNEU1 cells (data not shown). In contrast, this feature
was lost in RH30.sup.NEU1 cells (data not shown).
[0244] Finally, the activity of lysosomal .beta.-Hex was assayed in
the culture medium from each modified cell line and control. The
media from the low-NEU1 cells contained significantly more
.beta.-Hex activity (data not shown). Together, these results
demonstrate that the NEU1 status of a cell is sufficient to
determine its LEX phenotype.
Increased Lysosomal Exocytosis Correlates with Doxorubicin
Resistance
[0245] We next looked at the functional ramifications of NEU1
control over LEX in relation to the response of the parental cell
lines to chemotherapeutic drugs. RH41 and RH30 cells have been
shown to respond to a variety of antineoplastic therapy in markedly
different way (Houghton et al., 2007; Petak et al., 2000). We found
that upon exposure to doxorubicin (DOXO), RH41 cells readily
apoptosed; RH30 cells were resistant to treatment (data not shown).
To link these phenotypes to the level of NEU1 activity in these
cells, we first confirmed that DOXO was concentrated in their
lysosomes. The native red fluorescence of the drug colocalized with
lysotracker green, revealing that DOXO-loaded lysosomes were
present in both parental RMS cell lines, albeit differently
distributed throughout the cytoplasm (data not shown).
Specifically, after 2 hours of treatment, DOXO-loaded lysosomes in
RH41 cells clustered in the perinuclear region, but those in RH30
cells did not (data not shown). Overnight live imaging of lysosome
trafficking upon DOXO exposure confirmed these trends (data not
shown). The intracellular visualization of DOXO at 4 hours
confirmed that the drug was primarily concentrated in the nuclei of
RH41 cells, while in the RH30 cells a fraction of the drug remained
lysosomal (data not shown).
[0246] The increase in LEX observed in the low-NEU1 RH30 cells
could promote the efflux of DOXO, hence making the cells
insensitive to treatment. This prediction was further supported by
the fact that these cells do not express the multidrug-resistance
protein 1 (p-glycoprotein 1) that functions in a well-known
cellular mechanism for evading drug toxicity (Cocker et al., 2000).
By capturing and quantifying the effluxed red fluorescence from the
RH30 culture medium, we showed that DOXO was indeed released from
these cells (data not shown).
[0247] Finally, we generated DOXO dose response curves in both
parental cell lines and the modified lines to examine the
differences in their apoptotic responses. Immunoblots of the
cleaved poly (ADP-ribose) polymerase (PARP), a canonical apoptotic
marker, were used for this purpose (data not shown). The
RH41.sup.shNEU1 cells were more resistant to apoptosis than were
the corresponding unmodified cells (data not shown). In contrast,
the RH30.sup.NEU1 cells were more sensitive to DOXO than were their
unmodified controls (data not shown). In view of these results, we
tested whether inhibiting LEX with the calcium channel blocker
verapamil would sensitize the parental RH30 cells.
[0248] Verapamil is a p-glycoprotein inhibitor, but it also
inhibits drug efflux in the absence of p-glycoprotein, which
suggests an alternative efflux mechanism (Chiu et al., 2010).
Because LEX depends on calcium influx, we hypothesized that this
commonly used calcium channel blocker would inhibit this process.
Upon co-treatment of the RH30 cells with verapamil and DOXO, the
lysosomes accumulated the drug and clustered in the perinuclear
region (data not shown). The cells then underwent apoptosis, as
measured by PARP cleavage and morphological analysis (data not
shown).
NEU1-Dependent Exacerbation of Lysosomal Exocytosis Increases the
Invasive Capacity of Cancer Cells
[0249] The second feature of cancer cells that we predicted would
be affected by excessive LEX is invasiveness, because degradation
of the ECM would compromise the tissue's ability to contain the
tumor. The release of active lysosomal resident proteases,
particularly cathepsin B, correlates with basement membrane
perforation and metastasis (Khan et al., 1998a; Khan et al., 1998b;
Matarrese et al.).
[0250] We determined that NEU1 levels, and in turn extent of LEX,
in tumor cells are linked to their invasive properties. For this
purpose, we used the parental RH41 and RH30 lines as representative
of high- and low-NEU1 cells, respectively. The ex vivo invasive
potentials of these cells were measured using denucleated
peritoneal basement membranes (Marshall et al., 2011) obtained from
either wild-type or Neu1-knockout mice (data not shown). Because
the Neu1-knockout mouse is a model of constitutive, excessive LEX,
its tissues and ECM already have been subjected to progressive
environmental stresses that mimic tumor-adjacent tissue (Yogalingam
et al., 2008; Zanoteli et al., 2010). Compared to high-NEU1 RH41,
the low-NEU1 RH30 line more successfully invaded the wild-type
substrate (data not shown). However, the peritonea from
Neu1-knockout mice were significantly more vulnerable to invasion
from either RMS cell line than were the wild-type peritonea (data
not shown). Notably, the high-NEU1 RH41 cells seeded on a
Neu1-knockout peritoneum were as invasive as the RH30 cells on a
wild-type peritoneum, suggesting that intrinsic LEX had conditioned
the otherwise healthy tissue for cancerous invasion (data not
shown). The invasive properties of the RMS cell lines were further
evaluated in the Neu1-knockout peritoneum sections by
immunohistochemical visualization of the basement membrane
components, laminin and collagen IV, both of which are cathepsin B
substrates (Buck et al., 1992). Tissue exposed to RH30 cells
underwent more destruction/remodeling than did tissue exposed to
RH41 cells (data not shown).
[0251] The inverse relationship between NEU1 activity and invasive
potential of the RMS cells suggested that this is a general
mechanism that may be used by other cancers. We, therefore,
characterized other tumor cell lines, in terms of their invasive
potential and NEU1 status. We chose the Ewing sarcoma cell lines,
EW8 and SKNEP1, for their divergent NEU1 levels. Similar to the RMS
cell lines, the low-NEU1 SKNEP1 cells accumulated oversialylated
LAMP1, were resistant to DOXO, and more successfully invaded
matrigel and peritoneal basement membranes than did the high-NEU1
DOXO-sensitive EW8 cells (data not shown). We also analyzed the
colon carcinoma cell lines, SW480 and SW620, because of their
well-characterized derivation (Leibovitz et al., 1976): SW480 cells
are from a primary colon carcinoma; SW620 cells are from a
metastatic recurrence. The latter cells were also shown to be more
resistant to anti-neoplastic drugs than SW480 cells (Walker et al.,
2010), and we found that they downregulated NEU1 activity. This was
also paralleled by a drastic increase in LAMP1 levels compared to
that in SW480 (data not shown).
The NEU1 Status of RMS Cells is the Primary Determinant of
Differences in their Invasive Capacity
[0252] To determine how NEU1 levels influence the invasiveness of
RMS cells, we seeded the RH41.sup.shNEU1 and RH30.sup.NEU1 cells,
along with the empty-vector controls, on a matrigel substrate
consisting primarily of laminin and collagen IV. After the cells
were maintained in culture for 2 days, the matrigel plugs were
fixed and processed to visualize the ingress of cells into the
substrate. The cells with low-NEU1 activity (RH41.sup.shNEU1 and
RH30.sup.empty) successfully invaded the matrigel, whereas those
with high-NEU1 activity did not (data not shown). These results
established that the NEU1 status of these cancer cells is
sufficient to predict their ability to invade a standardized
substrate.
Neu1-Deficient Mice Generate More Aggressive Cancers in a
Tumor-Prone Model
[0253] Using Neu1-heterozygous mice crossed into a tumor-prone
model, the Arf-knockout mouse (Kamijo et al., 1999), we tested
whether excessive LEX affects malignant invasion in vivo. Two
groups of mice were compared for tumor outcome. The first group,
Arf.sup.-/-/Neu1.sup.+/+ mice, produced a range of tumors that
reflected the type of neoplasms previously reported in Arf single
knockouts (Kamijo et al., 1999). Typical tumors in Arf-knockout
mice younger than 10 months are poorly differentiated sarcomas,
though gliomas, lymphomas, and carcinomas have been observed in low
frequency (Kamijo et al., 1999). Tumors in these mice arose at the
average age of 9 months and were typically focal (data not shown).
The second group consisted of mice with an Arf.sup.-/-/Neu1.sup.+/+
genotype. We anticipated that the low-Neu1 activity in these mice
would encourage faster, more virulent growth of any developing
tumors. This was indeed the case; the mice developed tumors at an
average age of 6 months, which was significantly earlier than that
seen in the Arf single knockouts (p=0.029). In addition, tumor
growth was so aggressive that mice were quickly rendered moribund.
These tumors were often locally invasive and achieved large volumes
in a short time span. In one case, the tumor was fulminantly
metastatic (data not shown). Tumors in the Neu1-heterozygous mice
were sometimes pleomorphic, not resembling those commonly seen in
genetically-engineered mice. Two such malignancies had morphologic
and immunohistochemical features of the rhabdoid/epithelioid
sarcoma-like group of tumors. They contained rhabdoid-type cells
positive for both vimentin and cytokeratin 8, cytoskeletal markers
for mesenchymal and epithelial cells, respectively (data not
shown). This group of tumors has not, to our knowledge, been
reported in mice, and in humans is associated with aggressive
biological behavior and a poor prognosis (Oda and Tsuneyoshi,
2006).
[0254] One additional small cohort of mice,
Arf.sup.-/-/Neu1.sup.-/- double knockouts, was generated during
this breeding program. Very few mice of this genotype were born (17
of 122 total mice), and the mice succumbed to sialidosis-related
mortality early in life. For these reasons, we did not include this
cohort in the statistical analysis, though we report the outcomes
of the few mice that developed tumors, one metastatic, before the
sialidosis became fatal (data not shown).
The easily characterized tumors that arose in the
Arf.sup.-/-/Neu1.sup.+/+ mice provided an opportunity to
investigate the expression of Neu1 in tumors compared to their
cells of origin. We observed a loss of Neu1 immunostaining in
tumors compared to that in normal tissue from the same mouse. This
difference was seen in a carcinoma and a sarcoma (data not shown),
supporting the notion that downregulation of Neu1 offers selective
advantages to tumor growth.
Loss of NEU1 in Human Cancer
[0255] We reasoned that the pattern of Neu1 expression in the
Arf.sup.-/-/Neu1.sup.+/+ mice might be reproduced in patients with
cancer, who should have normal NEU1 activity. We first probed human
RMS tissue samples of the more common embryonic subtype for the
levels of NEU1 compared to healthy skeletal muscle controls. NEU1
was, in fact, downregulated in 10 of 12 (83.3%) RMS samples (data
not shown). Four of the 10 showed a complete absence of NEU1
staining (data not shown). To further characterize the
physiological impact of NEU1 downregulation, we assessed the levels
of its substrate LAMP1. LAMP1 immunohistochemistry revealed a
strong upregulation of this protein in 7 of 12 (58.3%) samples,
including all 4 of those with complete loss of NEU1 (data not
shown). We next decided to repeat this analysis on pancreatic
ductal adenocarinoma samples to gauge how common NEU1
downregulation is across cancer types. Again, 10 of 12 (83.3%)
samples showed a loss of NEU1 compared to the originating ductal
cells (data not shown). LAMP1 staining was also performed and
replicated the results seen in RMS, with 7 of 12 (58.3%) carcinoma
samples showing upregulation (data not shown). Collectively, these
results suggest that downregulation of NEU1 is a commonly employed
strategy in cancer cells, often coupled to accumulation of LAMP1
and concomitant exacerbation of LEX.
Discussion
[0256] In this study we provide evidence that the downregulation of
NEU1 and consequent enhanced LEX imparts at least two crucial
advantages to cancer cells: resistance to lysosomotropic
chemotherapeutic agents and the ability to become expansive and
infiltrative. These findings suggest a tumor-suppressor function
for NEU1.
[0257] The regulated expression of this pleiotropic lysosomal
enzyme affects two cellular processes, sialylation of
glycoconjugates and the extent and type of lysosomal trafficking,
both of which have been consistently invoked to explain tumor cell
behavior in vivo (Hendrix et al., 2010; Varki, 2009). Thus far,
oversialylation has been largely attributed to upregulation of the
biosynthetic enzymes sialyltransferases (Dall'Olio and Chiricolo,
2001). For instance, ST6GalNAc-I
[(.alpha.-N-acetyl-neuraminyl-2,3-.beta.-galactosyl-1,3)-N-acetylgalactos-
aminide .alpha.-2,6-sialyltransferase-I] is thought to contribute
to the generation of the mucin-associated sialyl-Tn antigen, a
marker of metastatic carcinoma (Heimburg-Molinaro et al., 2011;
Marcos et al., 2011), and ST6GalNAc-V imparts metastatic potential
to breast cancer (Bos et al., 2009). Both of these
sialyltransferases catalyze the transfer of sialic acids in
.alpha.2,6-linkage to GalNAc (N-acetyl-galactosamine) residues
found in glycoproteins and glycolipids, a process that may be
readily reversed by NEU1. We propose that diminished or deficient
NEU1 activity impinges on cancer cells in a manner that is
equivalent to sialyltransferase overexpression, thereby causing
impaired sialic acid catabolism of target NEU1 substrates. It is
noteworthy in this respect that a query of the Oncomine database
(Finak Breast www.oncomine.org/resource/main.html) revealed a
substantial upregulation (p<0.01) of at least 3
.alpha.2,6-sialyltransferases, including the ST6GalNAc-I enzyme, as
well as ST6GalNAc-III and ST6Gal-II. We found that these inversely
correlated with NEU1 expression, which was itself significantly
downregulated (p<0.01). The opposing expression levels of these
enzymes in cancer could cause an underappreciated double insult on
the cellular regulation of sialylation.
[0258] Our study identified over-sialylated LAMP1 as a critical
regulator of LEX in cancer, providing both a marker for excessive
LEX and a possible target for inhibiting this process. Deregulation
of other proteins involved in vesicular trafficking has been
observed in some cancers. Namely, upregulation of two LEX
effectors, Rab27B and BAIAP3, was shown to enhance the metastatic
growth and cell proliferation of breast cancer and desmoplastic
small round cell tumors, respectively (Hendrix et al., 2010; Palmer
et al., 2002), suggesting the involvement of an exocytic mechanism
in neoplasia (Chan and Weber, 2002). Here we present evidence to
definitively link LEX to the important cancer phenotypes of
invasiveness and drug resistance.
[0259] The suggestion that lysosomal trafficking influences
multidrug resistance has been proposed, with some reviews
explicitly calling for more research in this area (Castino et al.,
2003; Groth-Pedersen, 2010). An early report noted that DOXO is
concentrated into acidic vesicles that could exocytose their
contents (Klohs and Steinkampf, 1988). Later, vinblastine was
observed to specifically accumulate in lysosomes and be effluxed in
conjunction with lysosomal enzymes (Warren et al., 1991). The study
on how chemotherapeutics are released from tumor cells has largely
focused on the upregulation of p-glycoprotein or other ABC
transporters (Coley, 2010). However, specific inhibitors of these
pumps have often proven unsuccessful in clinical trials, suggesting
that other mechanisms of efflux are involved (Broxterman et al.,
2009; Coley, 2010). Again, our search of the Oncomine database
revealed supportive evidence for NEU1 mediating an important
resistance mechanism. While p-glycoprotein expression was not
significantly different (p=0.81) between responders and
non-responders to the 5-FU and the topoisomerase poison irinotecan
in a data set for metastatic colon cancer, NEU1 expression was
strongly downregulated (p=0.0064) in the resistant patients
(Graudens Colon data set at the website located at
oncomine.org/resource/main). Here we have established that the
DOXO-resistant RMS cell line effluxes DOXO, despite lacking the
p-glycoprotein. The same cells can then be made more sensitive to
DOXO by inhibiting LEX, either by upregulating NEU1 or using the
calcium channel blocker verapamil. Thus, lysosomotropic drug efflux
is under the control of NEU1, as a critical determinant of LEX.
[0260] NEU1-regulated LEX also has a direct influence on the
invasive/metastatic potential of cancer cells. First, otherwise
healthy tissue subjected to excessive LEX, as in the case of
Neu1-knockout mice, is more susceptible to cancer cell invasion.
Second, the level of NEU1 in cancer cells is inversely related to
their invasive potential. Third, the combined loss of NEU1 in the
tumor and the surrounding tissue in vivo creates the conditions for
highly aggressive cancers. The Arf.sup.-/-/Neu1.sup.+/- mice
developed various tumors, including highly aggressive, rare
rhabdoid/epithelioid-like sarcomas of uncertain histogenesis.
Because no mouse model has been identified for this type of cancer,
the spontaneous generation of these tumors offers a possible window
of opportunity into the study of these rare and deadly
malignancies.
[0261] In addition to low-NEU1 status being a potential risk factor
in the development of cancer, neoplastic transformation may place
selective pressure on tumor cells to downregulate NEU1, as
demonstrated by the loss of Neu1 expression in the tumors developed
by Arf single-knockout mice. NEU1 activity levels in the normal
human population range widely, with as-yet-unknown ramifications.
It is predictable that individuals with low NEU1 activity, i.e.,
those with the type I sialidosis, would be more vulnerable to
cancer than are healthy controls. This appears to be the case; Yagi
and colleagues recently reported different neoplastic malignancies
in three siblings with type I sialidosis and suggested a link
between these occurrences and NEU1 deficiency (Yagi et al.,
2011).
[0262] We show here that NEU1 is robustly downregulated in
malignant tissues from human RMS and pancreatic adenocarcinoma
compared to unmatched healthy tissue. Whether this is due to a
preexisting deficiency or to a transformation-related change is
unknown and deserves further attention. In either case, reduced
NEU1 expression may be an informative marker, both because of the
consistency of its downregulation and because of the physiological
ramifications of its loss. Coupled to accumulation of substrate,
NEU1 loss could function as a predictor of excessive LEX and its
important consequences of increased invasiveness and drug
resistance in tumors. Such information may help to shape treatment
decisions and to optimize the rational design of new drug
combination trials.
Experimental Procedures
Animals
[0263] Neu1.sup.+/- mice were bred with Arf.sup.-/- mice (kindly
provided by Dr. Charles Sherr). The colony was expanded for 1 year,
during which time the birth rates and tumor burdens were
documented. Full necropsies were performed by the St. Jude
Veterinary Pathology Core. All mouse experiments were performed
according to animal protocols approved by our institutional Animal
Care and Use Committee and NIH guidelines.
Cell Culture
[0264] The RMS cell lines RH41, RH30 and Ewing sarcoma cell lines
EW8, and SKNEP-1 were kindly provided by Drs. Gerard Grosveld and
Andrew Davidoff. These cell lines were characterized by the
Pediatric Preclinical Testing Program at St. Jude Children's
Research Hospital (Houghton et al., 2007). SW480 and SW620
adenocarcinoma colon cancer cell lines were obtained from ATCC.
Cells were maintained in DMEM or RPMI (Invitrogen) media
supplemented with Glutamax (Sigma), penicillin and streptomycin
(Invitrogen), and 10% cosmic calf serum (Hyclone).
Puromycin-resistant clones were selected and maintained in media
supplemented with puromycin (2 .mu.g/mL, Sigma).
Antibodies and Reagents
[0265] We used commercial antibodies: anti-LAMP1 (Sigma),
anti-laminin (Sigma), anti-.alpha./.beta. tubulin and anti-cleaved
PARP (Cell Signaling). Polyclonal anti-NEU1 antibody has been
previously described (Bonten et al., 2004). Verapamil (50 Sigma)
and DOXO (3 .mu.g/mL, LKT Laboratories) were added to the culture
media. Lysotracker Green DND-26 and Lysotracker Red DND-99 were
obtained from Invitrogen and applied per the manufacturer's
instructions.
Immunoblotting
[0266] Tissue and cell-pellet preparation, Western blotting, and
data analyses were performed as previously described (Zanoteli et
al., 2010).
Immunohistochemistry and Fluorescent Microscopy
[0267] Immunohistochemical analyses of mouse tissue sections and
microarray analyses of human tissue samples (US BioMax, Inc.) were
performed as previously described (Yogalingam et al., 2008). The
use of human tissue samples was approved by our Institutional
Review Board.
Enzyme Assays
[0268] NEU1 and .beta.-Hex enzymatic activities were measured with
the appropriate fluorimetric substrates (Sigma) and normalized to
BCA protein concentrations (Pierce Biotechnology) as described
previously (Yogalingam et al., 2008).
Stable Clone Cell Lines
[0269] RH30 cells were transduced with the pBABE-puro retroviral
vector (AddGene), either unmodified or containing the human NEU1
cDNA. Transduced cells were selected with puromycin (2 .mu.g/mL),
according to the predetermined sensitivity of the cell line, and
were maintained in 0.5 .mu.g/mL selection medium. RH41 cells were
transfected with a panel of NEU1 shRNA plasmids (RHS4533-NM000434,
Open Biosystems) or the accompanying empty vector control. The
cells were selected with puromycin and maintained under selection
medium.
Doxorubicin Efflux Assay
[0270] Cells were plated and maintained at 50% confluency. The next
day, they were treated with medium containing 3 .mu.g/mL DOXO for 2
hours. Treated cells were then washed 3 times with PBS and cultured
in DOXO-free medium for an additional 2 hours, to allow efflux of
the drug into fresh medium. The conditioned medium was then
centrifuged at 1000 rpm (867.times.g) for 5 minutes and a 500 .mu.L
aliquot was spun through an ULTRAFREE-MC 0.1 .mu.m Eppendorf
centrifugal filter (Millipore) to capture the effluxed drug. The
filter was then placed on a microscopic slide and images of the red
fluorescence filter were taken on a Nikon C1 microscope.
Invasion Assays
[0271] Peritoneal sections were harvested from wild-type or
Neu1-knockout mice of matched ages and mounted over transwell
inserts (Fisher), as previously described (Marshall et al., 2011).
A total of 250,000 RMS cells per line were then overlaid onto the
peritoneal preparation and kept in culture for 8 days.
[0272] In a separate set of experiments, 250,000 RMS cells per line
were seeded onto 200 matrigel in a transwell dish and maintained in
culture for 2 days, as previously described (Sabeh et al.,
2009).
Statistics
[0273] Data are expressed as mean.+-.standard deviation (SD) and
were evaluated using the Student's t-test for unpaired samples.
P-values less than 0.05 were considered statistically
significant.
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Example 5: Lysosomal Dysfunction and Excessive Lysosomal Exocytosis
Lead to Alzheimer's-Like Amyloidogenesis
Abstract
[0321] Lysosomal exocytosis is a regulated physiological process
responsible for the controlled secretion of metabolites from
specialized secretory cells and for the maintenance of plasma
membrane homeostasis in most cell types. The lysosomal sialidase
NEU1 is a pivotal negative regulator of this process; genetic
ablation of Neu1 in the mouse model of the childhood disease
sialidosis leads to exacerbated release of lysosomal content
extracellularly with deleterious effects for tissue and
extracellular matrix integrity. Here we show that Neu1.sup.-/- mice
develop pathological and molecular changes in the brain that are
reminiscent of Alzheimer's disease (AD). The synergistic action of
excessive lysosomal exocytosis of neuronal cells and lysosomal
accumulation of oversialylated Neu1 substrates, including the
amyloid precursor protein, contribute to the amyloidogenic cascade.
In addition, Neu1 downregulation, in a known model of AD,
accelerates the amyloidogenic process; conversely, up-regulation of
Neu1 in the same model reduces amyloid deposition and plaques
formation. These data may explain some of the pathological
mechanisms of AD and offer new therapeutic targets along a
previously unknown pathway.
Introduction
[0322] Lysosomes are the major site of compartmentalized
degradation of glycoproteins, glycolipids, as well as aged
organelles, and in this capacity they are pivotal for the
maintenance of cell homeostasis. Downregulation or deficiency of
any of the lysosomal constituents, whose coordinated activities
control overall lysosomal function, disrupts the balance between
synthesis and degradation with detrimental effects on multiple
tissues and organs. This is particularly true for brain, which is
exquisitely sensitive to metabolic changes. One of these
fundamental lysosomal enzymes is the sialidase NEU1, which
initiates the catabolism of a plethora of sialoglyconjugate
substrates by removing their terminal sialic acids. Aside from its
canonical degradative function, NEU1 was recently identified as the
enzyme that regulates the physiological process of lysosomal
exocytosis (LEX), a function that NEU1 exerts by controlling the
sialic acid content of one of its target substrates, the lysosomal
associated membrane protein, LAMP1. LEX is a Ca.sup.2+-dependent
regulated mechanism present in virtually all cell types. It begins
with the recruitment of a subset of lysosomes along the
cytoskeleton to the plasma membrane (PM), followed by their docking
at the PM, and fusion with the PM, which releases the lysosomal
luminal content into the extracellular space. The docking step of
the pathway is mediated by LAMP1. In absence of NEU1, a long-lived,
oversialylated LAMP1 specifies an increased number of lysosomes
poised to dock at the PM and engage in LEX upon Ca.sup.2+ influx.
The end result is the exacerbated release of lysosomal content
extracellularly, which results in abnormal remodeling of the
extracellular matrix (ECM) and changes in PM and ECM composition.
We determined that many of the systemic abnormalities downstream of
NEU1 deficiency in the mouse model of sialidosis could be
attributed to excessive LEX, although the downstream effects of
this phenotype might vary depending on the physiological
characteristics of the affected tissue.
[0323] Here we wished to investigate whether deregulated LEX could
contribute to the progressive, neuropathological manifestations of
the Neu1.sup.-/- mice, which reflect those in children with
sialidosis.
Results:
[0324] We first examined the pattern of expression of Neu1 in the
normal brain and demonstrated that the enzyme was widely
distributed throughout the parenchyma, with the highest expression
in the hippocampus (data not shown). In line with this expression
pattern, Lamp1 accumulated in an oversialylated state (data not
shown), a feature that correlated with excessive LEX in other cells
and tissues of the KO mice. Lamp1 was particularly abundant in
activated microglia and in the pyramidal neurons of the
Neu1.sup.-/- hippocampus (data not shown), suggesting that these
cells could exhibit excessive LEX. We tested this possibility by
measuring the levels of active lysosomal enzymes in the medium of
primary microglia and neurosphere cultures, isolated from
Neu1.sup.-/-/ARF and WT.sup./ARF mice to increase the number of
pluripotent cells and enhance cell viability. Neurospheres from
both genotypes had similar cell composition, but the activity of
lysosomal .beta.-hexosaminidase (.beta.-hex) was significantly
increased only in the media of the Neu1.sup.-/- microglia and
Neu1.sup.-/-/ARF neurospheres (data not shown), confirming the
occurrence in these cells of excessive LEX. Notably, the levels of
LEX were similar in WT and Neu1.sup.-/- primary astrocytes (data
not shown), hence we attributed the enhanced exocytic activity
measured in the Neu1 deficient neurospheres to the neuronal
population of these cultures.
[0325] We argued that the increased levels of LEX in Neu1.sup.-/-
neurons and microglia could dramatically affect the architecture
and composition of the brain parenchyma. In fact, histopathological
examination of the Neu1.sup.-/- brain identified numerous, abnormal
eosinophilic bodies, particularly abundant in the CA3 subregion of
the hippocampus (data not shown). They were heterogeneous in size
and shape, and mostly contained amorphous, granular proteinaceous
material, closely resembling the amyloid. These bodies were
positive for the histological markers thioflavin S and modified
Bielschowsky silver stain (data not shown). Moreover, clusters of
these deposits were detected specifically in the CA3 of the
Neu1.sup.-/- hippocampus by systemic injection of Methoxy-X04, a
finding that confirmed the occurrence of an amyloidogenic process
downstream of Neu1 deficiency (data not shown). At the
ultrastructural level, the amyloid deposits were identified as
swollen dystrophic neurites containing numerous vesicles of
abnormal morphology and content (data not shown). Combined these
phenotypic alterations were reminiscent of those characteristic of
Alzheimer's disease (AD). AD is considered a disease of protein
aggregates whose composition consists primarily of amyloid
precursor protein (APP) abnormally processed into amyloid
.beta.-peptides (A.beta.) and other proteolytic fragments.
[0326] To characterize the amyloid in the brain of Neu1.sup.-/-
mice, we used antibodies cross-reacting with full length APP and
found a progressive and time dependent accumulation of this protein
in the pyramidal neurons of the CA3 region (data not shown).
Ubiquitin and neurofilaments antibodies also immunostained most of
the APP+ dystrophic neurites, suggesting extensive cytoskeletal
abnormalities in these structures (data not shown). Accumulation of
APP was confirmed by immunoblots of hippocampal lysates that
demonstrated a marked increase of this protein in Neu1.sup.-/-
samples (data not shown). Because it is well established that
increased expression of APP in both AD and Down syndrome patients
represents a risk factor for the development of the disease, we
inferred that NEU1 loss of function could predispose to an AD-like
phenotype.
[0327] APP is a type-I membrane glycoprotein, which is glycosylated
and sialylated; changes in its glycan makeup have been linked to
aberrant processing of the protein leading to increased production
and secretion of toxic A.beta. peptides. We hypothesized that APP
could be a natural substrate of Neu1 and, if so, would accumulate
in an oversialylated state in absence of Neu1 activity. Indeed,
analysis of APP in Neu1.sup.-/- hippocampal lysates with sambucus
nigra lectin (SNA) confirmed the presence of excess amounts of
.alpha.-2,6 linked sialic acids on the protein (data not shown). In
vitro enzymatic removal of all N- and O-glycans released a core-APP
protein that was identical in size in the Neu1.sup.-/- and WT
samples, indicating that APP conformational changes in the
Neu1.sup.-/- brain were due to impaired removal of its sialic acids
(data not shown). Accumulated APP was also detected in crude
lysosomal fractions (CLF) isolated from the KO hippocampi, together
with two other substrates of Neu1 (data not shown), Lamp1 and
cathepsin B. These results identify APP as a novel substrate of
Neu1 that could be at least in part cleaved in the lysosomal
compartment.
[0328] A crucial step in the amyloidogenic processing of APP is the
generation of carboxy terminal fragments (CTFs), which are
subsequently cleaved into ft-amyloid. We therefore assessed their
levels in Neu1.sup.-/- samples, as predictive measure of abnormal
Aft processing. CTFs levels were increased in both Neu1.sup.-/-
hippocampal samples and in CLF compared to those in WT samples
(data not shown). Furthermore, .beta.-amyloid was abnormally
present in Neu1.sup.-/- CLF (data not shown) suggesting that
oversialylated APP is processed in the lysosomal compartment. These
observations were further supported by the detection of elevated
amounts of the amyloid peptide A.beta.42 (A.beta.) both in the
culture media of Neu1.sup.-/-/ARF neurospheres, and in the KO
cerebrospinal fluid (data not shown). To ascertain the role of LEX
in this amyloidogenic process, we cultured Neu1.sup.-/-/ARF and
Neu1.sup.WT/ARF neurospheres in presence of a human
TAMRA-conjugated, fluorescent A.beta.42 (T-A.beta.). T-A.beta. was
readily taken up by the cells and rerouted to late
endosomes/lysosomes (data not shown). This fraction of the
internalized peptide was then trafficked from the lysosomes to the
PM via LEX, as determined by live imaging of lysotracker-labeled
lysosomes with total internal reflection microscopy (data not
shown). By counting the number of lysotracker+ organelles proximal
to the PM we showed that Neu1.sup.-/-/ARF cells had significantly
higher number of T-A.beta.-containing lysosomes clustered at the PM
(data not shown). Moreover, when both neurosphere cultures were
maintained in T-A.beta. free medium for 24 h following exposure to
the peptide we were able to capture the T-A.beta. fluorescence
released extracellularly, and consequently we measured increased
amounts of T-A.beta. exocytosed into the medium of Neu1.sup.-/-/ARF
cells compared to WT cells (data not shown). Thus, A.beta. is
abnormally secreted in absence of Neu1 and is released via LEX.
[0329] Together these results suggest that Neu1 loss of function
and consequent exacerbation of LEX are predisposing factors to
.beta.-amyloidogenesis. To further verify this assumption, we
analyzed the effects of Neu1 ablation on amyloid .beta. levels and
plaque formation in vivo by using a well characterized transgenic
model of AD (5XFAD) that we crossed into the Neu1.sup.-/-
background. We first tested the expression levels of Neu1 in the
5XFAD mouse line by immunohistochemistry. We observed a marked
downregulation of the enzyme, especially noticeable in the
hippocampus, which was accompanied by increased expression of Lamp1
(data not shown). We also measured reduced Neu1 enzyme activity in
primary neurospheres isolated from the 5XFAD hippocampi (data not
shown). To further these observations, we demonstrated that the
levels of APP were increased in hippocampal lysates isolated from
5XFAD/Neu1.sup.-/- compared to those in 5XFAD/WT (data not shown).
Moreover, APP processing in these mice resulted in the accumulation
of amyloid-.beta. (data not shown), a finding that supports the
idea that downregulation or loss of Neu1 in 5XFAD mice accelerates
the .beta.-amyloidogenic process likely via deregulated LEX.
[0330] Finally, to test whether Neu1 deficiency (and excessive LEX)
could represent a therapeutic target to revert an AD-like
phenotype, we sought to exogenously increase Neu1 activity in the
brain of 5XFAD mice. The latter could be achieved by augmenting the
intracellular expression of the Neu1 chaperone Protective Protein
Cathepsin A (PPCA). We therefore performed stereotactic injection
of an adeno-associated virus containing both human NEU1 and PPCA
(AAVNEU1/AAVPPCA) into the hippocampal region of the 5XFAD mice.
This vector combination directed sustained expression of the
transgenes in vitro (data not shown). Four weeks after injection,
high expression of both PPCA and NEU1 was detected in brain
sections of mice treated with the recombinant AAV vectors (data not
shown). Remarkably, the abundant number of amyloid plaques seen in
the untreated 5XFAD mice was reduced by 44.3.+-.6.2% in the AAV
injected mice, compared to the same line injected only with carrier
solution (data not shown). Immunoblots of hippocampal lysates from
the injected 5XFAD mice confirmed that both APP and .beta.-amyloid
levels were reduced (data not shown).
Conclusions:
[0331] In conclusion our results reveal an unsuspected mechanism of
control over APP processing by a lysosomal enzyme. We propose a
two-hit model to explain the amyloidogenic process downstream of
Neu1 loss of function: the accumulation of an oversialylated APP
which is abnormally processed, followed by the release of APP end
products via excessive LEX. In this scenario the activated
microglia with increased LEX could engage in a feed forward
pathogenic cascade by releasing active lysosomal enzymes
extracellularly that may cleave APP, producing toxic A.beta.
peptides. Neu1 position in the amyloidogenic pathway and its role
as central regulator of LEX may be exploited for new potential
therapeutic targets to treat/delay plaque deposition and amyloid
formation in AD.
Sequence CWU 1
1
411644DNAHomo sapiensmisc_feature1427n = A,T,C or G 1ccaagcttag
atcttggagt ctagctgcca gggtcgcggc agctgcgggg agagatgact 60ggggagcgac
ccagcacggc gctcccggac agacgctggg ggccgcggat tctgggcttc
120tggggaggct gtagggtttg ggtgtttgcc gcgatcttcc tgctgctgtc
tctggcagcc 180tcctggtcca aggctgagaa cgacttcggt ctggtgcagc
cgctggtgac catggagcaa 240ctgctgtggg tgagcgggag acagatcggc
tcagtggaca ccttccgcat cccgctcatc 300acagccactc cgcggggcac
tcttctcgcc tttgctgagg cgaggaaaat gtcctcatcc 360gatgaggggg
ccaagttcat cgccctgcgg aggtccatgg accagggcag cacatggtct
420cctacagcgt tcattgtcaa tgatggggat gtccccgatg ggctgaacct
tggggcagta 480gtgagcgatg ttgagacagg agtagtattt cttttctact
ccctttgtgc tcacaaggcc 540ggctgccagg tggcctctac catgttggta
tggagcaagg atgatggtgt ttcctggagc 600acaccccgga atctctccct
ggatattggc actgaagtgt ttgcccctgg accgggctct 660ggtattcaga
aacagcggga gccacggaag ggccgcctca tcgtgtgtgg ccatgggacg
720ctggagcggg acggagtctt ctgtctcctc agcgatgatc atggtgcctc
ctggcgctac 780ggaagtgggg tcagcggcat cccctacggt cagcccaagc
aggaaaatga tttcaatcct 840gatgaatgcc agccctatga gctcccagat
ggctcagtcg tcatcaatgc ccgaaaccag 900aacaactacc actgccactg
ccgaattgtc ctccgcagct atgatgcctg tgatacacta 960aggccccgtg
atgtgacctt cgaccctgag ctcgtggacc ctgtggtagc tgcaggagct
1020gtagtcacca gctccggcat tgtcttcttc tccaacccag cacatccaga
gttccgagtg 1080aacctgaccc tgcgatggag cttcagcaat ggtacctcat
ggcggaaaga gacagtccag 1140ctatggccag gccccagtgg ctattcatcc
ctggcaaccc tggagggcag catggatgga 1200gaggagcagg ccccccagct
ctacgtcctg tatgagaaag gccggaacca ctacacagag 1260agcatctccg
tggccaaaat cagtgtctat gggacactct gagctgtgcc actgccacag
1320gggtattctg ccttcaggac tctgccttca ggaagacggg tctgtagagg
gtctgctgga 1380gacgcctgaa agacagttcc atcttccttt agactccagc
cttggcnaca tcaccttccc 1440tttaccaggg aaatcacttc ctttaggact
gaaagctagg cgtcctctcc cacaacaaag 1500tcctgccctc atctgagaat
actgtctttc catatggcta agtgtggccc caccaccctc 1560tctgccctcc
cgggacattg attggtcctg tcttgggcag gtctagtgag ctgtagaaat
1620gaatcaatgt gaactcaggg aact 16442415PRTHomo sapiens 2Met Thr Gly
Glu Arg Pro Ser Thr Ala Leu Pro Asp Arg Arg Trp Gly1 5 10 15Pro Arg
Ile Leu Gly Phe Trp Gly Gly Cys Arg Val Trp Val Phe Ala 20 25 30Ala
Ile Phe Leu Leu Leu Ser Leu Ala Ala Ser Trp Ser Lys Ala Glu 35 40
45Asn Asp Phe Gly Leu Val Gln Pro Leu Val Thr Met Glu Gln Leu Leu
50 55 60Trp Val Ser Gly Arg Gln Ile Gly Ser Val Asp Thr Phe Arg Ile
Pro65 70 75 80Leu Ile Thr Ala Thr Pro Arg Gly Thr Leu Leu Ala Phe
Ala Glu Ala 85 90 95Arg Lys Met Ser Ser Ser Asp Glu Gly Ala Lys Phe
Ile Ala Leu Arg 100 105 110Arg Ser Met Asp Gln Gly Ser Thr Trp Ser
Pro Thr Ala Phe Ile Val 115 120 125Asn Asp Gly Asp Val Pro Asp Gly
Leu Asn Leu Gly Ala Val Val Ser 130 135 140Asp Val Glu Thr Gly Val
Val Phe Leu Phe Tyr Ser Leu Cys Ala His145 150 155 160Lys Ala Gly
Cys Gln Val Ala Ser Thr Met Leu Val Trp Ser Lys Asp 165 170 175Asp
Gly Val Ser Trp Ser Thr Pro Arg Asn Leu Ser Leu Asp Ile Gly 180 185
190Thr Glu Val Phe Ala Pro Gly Pro Gly Ser Gly Ile Gln Lys Gln Arg
195 200 205Glu Pro Arg Lys Gly Arg Leu Ile Val Cys Gly His Gly Thr
Leu Glu 210 215 220Arg Asp Gly Val Phe Cys Leu Leu Ser Asp Asp His
Gly Ala Ser Trp225 230 235 240Arg Tyr Gly Ser Gly Val Ser Gly Ile
Pro Tyr Gly Gln Pro Lys Gln 245 250 255Glu Asn Asp Phe Asn Pro Asp
Glu Cys Gln Pro Tyr Glu Leu Pro Asp 260 265 270Gly Ser Val Val Ile
Asn Ala Arg Asn Gln Asn Asn Tyr His Cys His 275 280 285Cys Arg Ile
Val Leu Arg Ser Tyr Asp Ala Cys Asp Thr Leu Arg Pro 290 295 300Arg
Asp Val Thr Phe Asp Pro Glu Leu Val Asp Pro Val Val Ala Ala305 310
315 320Gly Ala Val Val Thr Ser Ser Gly Ile Val Phe Phe Ser Asn Pro
Ala 325 330 335His Pro Glu Phe Arg Val Asn Leu Thr Leu Arg Trp Ser
Phe Ser Asn 340 345 350Gly Thr Ser Trp Arg Lys Glu Thr Val Gln Leu
Trp Pro Gly Pro Ser 355 360 365Gly Tyr Ser Ser Leu Ala Thr Leu Glu
Gly Ser Met Asp Gly Glu Glu 370 375 380Gln Ala Pro Gln Leu Tyr Val
Leu Tyr Glu Lys Gly Arg Asn His Tyr385 390 395 400Thr Glu Ser Ile
Ser Val Ala Lys Ile Ser Val Tyr Gly Thr Leu 405 410 41531815DNAHomo
sapiens 3ggggagatga tccgagccgc gccgccgccg ctgttcctgc tgctgctgct
gctgctgctg 60ctagtgtcct gggcgtcccg aggcgaggca gcccccgacc aggacgagat
ccagcgcctc 120cccgggctgg ccaagcagcc gtctttccgc cagtactccg
gctacctcaa aagctccggc 180tccaagcacc tccactactg gtttgtggag
tcccagaagg atcccgagaa cagccctgtg 240gtgctttggc tcaatggggg
tcccggctgc agctcactag atgggctcct cacagagcat 300ggccccttcc
tggtccagcc agatggtgtc accctggagt acaaccccta ttcttggaat
360ctgattgcca atgtgttata cctggagtcc ccagctgggg tgggcttctc
ctactccgat 420gacaagtttt atgcaactaa tgacactgag gtcgcccaga
gcaattttga ggcccttcaa 480gatttcttcc gcctctttcc ggagtacaag
aacaacaaac ttttcctgac cggggagagc 540tatgctggca tctacatccc
caccctggcc gtgctggtca tgcaggatcc cagcatgaac 600cttcaggggc
tggctgtggg caatggactc tcctcctatg agcagaatga caactccctg
660gtctactttg cctactacca tggccttctg gggaacaggc tttggtcttc
tctccagacc 720cactgctgct ctcaaaacaa gtgtaacttc tatgacaaca
aagacctgga atgcgtgacc 780aatcttcagg aagtggcccg catcgtgggc
aactctggcc tcaacatcta caatctctat 840gccccgtgtg ctggaggggt
gcccagccat tttaggtatg agaaggacac tgttgtggtc 900caggatttgg
gcaacatctt cactcgcctg ccactcaagc ggatgtggca tcaggcactg
960ctgcgctcag gggataaagt gcgcatggac cccccctgca ccaacacaac
agctgcttcc 1020acctacctca acaacccgta cgtgcggaag gccctcaaca
tcccggagca gctgccacaa 1080tgggacatgt gcaactttct ggtaaactta
cagtaccgcc gtctctaccg aagcatgaac 1140tcccagtatc tgaagctgct
tagctcacag aaataccaga tcctattata taatggagat 1200gtagacatgg
cctgcaattt catgggggat gagtggtttg tggattccct caaccagaag
1260atggaggtgc agcgccggcc ctggttagtg aagtacgggg acagcgggga
gcagattgcc 1320ggcttcgtga aggagttctc ccacatcgcc tttctcacga
tcaagggcgc cggccacatg 1380gttcccaccg acaagcccct cgctgccttc
accatgttct cccgcttcct gaacaagcag 1440ccatactgat gaccacagca
accagctcca cggcctgatg cagcccctcc cagcctctcc 1500cgctaggaga
gtcctcttct aagcaaagtg cccctgcagg cgggttctgc cgccaggact
1560gcccccttcc cagagccctg tacatcccag actgggccca gggtctccca
tagacagcct 1620gggggcaagt tagcacttta ttcccgcagc agttcctgaa
tggggtggcc tggccccttc 1680tctgcttaaa gaatgccctt tatgatgcac
tgattccatc ccaggaaccc aacagagctc 1740aggacagccc acagggaggt
ggtggacgga ctgtaattga tagattgatt atggaattaa 1800attgggtaca gcttc
18154480PRTHomo sapiens 4Met Ile Arg Ala Ala Pro Pro Pro Leu Phe
Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Leu Val Ser Trp Ala Ser Arg
Gly Glu Ala Ala Pro Asp Gln 20 25 30Asp Glu Ile Gln Arg Leu Pro Gly
Leu Ala Lys Gln Pro Ser Phe Arg 35 40 45Gln Tyr Ser Gly Tyr Leu Lys
Ser Ser Gly Ser Lys His Leu His Tyr 50 55 60Trp Phe Val Glu Ser Gln
Lys Asp Pro Glu Asn Ser Pro Val Val Leu65 70 75 80Trp Leu Asn Gly
Gly Pro Gly Cys Ser Ser Leu Asp Gly Leu Leu Thr 85 90 95Glu His Gly
Pro Phe Leu Val Gln Pro Asp Gly Val Thr Leu Glu Tyr 100 105 110Asn
Pro Tyr Ser Trp Asn Leu Ile Ala Asn Val Leu Tyr Leu Glu Ser 115 120
125Pro Ala Gly Val Gly Phe Ser Tyr Ser Asp Asp Lys Phe Tyr Ala Thr
130 135 140Asn Asp Thr Glu Val Ala Gln Ser Asn Phe Glu Ala Leu Gln
Asp Phe145 150 155 160Phe Arg Leu Phe Pro Glu Tyr Lys Asn Asn Lys
Leu Phe Leu Thr Gly 165 170 175Glu Ser Tyr Ala Gly Ile Tyr Ile Pro
Thr Leu Ala Val Leu Val Met 180 185 190Gln Asp Pro Ser Met Asn Leu
Gln Gly Leu Ala Val Gly Asn Gly Leu 195 200 205Ser Ser Tyr Glu Gln
Asn Asp Asn Ser Leu Val Tyr Phe Ala Tyr Tyr 210 215 220His Gly Leu
Leu Gly Asn Arg Leu Trp Ser Ser Leu Gln Thr His Cys225 230 235
240Cys Ser Gln Asn Lys Cys Asn Phe Tyr Asp Asn Lys Asp Leu Glu Cys
245 250 255Val Thr Asn Leu Gln Glu Val Ala Arg Ile Val Gly Asn Ser
Gly Leu 260 265 270Asn Ile Tyr Asn Leu Tyr Ala Pro Cys Ala Gly Gly
Val Pro Ser His 275 280 285Phe Arg Tyr Glu Lys Asp Thr Val Val Val
Gln Asp Leu Gly Asn Ile 290 295 300Phe Thr Arg Leu Pro Leu Lys Arg
Met Trp His Gln Ala Leu Leu Arg305 310 315 320Ser Gly Asp Lys Val
Arg Met Asp Pro Pro Cys Thr Asn Thr Thr Ala 325 330 335Ala Ser Thr
Tyr Leu Asn Asn Pro Tyr Val Arg Lys Ala Leu Asn Ile 340 345 350Pro
Glu Gln Leu Pro Gln Trp Asp Met Cys Asn Phe Leu Val Asn Leu 355 360
365Gln Tyr Arg Arg Leu Tyr Arg Ser Met Asn Ser Gln Tyr Leu Lys Leu
370 375 380Leu Ser Ser Gln Lys Tyr Gln Ile Leu Leu Tyr Asn Gly Asp
Val Asp385 390 395 400Met Ala Cys Asn Phe Met Gly Asp Glu Trp Phe
Val Asp Ser Leu Asn 405 410 415Gln Lys Met Glu Val Gln Arg Arg Pro
Trp Leu Val Lys Tyr Gly Asp 420 425 430Ser Gly Glu Gln Ile Ala Gly
Phe Val Lys Glu Phe Ser His Ile Ala 435 440 445Phe Leu Thr Ile Lys
Gly Ala Gly His Met Val Pro Thr Asp Lys Pro 450 455 460Leu Ala Ala
Phe Thr Met Phe Ser Arg Phe Leu Asn Lys Gln Pro Tyr465 470 475
480
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References