U.S. patent application number 14/383687 was filed with the patent office on 2015-01-15 for modulation of breast cancer growth by modulation of xbp1 activity.
The applicant listed for this patent is Cornell University. Invention is credited to Xi Chen, Laurie H. Glimcher.
Application Number | 20150018406 14/383687 |
Document ID | / |
Family ID | 47902380 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150018406 |
Kind Code |
A1 |
Glimcher; Laurie H. ; et
al. |
January 15, 2015 |
MODULATION OF BREAST CANCER GROWTH BY MODULATION OF XBP1
ACTIVITY
Abstract
Described herein is a previously unknown function of XBP1 in
triple-negative breast cancer (TNBC). It is shown that XBP1 is
preferentially spliced and activated in TNBC, and that deletion of
XBP1 significantly blocks triple negative breast tumor growth.
Strikingly, XBP1 is required for the self-renewal of breast tumor
initiating cells (TICs). Genome-wide mapping of the XBP1
transcriptional regulatory network identified a fundamental role
for XBP1 in regulating the response to hypoxia via the
transcription factor hypoxia-inducible factor 1a (HIF1a).
Importantly, activation of this pathway appears to carry prognostic
implications, as expression of the XBP1-dependent signature is
associated with shorter survival times in human TNBC.
Inventors: |
Glimcher; Laurie H.; (New
York, NY) ; Chen; Xi; (Harrison, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell University |
Ithaca |
NY |
US |
|
|
Family ID: |
47902380 |
Appl. No.: |
14/383687 |
Filed: |
March 11, 2013 |
PCT Filed: |
March 11, 2013 |
PCT NO: |
PCT/US2013/030251 |
371 Date: |
September 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61609130 |
Mar 9, 2012 |
|
|
|
Current U.S.
Class: |
514/44A ;
506/9 |
Current CPC
Class: |
C12N 2310/11 20130101;
A61K 45/06 20130101; G01N 33/57415 20130101; C12N 15/113 20130101;
C12N 2310/14 20130101; C12N 15/1137 20130101; C12N 2310/531
20130101; C12N 15/1135 20130101; C12Q 2600/136 20130101; C12Y
207/11001 20130101; C12N 2320/30 20130101; A61P 35/00 20180101;
C12Q 1/6886 20130101; G01N 2500/04 20130101; A61K 31/7088 20130101;
G01N 33/5011 20130101; C12Q 2600/118 20130101; C12N 2320/31
20130101; G01N 2800/52 20130101 |
Class at
Publication: |
514/44.A ;
506/9 |
International
Class: |
C12N 15/113 20060101
C12N015/113; G01N 33/50 20060101 G01N033/50; G01N 33/574 20060101
G01N033/574; A61K 45/06 20060101 A61K045/06; A61K 31/7088 20060101
A61K031/7088 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] This invention was made with government support under
CA112663 and AI032412 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of treating triple negative breast cancer (TNBC) in a
subject, the method comprising administering to the subject a
direct or indirect inhibitor of XBP1 in an amount effective to
inhibit growth of cancer cells in said subject, such that TNBC in
the subject is treated.
2. The method of claim 1, wherein the subject has an advanced stage
of breast cancer.
3. The method of claim 1, wherein the inhibitor of XBP1 is a direct
inhibitor.
4. The method of claim 3, wherein the inhibitor of XBP1 is selected
from the group consisting of a nucleic acid molecule that is
antisense to an XBP1-encoding nucleic acid molecule, an XBP1 shRNA,
an XBP siRNA, a microRNA that targets XBP1, a dominant negative
XBP1 molecule and a small molecule inhibitor of XBP1.
5. The method of claim 4, wherein the inhibitor of XBP1 is an XBP1
shRNA or an XBP siRNA.
6. The method of claim 1, wherein the inhibitor of XBP1 is an
indirect inhibitor.
7. The method of claim 6, wherein the inhibitor of XBP1 is an agent
that inhibits IRE1 or an agent that inhibits an endonuclease that
produces XBP1s.
8. The method of claim 1, wherein the inhibitor of XBP1 is
administered to breast tissue of said subject.
9. The method of claim 8, wherein the inhibitor of XBP1 is
administered directly to a tumor in said tissue.
10. The method of claim 1, wherein the inhibitor of XBP1 is
administered in combination with a second cancer therapeutic
agent.
11. The method of claim 10, wherein the inhibitor of XBP1 is
administered in combination with a chemotherapeutic agent.
12. The method of claim 1, wherein the treatment promotes longer
relapse-free survival of the subject.
13. A method for determining a prognosis status for a subject with
triple negative breast cancer (TNBC), the method comprising: a)
determining an XBP1 gene signature for the TNBC of the subject; and
b) correlating the XBP1 gene signature with a prognosis status for
the subject, wherein higher expression of the XBP1 gene signature,
relative to a control, correlates with shorter relapse-free
survival of the subject and lower expression of the XBP1 gene
signature, relative to a control, correlates with longer
relapse-free survival of the subject.
14. The method of claim 13, wherein higher expression of the XBP1
gene signature, relative to a control, correlates with shorter
relapse-free survival of the subject.
15. The method of claim 14, wherein the XBP1 gene signature
comprises a plurality of genes set forth in Table 1.
16. The method of claim 15, wherein at least 5% of the genes set
forth in Table 1 are expressed at a higher level relative to
control.
17. The method of claim 15, wherein at least 20% of the genes set
forth in Table 1 are expressed at a higher level relative to
control.
18. The method of claim 13, wherein the gene signature is
determined for a population of cells isolated from a breast tissue
tumor breast of said subject.
19. The method of claim 13, wherein the gene signature is
determined for a population of cancer cells isolated from said
subject.
20. The method of claim 13, wherein the subject is negative for one
or more of estrogen receptor (ER), progesterone receptor (PR) or
Her2/neu.
21. The method of claim 13, wherein the subject is positive for
BRCA1.
22. The method of claim 21, wherein the subject is further
subjected to histopathological analysis of tumors.
23. A method of identifying a compound useful in inhibiting the
growth of triple negative breast cancer (TNBC) cells, the method
comprising: a) providing an indicator composition comprising XBP1
and HIF1.alpha., or biologically active portions thereof; b)
contacting the indicator composition with each member of a library
of test compounds; c) selecting from the library of test compounds
a compound of interest that decreases the interaction of XBP1 and
HIF1.alpha., or biologically active portions thereof, wherein the
ability of a compound to inhibit growth of TNBC cells is indicated
by a decrease in the interaction as compared to the amount of
interaction in the absence of the compound.
24. The method of claim 20, wherein the subject is positive for
BRCA1.
Description
BACKGROUND OF THE INVENTION
[0002] During tumor development and progression, cancer cells
encounter cytotoxic conditions such as hypoxia, nutrient
deprivation, and low pH due to inadequate vascularization (Hanahan,
D., et al. 2011. Cell 144, 646-674). To maintain survival and
growth in the face of these physiologic stressors, a set of
adaptive response pathways are induced. One adaptive pathway well
studied in other contexts is the unfolded protein response (UPR),
which is induced by factors affecting the endoplasmic reticulum
(ER) such as changes in glycosylation, redox status, glucose
availability, calcium homeostasis or the accumulation of unfolded
or misfolded proteins (Hetz, C., et al. 2001. Physiol Rev 91,
1219-1243). Notably, features of the tumor microenvironment, such
as hypoxia and nutrient deprivation, can disrupt ER homeostasis by
the perturbation of aerobic processes such as oligosaccharide
modification, disulphide bond formation, isomerization, and protein
quality control and export (Wouters, B. G., et al. 2008. Nat Rev
Cancer 8, 851-864).
[0003] In mammalian cells, the UPR is mediated by three
ER-localized transmembrane protein sensors: Inositol-requiring
transmembrane kinase/endonuclease-1 (IRE1), PKR-like ER kinase
(PERK) and activating transcription factor 6 (ATF6) (Walter, P., et
al. 2011. Science 334, 1081-1086). Of these, IRE1 is the most
evolutionarily conserved branch. An increase in the load of folding
proteins in the ER activates IRE1, an ER-resident kinase and
endoribonuclease that acts as an ER-stress sensor (Walter, P., et
al. 2011. Science 334, 1081-1086). Activated IRE1 removes a 26 bp
intron from XBP1 mRNA and results in a frame shift in the coding
sequence, with the spliced form encoding a 226 amino acid
transcriptional activation domain (Calfon, M., et al. 2002. Nature
415, 92-96; Yoshida, H., et al. 2001. Cell 107, 881-891). In
contrast to the unspliced XBP1 (XBP1u), which is unstable and
quickly degraded, spliced XBP1 (XBP1s) is stable and is a potent
inducer of target genes that orchestrate the cellular response to
ER stress (Hetz, C., et al. 2011. Physiol Rev 91, 1219-1243). XBP1
deficient mice display severe abnormalities in differentiation of
several lineages of specialized secretory cells, including plasma
cells (Reimold, A. M., et al. 2001. Nature 412, 300-307), exocrine
pancreas cells (Lee, A. H., et al. 2005. EMBO J. 24, 4368-4380) and
intestinal epithelial cells (Kaser, A., et al. 2008. Cell 134,
743-756). As the mammary gland is a secretory tissue that undergoes
extensive secretory compartment expansion during the transition
from pregnancy to lactation, the function of XBP1 in the normal
mammary gland and in breast cancer is of special interest. XBP1
expression was reported to be regulated by estrogen receptor and
induced in primary human breast cancer (Fujimoto, T., et al. 2003.
Breast Cancer 10, 301-306), however, the functional role of the UPR
and XBP1 in the normal and malignant mammary gland is largely
unknown.
SUMMARY OF THE INVENTION
[0004] The unfolded protein response (UPR) is essential for tumor
cells to survive the pathologic stresses intrinsic to the tumor
microenvironment. The instant invention is based, at least in part,
on the new finding of an unexpected function of XBP1 (X box binding
protein 1), a key component of the UPR, in human triple negative
breast cancer (TNBC). In particular, the instant inventors have
discovered that XBP1 promotes TNBC and does so by controlling the
hypoxia response. Triple negative breast cancer (TNBC) is a highly
aggressive malignancy with limited treatment options and
TNBC-targeted therapies do not yet exist. Here, it is reported that
XBP1, a key component of the Unfolded Protein Response (UPR), is
activated in TNBC and plays a pivotal role in the tumorigenicity
and progression of this human breast cancer subtype. The instant
inventors show that XBP1 is required for the transformation of
immortalized mammary epithelial cells. Silencing of XBP1
significantly suppressed the growth and invasiveness of TNBCs.
Activation of the XBP1 pathway is associated with poor prognosis in
human TNBC patients. Intriguingly, XBP1 is preferentially activated
in tumor initiating cells (TICs) and is essential for sustaining
TIC self-renewal. Moreover, overexpression of the active form of
XBP1 (XBP1s) in non-TICs is sufficient to confer stem-like or
tumor-initiating properties on them, while depletion of XBP1
inhibited tumor relapse due to a preferential depletion of TICs (by
reducing the population of chemotherapy-resistant TICs).
[0005] Genome-wide mapping of the XBP1 transcriptional regulatory
network revealed that XBP1 regulates the hypoxia response through
controlling HIF1.alpha. transcriptional activity and the expression
of HIF1.alpha. targets. The instant inventors have identified a
genetic fingerprint (gene expression signature) indicative of XBP1
pathway activation that is associated with poor prognosis in human
TNBC patients. These findings, for the first time, reveal a key
function for this branch of the UPR in TNBC (linking the UPR
pathway with TNBC and TIC), opening new avenues for therapeutics
for TNBC patients.
DESCRIPTION OF THE FIGURES
[0006] FIG. 1. The UPR is activated in human breast cancer.
[0007] (A) A TMA containing normal breast tissue or breast cancer
tissue sections was subjected to IHC for phospho-PERK (Thr980) (DAB
staining, brown). Representative pictures are shown from normal and
human breast cancer tissues.
[0008] (B). Comparison of PERK phosphorylation in normal breast
tissue samples and breast cancer samples. 66 normal human breast
tissues and 40 human breast cancer tissues were evaluated.
[0009] (C) The TMA were subjected to IHC for phospho-EIF2.alpha.
(Ser51) (DAB staining, brown). Representative pictures are shown
from normal and human breast cancer tissues.
[0010] (D) Comparison of EIF2.alpha. phosphorylation in normal
breast tissue samples and breast cancer samples. 59 normal human
breast tissues and 41 human breast cancer tissues were
evaluated.
[0011] FIG. 2. XBP1 is required for transformation of immortalized
mammary epithelial cells
[0012] (A) XBP1 silencing blocks the phenotypic transformation of
MCF10A ER-Src cells. MCF10A ER-Src cells were infected with
lentivirus encoding XBP1 shRNA (shXBP1) or control shRNA (shCtrl),
and treated with tamoxifen (TAM) for 36 hr. Phase-contrast images
are shown.
[0013] (B) Quantification of invasive cells in untreated and
TAM-treated MCF10A ER-Src cells in the presence or absence of
control or XBP1 shRNA.
[0014] (C) Quantification of soft agar colony formation in
untreated and TAM-treated MCF10A ER-Src cells in the presence or
absence of control or XBP1 shRNA. Experiments were performed in
triplicate and data are shown as mean.+-.SD.
[0015] (D) Tumor growth (mean.+-.SD) of untreated, control shRNA,
and XBP1 shRNA treated MCF10A ER-Src (TAM treated) cells. TX:
treatment with shRNA.
[0016] (E) MCF10A ER-Src cells were infected with retrovirus
encoding XBP1s or empty vector. Phase-contrast images are
shown.
[0017] (F) Quantification of soft agar colonies in MCF10A ER-Src
cells infected with empty vector or spliced XBP1 (XBP1s) expressing
retroviruses. Phase-contrast images are shown in the lower
panel.
[0018] All experiments were performed in triplicate and data are
shown as mean.+-.SD.
[0019] FIG. 3. XBP1 inhibition blocks breast cancer cell growth and
invasiveness in vitro and in vivo.
[0020] (A) RT-PCR analysis of XBP1 splicing in different luminal
and basal-like cell lines. XBP1u: unspliced XBP1, XBP1s: spliced
XBP1.
[0021] (B) Quantification of soft agar colony formation in
untreated and control shRNA or XBP1 shRNA infected breast cancer
cells.
[0022] (C) Quantification of invasive cells in untreated and
control shRNA or XBP1 shRNA infected breast cancer cells.
**p,0.01
[0023] (D) Quantitative RT-PCR analysis of XBP1 expression in
MDA-MB-231 cells infected with doxycycline (DOX) inducible
lentiviruses encoding shRNAs against XBP1 or scrambled LACZ
control, in the presence or absence of doxycycline for 48 h. Data
are presented relative to .beta.-actin. Experiments were performed
in triplicate and data are shown as mean.+-.SD.
[0024] (E) Representative bioluminescent images of orthotopic
tumors formed by MDA-MB-231 cells as in (D) that were then
superinfected with a retrovirus encoding firefly luciferase. A
total of 1.5.times.10.sup.6 cells were injected into the fourth
mammary glands of NOD/SCID/IL2R.gamma.-/- mice. Bioluminescent
images were obtained 5 days later and serially after mice were
begun on chow containing doxycycline (day 19). Pictures shown are
the day 19 image (Before Dox) and day 64 image (After Dox).
[0025] (F) Quantitation of imaging studies as in (E). *p<0.05,
**p<0.01.
[0026] (G) Tumor incidence of TNBC patient-derived BCM-2147 tumor
treated with scrambled siRNA (n=11) or XBP1 siRNA (n=9). Tumor
incidence is reported at 10 weeks post-transplantation. Statistical
significance was determined by Barnard's test. (Barnard, G. A.,
1945. Nature 156, 177; Barnard, G. A., 1947. Biometrika 34,
123-138).
[0027] (H) Tumor growth (mean.+-.SD) of BCM-2147 tumors as in (G).
*p<0.05, **p<0.01.
[0028] (I) Knockdown efficiency of XBP1 in MDA-MB-231 derived
xenograft tumor (as in FIG. 3E). Quantitative RT-PCR analysis of
XBP1 expression in shCtrl or shXBP1 xenograft tumor. Data are
presented relative to .beta.-actin. There are 5 mice in each group
and data are shown as mean.+-.SD.
[0029] (J) Knockdown efficiency of XBP1 in MDA-MB-231 cells with
two shRNA constructs targeting different regions of XBP1.
[0030] (K) Bioluminescent images of orthotopic tumors formed by
luciferase-expressing MDA-MB-231 cells infected with different
lentiviruses. A total of 1.5.times.10 cells were injected into the
fourth mammary glands of nude mice. Bioluminescent images were
obtained 1 week later and serially after mice were begun on chow
containing doxycycline (Dox) (day 10). Pictures shown are the day
10 images (Before Dox) and day 45 images (After Dox).
[0031] (L) Tumor growth (mean.+-.SD) of untreated or control shRNA,
and XBP1 shRNA treated MDA-MB-436 cells. **p<0.01.
[0032] (M) Tumor growth (mean.+-.SD) of untreated or control shRNA,
and XBP1 shRNA treated HBL-100 cells. **p<0.01. TX: treatment
with shRNA.
[0033] FIG. 4. XBP1 is required to sustain cancer stem cell
self-renewal
[0034] (A) RT-PCR analysis of XBP1 splicing in untreated and TAM
treated NTICs (CD44.sup.low/CD24.sup.high) and TICs
(CD44.sup.high/CD24.sup.low). XBP1u: unspliced XBP1, XBP1s: spliced
XBP1.
[0035] (B) Flow cytometry analyzing CD44 and CD24 expression of
untreated and TAM treated (36 h) MCF10A ER-Src cells infected with
control GFP shRNA or XBP1 shRNA encoding lentivirus.
[0036] (C) Number of mammospheres per 1,000 cells generated by TAM
treated MCF10A ER-Src cells uninfected, or infected with control
shRNA or XBP1 shRNA encoding lentivirus.
[0037] (D) The indicated number of TAM-treated MCF10A-ER-Src cells
infected with control shRNA or XBP1 shRNA were injected into
NOD/SCID/IL2Ra-/- mice and the tumor incidence was reported at 12
weeks post-transplantation.
[0038] (E) RT-PCR analysis of XBP1 splicing in NTICs and TICs
purified from TNBC patient. XBP1u: unspliced XBP1, XBP1s: spliced
XBP1.
[0039] (F) Number of mammospheres per 1,000 cells generated from
untreated and control shRNA or XBP1 shRNA encoding lentivirus
infected primary tissue samples from five patients with TNBC
[0040] (G) 10 NTIC sorted from two human TNBC patients or NTIC
overexpressing XBP1s were injected into NOD/SCID/IL2R.gamma.-/-
mice and the incidence of tumors was monitored.
[0041] (H) Knockdown efficiency of XBP1 in MCF10A-ER-Src cells.
[0042] (I) Percentage of TICs (CD44high/CD24low) in TAM treated
MCF10A-ER-Src cells infected with control shRNA or XBP1 shRNA
encoding lentivirus.
[0043] (J) Cell viability assay (Cell-titer Glo) on TICs
(CD44high/CD24low) isolated from transformed MCF10A-ER-Src cells
infected with control shRNA or XBP1 shRNA encoding lentivirus (72 h
after infection). Data were normalized to the control (cell
infected with shCtrl). Experiments were performed in triplicate and
data are shown as mean.+-.SD.
[0044] (K) Cell viability assay (Cell-titer Glo) on NTICs
(CD44low/CD24high). Data analysis is the same as (J).
[0045] (L) Tumor growth (mean.+-.SD) of MDA-MB-231 cells untreated
or treated with doxorubicin, or doxorubicin (dox)+control shRNA, or
doxorubicin+XBP1 shRNA. TX: treatment with Dox or Dox+shRNA.
[0046] (M) Number of mammospheres per 1,000 cells generated from
day 20 xenograft tumors under different treatments as indicated.
Data are shown as mean.+-.SD.
[0047] FIG. 5. XBP1 interacts with HIF1.alpha. and co-occupies
promoters of HIF1.alpha. target genes.
[0048] (A) Motif enrichment analysis in the XBP1 binding sites. The
average HIF1.alpha. motif enrichment signal is shown for the 1 kb
region surrounding the center of the XBP1 binding site.
[0049] (B) FLAG-tagged HIF1.alpha. and XBP1s were co-expressed in
293T cells and the cells were treated in 0.1% O2 for 16 h. Co-IP
was performed with M2 anti-FLAG antibody. Western blot was carried
out with anti-XBP1s antibody or anti-FLAG antibody. Empty vector
was used as negative control.
[0050] (C) Nuclear extracts from MDA-MB-231 cells treated with TM
(1 ug/ml, 6 h) in 0.1% O2 (16 h) were subjected to co-IP with
anti-HIF1.alpha. antibody or rabbit IgG. Western blot was carried
out with anti-XBP1s antibody or anti-HIF1.alpha. antibody.
[0051] (D-F) Schematic diagram of the primer locations across the
JMJD1A promoter (D). XBP1 and HIF1.alpha. cobind to JMJD1A, DDIT4,
VEGFA, and PDK1 promoters under hypoxic conditions. A ChIP assay
was performed using anti-XBP1 polyclonal antibody (D-E) or
anti-HIF1.alpha. polyclonal antibody (D, F) to detect enriched
fragments. Fold enrichment is the relative abundance of DNA
fragments at the amplified region over a control amplified region.
GST antibody was used as mock ChIP control (D-F). Primer locations
correspond to (D).
[0052] (G) Schematic of the luciferase reporter constructs
containing three copies of HRE (3.times.HRE)
[0053] (H) 3.times.HRE reporter was co-transfected with XBP1s
expression plasmid or empty vector into MDA-MB-231 cells and
luciferase activity measured.
[0054] (I) 3.times.HRE reporter was co-transfected with doxycycline
(DOX) inducible constructs encoding two shRNAs targeting different
regions of XBP1 or scrambled LACZ control into MDA-MB-231 cells.
Cells were treated in 0.1% O.sub.2 for 24 h in the presence or
absence of doxycycline, and luciferase activity assayed. All
luciferase activity was measured relative to the renilla luciferase
internal control. Experiments were performed in triplicate and data
are shown as mean.+-.SD. *p<0.05, **p<0.01.
[0055] (J) Western blotting analysis of XBP1s expression in nuclear
extract of MDA-MB-231 cells cultured under unstressed or stressed
condition (0.1% O2 and glucose deprivation) for 16 h. Lamin B was
used as loading control.
[0056] (K) Distribution of XBP1 binding sites. Locations of XBP1
binding sites relative to the nearest tran transcription units. The
percentages of binding sites at the respective locations are
shown
[0057] (L) Identification of XBP1 motif in ChIP-seq. Matrices
predicted by the de novo motif-discovery algorithm Seqpos.
p=1.times.10.sup.-30.
[0058] (M) Nuclear extracts from Hs578T cells treated with TM (1
ug/ml, 6 h) in 0.1% O2 (16 h) were subjected to co-IP with
anti-HIF1.alpha. antibody or rabbit IgG. Western blot was carried
out with anti-XBP1s antibody or anti-HIF1.alpha. antibody.
[0059] (N) XBP1 and HIF1.alpha. co-bind to the JMJD2C promoter
under hypoxic conditions.
[0060] FIG. 6. XBP1 regulates the hypoxia response.
[0061] (A) Plot from GSEA showing enrichment of the HIF1.alpha.
mediated hypoxia response pathway in XBP1-upreuglated genes.
[0062] (B) Gene expression microarray heatmap showing that genes
involved in the HIF1.alpha. mediated hypoxia responses were
differentially expressed after XBP1 knockdown.
[0063] (C-D) Quantitative RT-PCR analysis of VEGFA, PDK1, GLUT1,
JMJD1A and DDIT4 expression after knockdown of XBP1 in MDA-MB-231
under hypoxic conditions (C) or MDA-MB-231 derived xenograft tumors
(d, n=5). Results are presented relative to .beta.-actin
expression. Experiments were performed in triplicate and data are
shown as mean.+-.SD. *p<0.05, **p<0.01.
[0064] (E) Plot showing the genome-wide association between the
strength of the XBP1 binding and the occurrence of the HIF1.alpha.
motif. The signal of XBP1 ChIP-seq peaks was shown as a heatmap
using red (the strongest signal) and white (the weakest signal)
color scheme. Each row shows .+-.300 bp centered on the XBP1
ChIP-seq peak summits. Rows are ranked by XBP1 occupancy. The
horizontal blue lines denote the presence of the HIF1.alpha.
motif.
[0065] (F-G) Chromatin extracts from control MDA-MB-231 cells or
XBP1 knockdown MDA-MB-231 cells (treated with 0.1% O.sub.2 for 24
h) were subjected to ChIP using anti-HIF1.alpha. antibody (F), and
anti-RNA polymerase II antibody (G). The primers used to detect
ChIP-enriched DNA in (F-G) were the peak pair of primers in JMJD1A,
DDIT4, NDRG1, PDK1 and VEGFA promoters (Table 2). Primers in the
.beta.-actin region/promoter were used as control. Data are
presented as the mean.+-.SD.
[0066] (H) Quantitative RT-PCR analysis of VEGFA, PDK1, GLUT1,
MCT4, JMJD1A and XBP1 expression after knockdown of XBP1 in Hs578T
cells treated with 0.1% O2 for 24 h. Results are presented relative
to .beta.-actin expression. Experiments were performed in
triplicate and data are shown as mean.+-.SD. *p<0.05,
**p<0.01.
[0067] (I) Chromatin extracts from control MDA-MB-231 cells or XBP1
knockdown MDA-MB-231 cells (treated with 0.1% O2 for 24 h) were
subjected to ChIP using anti-XBP1s antibody. Data are presented as
the mean.+-.SD.
[0068] (J) Immunoblotting analysis of control MDA-MB-231 cell
lysates and XBP1 knockdown lysates (treated with 0.1% O2 for 24 h)
were performed using anti-HIF1.alpha. or anti-HSP90 antibody.
[0069] FIG. 7. XBP1 genetic signature is associated with human
breast cancer prognosis.
[0070] (A) Heatmap showing the expression profile of genes bound by
XBP1 and differentially expressed after XBP1 knockdown
[0071] (B-C) Kaplan-Meier graphs demonstrating a significant
association elevated expression of the XBP1 signature with shorter
relapse-free survival in two cohorts of triple negative breast
cancer patients (B and C). The log-rank test P values are
shown.
[0072] (D). Kaplan-Meier graphs showing the significant association
of expression of HIF1.alpha. gene signature with shorter
relapse-free survival in a cohort of 383 TNBC patients. The
log-rank test P values are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The unfolded protein response (UPR) is essential for tumor
cells to survive the pathologic stresses intrinsic to the tumor
microenvironment. Here, it is reported an unexpected function of
XBP1 (X box binding protein1), a key component of the UPR, in human
triple negative breast cancer (TNBC). It is shown that XBP1 is
required for the transformation of immortalized mammary epithelial
cells. Silencing of XBP1 significantly suppressed the growth and
invasiveness of TNBCs. Activation of the XBP1 pathway is associated
with poor prognosis in human TNBC patients. Intriguingly, XBP1 is
preferentially activated in tumor initiating cells (TICs) and is
essential for sustaining TIC self-renewal. Moreover, overexpression
of the active form of XBP1 in non-TICs is sufficient to confer
stem-like properties on them, while depletion of XBP1 inhibited
tumor relapse due to a preferential depletion of TICs. Genome-wide
mapping of the XBP1 transcriptional regulatory network revealed
that XBP1 regulates the hypoxia response through controlling
HIF1.alpha. transcriptional activity and the expression of
HIF1.alpha. targets. The instant inventors have identified a
genetic fingerprint indicative of XBP1 pathway activation that is
associated with poor prognosis in human TNBC patients. These
findings, for the first time, link the UPR pathway with TNBC and
TIC, opening new avenues for therapeutics for TNBC patients.
[0074] Accordingly, in one aspect, the invention pertains to a
method of inhibiting growth of triple negative breast cancer (TNBC)
in a subject, the method comprising administering to the subject a
direct or indirect inhibitor of XBP1 such that growth of the TNBC
in the subject is inhibited. Non-limiting examples of direct
inhibitors of XBP1 include a nucleic acid molecule that is
antisense to an XBP1-encoding nucleic acid molecule, an XBP1 shRNA,
an XBP siRNA, a microRNA that targets XBP1, a dominant negative
XBP1 molecule and small molecule inhibitors of XBP1. Non-limiting
examples of indirect inhibitors of XBP1 include agents that target
IRE1, an endonuclease essential for proper splicing and activation
of XBP1, such that inhibition of IRE1 leads to inhibition of the
production of the spliced, active form of XBP1. Non-limiting
examples of IRE1 inhibitors include a nucleic acid molecule that is
antisense to an IRE1-encoding nucleic acid molecule, an IRE1 shRNA,
an IRE1 siRNA, a microRNA that targets IRE1, a dominant negative
IRE1 molecule and small molecule inhibitors of IRE1.
[0075] In another aspect, the invention pertains to a method of
identifying a compound useful in inhibiting the growth of triple
negative breast cancer (TNBC) cells, the method comprising:
[0076] a) providing an indicator composition comprising XBP1 and
HIF1.alpha., or biologically active portions thereof;
[0077] b) contacting the indicator composition with each member of
a library of test compounds;
[0078] c) selecting from the library of test compounds a compound
of interest that decreases the interaction of XBP1 and HIF1.alpha.,
or biologically active portions thereof, wherein the ability of a
compound to inhibit growth of TNBC cells is indicated by a decrease
in the interaction as compared to the amount of interaction in the
absence of the compound.
[0079] The indicator composition can be, for example, a cell-free
preparation comprising XBP1 and HIF1 cc, or biologically active
portions thereof (e.g., isolated recombinant proteins), or a cell
comprising XBP1 and HIF1.alpha., or biologically active portions
thereof (e.g., a recombinant cell transfected to express XBP1 and
HIF1.alpha. proteins). The read-out for the method to determine the
amount of interaction between XBP1 and HIF1.alpha. can be, for
example, a direct read-out that measures the amount of binding
between XBP1 and HIF1.alpha. (e.g., one or both proteins can be
labeled or tagged), such as co-immunnoprecipitation, or an indirect
read-out that measures the amount of transcriptional activity of
the XBP1/HIF1.alpha. complex, such as use of a reporter gene
responsive to the XBP1/HIF1.alpha. complex and measurement of the
level of the reporter.
[0080] In yet another aspect, the invention pertains to a method
for determining a prognosis status for a subject with triple
negative breast cancer (TNBC), the method comprising:
[0081] a) determining an XBP1 gene signature for the TNBC of the
subject; and
[0082] b) correlating the XBP1 gene signature with a prognosis
status for the subject, wherein higher expression of the XBP1 gene
signature, relative to a control, correlates with shorter
relapse-free survival of the subject and lower expression of the
XBP1 gene signature, relative to a control, correlates with longer
relapse-free survival of the subject.
[0083] The XBP1 gene signature can comprise, for example, a
plurality of genes regulated by XBP1 in TNBC, such as a plurality
of genes selected from the 133 genes shown in Table 1.
[0084] The contents of all references, patents, and published
patent applications cited throughout this application, as well as
the figures and the sequence listing, are hereby incorporated by
reference.
[0085] Various aspects of the invention are described in further
detail in the following subsections:
[0086] I. XBP1 and Triple Negative Breast Cancer
[0087] During tumor development and progression, cancer cells
encounter cytotoxic conditions such as hypoxia, nutrient
deprivation, and low pH due to inadequate vascularization (Hanahan,
D., et al. 2011. Cell 144, 646-74). To maintain survival and growth
in the face of these physiologic stressors, a set of adaptive
response pathways are induced. One adaptive pathway well studied in
other contexts is the unfolded protein response (UPR), which is
induced by factors affecting the endoplasmic reticulum (ER) such as
changes in glycosylation, redox status, glucose availability,
calcium homeostasis or the accumulation of unfolded or misfolded
proteins (Hetz, C., et al. 2011. Physiol Rev 91, 1219-43). Notably,
features of the tumor microenvironment, such as hypoxia and
nutrient deprivation, can disrupt ER homeostasis by the
perturbation of aerobic processes such as oligosaccharide
modification, disulphide bond formation, isomerization, and protein
quality control and export (Wouters, B. G., et al. 2008. Nat Rev
Cancer 8, 851-64). In mammalian cells, the UPR is mediated by three
ER-localized transmembrane protein sensors: Inositol-requiring
transmembrane kinase/endonuclease-1 (IRE1), PKR-like ER kinase
(PERK) and activating transcription factor 6 (ATF6) (Walter, P., et
al. 2011. Science 334, 1081-6). Of these, IRE1 is the most
evolutionarily conserved branch. An increase in the load of folding
proteins in the ER activates IRE1, an ER-resident kinase and
endoribonuclease that acts as an ER-stress sensor4. Activated IRE1
removes a 26 bp intron from XBP1 mRNA and results in a frame shift
in the coding sequence, with the spliced form encoding a 226 amino
acid transcriptional activation domain (Calfon, M., et al. 2002.
Nature 415, 92-6; Yoshida, H., et al. 2001. Cell 107, 881-91). In
contrast to the unspliced XBP1 (XBP1u), which is unstable and
quickly degraded, spliced XBP1 (XBP1s) is stable and is a potent
inducer of target genes that orchestrate the cellular response to
ER stress (Hetz, C., et al. 2011. Physiol Rev 91, 1219-43). Several
studies have reported on the activation of the UPR in various human
tumors and its relevance to combinatorial therapy (Ma, Y., et al.
2004. Nat Rev Cancer 4, 966-77; De Raedt, T., et al. 2011. Cancer
Cell 20, 400-13; Mahoney, D. J., et al. 2011. Cancer Cell 20,
443-56; Healy, S. J., et al. 2009. Eur J Pharmacol 625, 234-46;
Carrasco, D. R., et al. 2007. Cancer Cell 11, 349-60). However, the
role of the UPR and XBP1 in the malignant mammary cell is largely
unknown.
[0088] As described in detail above, the UPR is a major cellular
stress response pathway activated in tumors that allows them to
adapt to the stresses of the tumor microenvironment. Several
studies have reported on the activation of the UPR in various human
tumors and its relevance to combinatorial therapy (Carrasco, D. R.,
et al. 2007. Cancer Cell 11, 349-36; De Raedt, T., et al. 2011.
Cancer Cell 20, 400-413; Healy, S. J., et al. 2009. Eur J Pharmacol
625, 234-246; Ma, Y., et al. 2004. Nat Rev Cancer 4, 966-977;
Mahoney, D. J., et al. 2011. Cancer Cell 20, 443-456). However, the
role of the UPR in breast cancer pathogenesis remains elusive.
Here, the instant inventors have identified a previously unknown
function of XBP1 in triple-negative breast cancer (TNBC). It is
demonstrated that XBP1 is spliced and activated in TNBC, and that
deletion of XBP1 significantly blocks triple negative breast tumor
growth. Here, it is demonstrates that XBP1, a key component of the
most evolutionarily conserved branch of the UPR, is essential for
the transformation of mammary epithelial cells and is
preferentially activated in tumor initiating cells (TICs) where it
is essential for sustaining TIC self-renewal. Furthermore, XBP1
silencing suppressed tumor relapse along with depleting the breast
tumor initiating cells (TICs). Genome-wide mapping of the XBP1
transcriptional regulatory network identified its key downstream
target to be the hypoxia response via the transcription factor
hypoxia-inducible factor 1.alpha. (HIF1.alpha.). XBP1 regulates
HIF1.alpha. transcriptional activity by controlling HIF1.alpha.
binding to promoter DNA and by the recruitment of RNA polymerase
II. We generated a genetic fingerprint indicative of XBP1 pathway
activation that we found to be associated with poor prognosis in
human TNBC patients. Moreover, activation of the hypoxia response
pathway appears to carry prognostic implications, as expression of
the XBP1-dependent signature is associated with shorter survival
times in patients with TNBC.
[0089] XBP1 was reported to be highly expressed in ER+ breast
tumors and to activate ER.alpha. in a ligand-independent manner
(Ding, L., et al. 2003. Nucleic Acids Res 31, 5266-5274; Fujimoto,
T., et al. 2003. Breast Cancer 10, 301-306). Splicing of XBP1
confers estrogen independence and anti-estrogen resistance to
breast cancer cell lines (Gomez, B. P., et al. 2007. Faseb J 21,
4013-4027). Here, by manipulating the expression of XBP1 in a panel
of breast cancer cell lines and in a human xenograft model, we
discovered a key function for XBP1 in TNBC. TNBC is a subtype of
breast tumors characterized by a of the absence of expression of
ER, PR and HER2, signaling receptors known to fuel most breast
cancers. TNBC is extremely aggressive and more likely to recur and
metastasize than the other subtypes (Foulkes, W. D., et al. 2010. N
Engl J Med 363, 1938-1948). While ER+, PR+ or Her2 tumors respond
well to ER antagonist, aromatase inhibitor, or Her2-targeted
therapies, TNBC is unresponsive to most receptor targeted
treatments. TNBC is a highly heterogeneous group of cancers, the
genes linked to TNBC are not well understood and thus, targeted
therapies do not yet exist. We found that XBP1 was preferentially
activated in TNBC cells, and that silencing of XBP1 was very
effective in suppressing the tumorigenicity and progression of
TNBCs.
[0090] A TNBC
[0091] Triple-negative breast cancer (TNBC) refers to any breast
cancer that does not express the genes for estrogen receptor (ER),
progesterone receptor (PR) or Her2/neu. Triple negative is
sometimes used as a surrogate term for basal-like; however, more
detailed classification may provide better guidance for treatment
and better estimates for prognosis. (Hudis, C. A., et al. 2011. The
Oncologist 16, 1-11). Triple-negative breast cancer (TNBC) is
breast cancer characterized by malignant tumors. As used herein,
the term "malignant" refers to a non-benign tumor or a cancer. In
one embodiment a malignancy expands to other parts of the body as
well (metastasizes). A malignant tumor is usually life-threatening,
causing death if it remains untreated. If treated, the spread of a
malignant tumor can be slowed or even arrested. Depending on the
amount of tissue damage prior to treatment, tissue or organ
function can be compromised.
[0092] Triple negative breast cancers have a relapse pattern that
is very different from hormone-positive breast cancers: the risk of
relapse is much higher for the first 3-5 years but drops sharply
and substantially below that of hormone-positive breast cancers
after that. This relapse pattern has been recognized for all types
of triple negative cancers for which sufficient data exists
although the absolute relapse and survival rates differ across
subtypes. (Hudis, C. A., et al. 2011. The Oncologist 16, 1-11;
Cheang, M. C. U., et al. 2008. Clinical Cancer Research 14 (5),
1368-1376).
[0093] Triple-negative breast cancers are sometimes classified into
"basal-type" and other cancers; however, there is no standard
classification scheme. Basal type cancers are frequently defined by
cytokeratin 5/6 and EGFR staining. However no clear criteria or
cutoff values have been standardized yet. (Hudis, C. A., et al.
(2011). The Oncologist 16, 1-11). About 75% of basal-type breast
cancers are triple negative. Some TNBC overexpresses epidermal
growth factor receptor (EGFR). Some TNBC over expresses
transmembrane glycoprotein NMB (GPNMB). On histologic examination
triple negative breast tumors mostly fall into the categories
secretory carcinoma or adenoid cystic types (both considered less
aggressive), medullary cancers and grade 3 invasive ductal
carcinomas with no specific subtype, and highly aggressive
metastatic cancers. (Hudis, C. A. et al. 2011. The Oncologist 16,
1-11). Medullary TNBC in younger women are frequently
BRCA1-related. Rare forms of triple negative breast cancer are
apocrine and squamous carcinoma. Inflammatory breast cancer is also
frequently triple negative.
[0094] B. UPR
[0095] The term "Unfolded Protein Response" (UPR) or the "Unfolded
Protein Response pathway" refers to an adaptive response to the
accumulation of unfolded proteins in the ER and includes the
transcriptional activation of genes encoding chaperones and folding
catalysts and protein degrading complexes as well as translational
attenuation to limit further accumulation of unfolded proteins.
Both surface and secreted proteins are synthesized in the
endoplasmic reticulum (ER) where they need to fold and assemble
prior to being transported.
[0096] Since the ER and the nucleus are located in separate
compartments of the cell, the unfolded protein signal must be
sensed in the lumen of the ER and transferred across the ER
membrane and be received by the transcription machinery in the
nucleus. The unfolded protein response (UPR) performs this function
for the cell. Activation of the UPR can be caused by treatment of
cells with reducing agents like DTT, by inhibitors of core
glycosylation like tunicamycin or by Ca-ionophores that deplete the
ER calcium stores. First discovered in yeast, the UPR has now been
described in C. elegans as well as in mammalian cells. In mammals,
the UPR signal cascade is mediated by three types of ER
transmembrane proteins: the protein-kinase and site-specific
endoribonuclease IRE-1; the eukaryotic translation initiation
factor 2 kinase, PERK/PEK; and the transcriptional activator ATF6.
If the UPR cannot adapt to the presence of unfolded proteins in the
ER, an apoptotic response is initiated leading to the activation of
JNK protein kinase and caspases 7, 12, and 3. The most proximal
signal from the lumen of the ER is received by a transmembrane
endoribonuclease and kinase called IRE-1. Following ER stress,
IRE-1 is essential for survival because it initiates splicing of
the XBP-1 mRNA the spliced version of which, as shown herein,
activates the UPR.
[0097] C. XBP1
[0098] The term "XBP-1" refers to a X-box binding human protein
that is a DNA binding protein and has an amino acid sequence as
described in, for example, Liou, H. C., et. al. 1990. Science 247,
1581-1584 and Yoshimura, T., et al. 1990. EMBO J. 9, 2537-2542, and
other mammalian homologs thereof, such as described in Kishimoto
T., et al. 1996. Biochem. Biophys. Res. Commun. 223, 746-751 (rat
homologue). Exemplary proteins intended to be encompassed by the
term "XBP-1" include those having amino acid sequences disclosed in
GenBank with accession numbers A36299 [gi:105867], NP.sub.--005071
[gi:4827058], P17861 [gi:139787], CAA39149 [gi:287645], and
BAA82600 [gi:5596360] or e.g., encoded by nucleic acid molecules
such as those disclosed in GenBank with accession numbers AF027963
[gi: 13752783]; NM.sub.--013842 [gi:13775155]; or M31627
[gi:184485]. XBP-1 is also referred to in the art as TREB5 or HTF
(Yoshimura, T., et al. 1990. EMBO Journal. 9, 2537; Matsuzaki, Y.,
et al. 1995. J. Biochem. 117, 303). Like other members of the b-zip
family, XBP-1 has a basic region that mediates DNA-binding and an
adjacent leucine zipper structure that mediates protein
dimerization.
[0099] As described above, there are two forms of XBP-1 protein,
unspliced and spliced, which differ markedly in their sequence and
activity. Unless the form is referred to explicitly herein, the
term "XBP-1" as used herein includes both the spliced and unspliced
forms. Spliced XBP-1 ("XBP1s") directly controls the activation of
the UPR, while unspliced XBP-1 functions due to its ability to
negatively regulate spliced XBP-1.
[0100] As used herein, the term "spliced XBP-1" ("XBP1s") refers to
the spliced, processed form of the mammalian XBP-1 mRNA or the
corresponding protein. Human and murine XBP-1 mRNA contain an open
reading frame (ORF1) encoding bZIP proteins of 261 and 267 amino
acids, respectively. Both mRNA's also contain another ORF, ORF2,
partially overlapping but not in frame with ORF1. ORF2 encodes 222
amino acids in both human and murine cells. Human and murine ORF1
and ORF2 in the XBP-1 mRNA share 75% and 89% identity
respectively.
[0101] As used herein, the term "unspliced XBP-1" refers to the
unprocessed XBP-1 mRNA or the corresponding protein. As set forth
above, unspliced murineXBP-1 is 267 amino acids in length and
spliced murine XBP-1 is 371 amino acids in length. The sequence of
unspliced XBP-1 is known in the art and can be found, e.g., Liou,
H. C., et. al. 1990. Science 247, 1581-1584 and Yoshimura, T., et
al. 1990. EMBO J. 9, 2537-2542, or at GenBank accession numbers
NM.sub.--005080 [gi:14110394] or NM.sub.--013842 [gi:13775155].
[0102] II. XBP1 and Tumor Initiating Cells
[0103] TNBC typically contain a higher proportion of "stem-like"
breast cancer cells, also known as tumor initiating cells (TICs),
characterized by a CD44.sup.+CD24.sup.-/low surface phenotype of
and the expression of aldehyde dehydrogenase 1 (Al-Hajj, M., et al.
2003. Proc Natl Acad Sci USA 100, 3983-3988; Ginestier, C., et al.
2007. Cell Stem Cell 1, 555-567). TICs resemble stem cells, as they
are capable of both indefinite self-renewal and differentiation.
Relative to NTICs, TICs contribute to a significantly higher
incidence of recurrence and distant metastasis, and are responsible
for tumor initiation and maintenance (Smalley, M., et al. 2003. Nat
Rev Cancer 3, 832-844; Stingl, J., et al. 2007. Nat Rev Cancer 7,
791-799). Although conventional therapies have shown great promise
in killing the bulk of differentiated tumor cells, TICs are
resistant to chemotherapy (Stingl, J., et al. 2007. Nat Rev Cancer
7, 791-799). The development of effective therapies targeting the
TIC is urgently needed to treat breast cancer metastasis and
relapse. Although several self-renewal regulatory pathways
including the Notch, Wnt and Hedgehog pathways (Visvader, J. E., et
al. 2008. Nat Rev Cancer 8, 755-768), as well as microenvironmental
stress, such as hypoxia (Keith, B., et al. 2007. Cell 129, 465-472;
Schwab, L. P., et al. 2012. Breast Cancer Res 14, R6), are known to
be essential in promoting a stem-like phenotype, progress in
targeting TICs with novel therapeutics is still hindered by our
incomplete knowledge of the molecular pathways contributing to TIC
identity.
[0104] Here we have demonstrated that XBP1 is essential for the
self-renewal of breast TICs. In support of this claim, we showed
that XBP1 was selectively activated in TICs, XBP1 inhibition
blocked the formation of TICs, and depletion of XBP1 greatly
suppressed the growth of mammospheres derived from human TNBC
patients and various breast cancer cell lines, a key measure of TIC
function. Overexpression of XBP1s in non-TICs conferred stem-like
traits and tumorigenic potential at very low dilutions (10 cells).
Finally, XBP1 depletion in combination with chemotherapy blocked
xenograft tumor growth and relapse, which was attributed to the
decreased TIC population after combinatorial treatment. Ours is the
first study to demonstrate that compromising the ER stress response
significantly impairs TIC growth and self-renewal. We speculate
that the rapid proliferation of TICs requires robust ER protein
folding, assembly, and transport, functions which rely on XBP1
activation and which are compromised in its absence. XBP1 serves as
one of the major cellular adaptive mechanisms activated to protect
TICs in a non-dividing dormant state, and XBP1 confers on TICs
growth and survival advantages over non-TICs. The specific
acquisition of XBP1 activation in TICs is intriguing and provides
new insights into pathways that may be used to target this
subpopulation of cancer cells.
[0105] III. XBP1 Regulates the Hypoxia Response Through
HIF1.alpha.
[0106] Hypoxia is known to promote aggressive tumor phenotypes. A
growing body of evidence indicates that hypoxia is required for TIC
survival and tumor propagation in glioma, lymphoma and acute
myeloid leukemia (Heddleston, J. M., et al. 2009. Br J Cancer 102,
789-795; Jogi, A., et al. 2002. Proc Natl Acad Sci USA 99,
7021-7026; Li., Z., et al. 2009. Cancer Cell 15, 501-513; Wang, Y.,
et al. 2011. Cell Stem Cell 8, 399-411). HIF transcription factors
are crucial to the maintenance of the undifferentiated state of
stem cells residing in hypoxic niches. TNBCs also display increased
levels of hypoxia (Rakha, E. A., et al. 2009. Clin Cancer Res 15,
2302-2310; Tan, E. Y., et al. 2009. Br J Cancer 100, 405-411) and
HIF1.alpha. was recently demonstrated to be essential for their
maintenance of breast TICs. HIF1.alpha. promotes expansion of
breast TICs in vivo, and deletion of HIF 1.alpha. results in
reduced mammosphere formation, primary breast tumor growth and
pulmonary metastases in the MMTV-PyVT breast cancer mouse model
(Schwab, L. P., et al. 2012. Breast Cancer Res 14, R6). Increased
HIF1.alpha. levels are also associated with increased metastasis
and decreased survival in patients with breast cancer (Bos, R., et
al. 2003. Cancer 97, 1573-1581; Semenza, G. L., 2010. Cell 107,
1-3).
[0107] Our data reveal that XBP1 acts in breast TICs and TNBC
through regulating the response to hypoxia. HIF1.alpha. requires
XBP1 to sustain downstream target expression under hypoxic
conditions. XBP1 interacts with HIF1.alpha. to co-occupy a set of,
if not all, HIF1.alpha. targets. Depletion of XBP1 leads to
reduction in classic HIF1.alpha. targets expression and HRE
activity by blocking HIF1.alpha. binding to its target genes, which
subsequently affects the recruitment of RNA polymerase II to target
promoters. Hypoxia is a physiological inducer of the UPR in cancer
(Wouters, B. G., et al. 2008. Nat Rev Cancer 8, 851-864). In this
study, we found that XBP1 functions in a positive feedback loop to
sustain the hypoxia response via regulating HIF1.alpha.
transcriptional activity. This feed-forward circuit ensures maximum
HIF activity and an efficient adaptive response to the cytotoxic
microenvironment of solid tumors. HIF activity is tightly
controlled during tumor progression, through translational and
post-translational regulation of HIF1.alpha. but relatively little
is known about how HIF1.alpha. transcriptional activity is
controlled (Kaelin, W. G., Jr., et al. 2008. Mol Cell 30, 393-402).
Our study reveals an unexpected function for XBP1 as a HIF1.alpha.
transcriptional cofactor. We propose a model in which these two
critical pathways, the UPR and the hypoxia response, are physically
interconnected and act together to mount an appropriate adaptive
response that promotes the survival of TICs in the hostile tumor
microenvironment
[0108] IV. Therapeutic Targeting of the UPR in TNBC
[0109] We have highlighted the importance of the IRE1/XBP1 pathway
in TNBC growth and metastasis, in part through regulating TICs.
XBP1s expression is directly correlated with poor patient survival
in human TNBC patients. Strikingly, while XBP1 is selectively
activated in rapidly growing TICs, UPR pathways remain in a
quiescent state in most normal unstressed cells. Hence inhibition
of the UPR may offer a means to exclusively target tumor cells.
[0110] XBP1 is a transcription factor, and traditionally
transcription factors other than hormone receptors have been
difficult to target with small molecules. However, the upstream
kinase and endoribonuclease IRE1, which drives the splicing of XBP1
mRNA, is a viable drug target. Recently, two groups have identified
specific IRE1 endoribonuclease inhibitors (Papandreou, I., et al.
2011. Blood 117, 1311-1314; Volkmann, K., et al. 2011. J Biol Chem
286, 12743-12755). Intriguingly, these compounds efficiently
inhibit XBP1 splicing in vivo and dramatically impair tumor growth
in a xenograft model (Mahoney, D. J., et al. 2011. Cancer Cell 20,
443-456; Papandreou, I., et al. 2011. Blood 117, 1311-1314;
Volkmann, K., et al. 2011. J Biol Chem 286, 12743-12755). While
large-scale small molecule screens have provided potentially
promising candidates that target the IRE1/XBP1 pathway, attention
needs to be paid to the specificity and cytotoxity of these
compounds in vivo. Recent advances in solving the crystal structure
of IRE1 (Korennykh, A. V., et al. 2009. Nature 457, 687-693; Lee,
K. P., et al. 2008. Cell 132, 89-100; Zhou, J., et al. 2006. Proc
Natl Acad Sci USA 103, 14343-14348) should accelerate the design of
more potent and specific IRE1 inhibitors. The use of UPR inhibitors
in combination with standard chemotherapy may greatly enhance the
effectiveness of anti-tumor therapies.
[0111] The methods of the invention using inhibitory compounds
which inhibit the expression, processing, post-translational
modification, or activity of spliced XBP-1 or a molecule in a
biological pathway involving XBP-1 can be used in the treatment of
TNBC. In one embodiment of the invention, an inhibitory compound
can be used to inhibit (e.g., specifically inhibit) the expression,
processing, post-translational modification, or activity of spliced
XBP-1. In another embodiment, an inhibitory compound can be used to
inhibit (e.g., specifically inhibit) the expression, processing,
post-translational modification, or activity of unspliced
XBP-1.
[0112] Inhibitory compounds of the invention can be, for example,
intracellular binding molecules that act to specifically or
directly inhibit the expression, processing, post-translational
modification, or activity e.g., of XBP-1 or a molecule in a
biological pathway involving XBP-1 (e.g., HIF1.alpha.). As used
herein, the term "intracellular binding molecule" is intended to
include molecules that act intracellularly to inhibit the
processing expression or activity of a protein by binding to the
protein or to a nucleic acid (e.g., an mRNA molecule) that encodes
the protein. Examples of intracellular binding molecules, described
in further detail below, include antisense nucleic acids,
intracellular antibodies, peptidic compounds that inhibit the
interaction of XBP-1 or a molecule in a biological pathway
involving XBP-1 and a target molecule (e.g., HIF1.alpha.), and
chemical agents that specifically or directly inhibit XBP-1
activity or the activity of a molecule in a biological pathway
involving XBP-1 (e.g., HIF1.alpha.).
[0113] In one embodiment, an inhibitory compound of the invention
is an antisense nucleic acid molecule that is complementary to a
gene encoding XBP-1 or a molecule in a signal transduction pathway
involving XBP-1, e.g., a molecule with which XBP-1 interacts), or
to a portion of said gene, or a recombinant expression vector
encoding said antisense nucleic acid molecule. The use of antisense
nucleic acids to downregulate the expression of a particular
protein in a cell is well known in the art (see e.g., Weintraub,
H., et al 1986. Reviews--Trends in Genetics, Vol. 1(1); Askari, F.
K., et al 1996. N. Eng. J. Med. 334, 316-318; Bennett, M. R., et
al. 1995. Circulation 92, 1981-1993; Mercola, D., et al. 1995.
Cancer Gene Ther. 2, 47-59; Rossi, J. J., 1995. Br. Med. Bull. 51,
217-225; Wagner, R. W., 1994. Nature 372, 333-335). An antisense
nucleic acid molecule comprises a nucleotide sequence that is
complementary to the coding strand of another nucleic acid molecule
(e.g., an mRNA sequence) and accordingly is capable of hydrogen
bonding to the coding strand of the other nucleic acid molecule.
Antisense sequences complementary to a sequence of an mRNA can be
complementary to a sequence found in the coding region of the mRNA,
the 5' or 3' untranslated region of the mRNA or a region bridging
the coding region and an untranslated region (e.g., at the junction
of the 5' untranslated region and the coding region). Furthermore,
an antisense nucleic acid can be complementary in sequence to a
regulatory region of the gene encoding the mRNA, for instance a
transcription initiation sequence or regulatory element.
Preferably, an antisense nucleic acid is designed so as to be
complementary to a region preceding or spanning the initiation
codon on the coding strand or in the 3' untranslated region of an
mRNA. Given the known nucleotide sequence for the coding strand of
the XBP-1 gene and thus the known sequence of the XBP-1 mRNA,
antisense nucleic acids of the invention can be designed according
to the rules of Watson and Crick base pairing. For example, the
antisense oligonucleotide can be complementary to the region
surrounding the translation start site of an XBP-1 An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid
of the invention can be constructed using chemical synthesis and
enzymatic ligation reactions using procedures known in the art. To
inhibit expression in cells, one or more antisense oligonucleotides
can be used.
[0114] Alternatively, an antisense nucleic acid can be produced
biologically using an expression vector into which all or a portion
of a cDNA has been subcloned in an antisense orientation (i.e.,
nucleic acid transcribed from the inserted nucleic acid will be of
an antisense orientation to a target nucleic acid of interest). The
antisense expression vector can be in the form of, for example, a
recombinant plasmid, phagemid or attenuated virus. The antisense
expression vector can be introduced into cells using a standard
transfection technique.
[0115] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a protein to thereby inhibit expression of the protein,
e.g., by inhibiting transcription and/or translation. An example of
a route of administration of an antisense nucleic acid molecule of
the invention includes direct injection at a tissue site.
Alternatively, an antisense nucleic acid molecule can be modified
to target selected cells and then administered systemically. For
example, for systemic administration, an antisense molecule can be
modified such that it specifically binds to a receptor or an
antigen expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecule to a peptide or an antibody which
binds to a cell surface receptor or antigen. The antisense nucleic
acid molecule can also be delivered to cells using the vectors
described herein.
[0116] In yet another embodiment, an antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gautier, C., et al. 1987. Nucleic Acids. Res. 15, 6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue, H., et al. 1987. Nucleic Acids
Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue, H., et
al. 1987. FEBS Lett. 215, 327-330).
[0117] In still another embodiment, an antisense nucleic acid
molecule of the invention is a ribozyme. Ribozymes are catalytic
RNA molecules with ribonuclease activity which are capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which
they have a complementary region. Thus, ribozymes (e.g., hammerhead
ribozymes (described in Haselhoff, J., et al. 1988. Nature 334,
585-591)) can be used to catalytically cleave mRNA transcripts to
thereby inhibit translation mRNAs. Alternatively, gene expression
can be inhibited by targeting nucleotide sequences complementary to
the regulatory region of a gene (e.g., an XBP-1 promoter and/or
enhancer) to form triple helical structures that prevent
transcription of a gene in target cells. See generally, Helene, C.,
1991. Anticancer Drug Des. 6(6), 569-84; Helene, C., et al. 1992.
Ann. N.Y. Acad. Sci. 660, 27-36; and Maher, L. J., 1992. Bioassays
14(12), 807-15.
[0118] In another embodiment, a compound that promotes RNAi can be
used to inhibit expression of XBP-1 or a molecule in a biological
pathway involving XBP-1. The term "RNA interference" or "RNAi", as
used herein, refers generally to a sequence-specific or selective
process by which a target molecule (e.g., a target gene, protein or
RNA) is downregulated. In specific embodiments, the process of "RNA
interference" or "RNAi" features degradation of RNA molecules,
e.g., RNA molecules within a cell, said degradation being triggered
by an RNA agent. Degradation is catalyzed by an enzymatic,
RNA-induced silencing complex (RISC). RNAi occurs in cells
naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi
proceeds via fragments cleaved from free dsRNA which direct the
degradative mechanism to other similar RNA sequences.
Alternatively, RNAi can be initiated by the hand of man, for
example, to silence the expression of target genes. RNA
interference (RNAi is a post-transcriptional, targeted
gene-silencing technique that uses double-stranded RNA (dsRNA) to
degrade messenger RNA (mRNA) containing the same sequence as the
dsRNA (Sharp, P. A., et al. 2000. Science 287, 5462:2431-3.;
Zamore, P. D., et al. 2000. Cell 101, 25-33. Tuschl, T., et al.
1999. Genes Dev. 13, 3191-3197; Cottrell T. R., et al. 2003. Trends
Microbiol. 11, 37-43; Bushman F., 2003. Mol Therapy 7, 9-10;
McManus M. T., et al. 2002. Nat Rev Genet. 3, 737-47). The process
occurs when an endogenous ribonuclease cleaves the longer dsRNA
into shorter, e.g., 21-23-nucleotide-long RNAs, termed small
interfering RNAs or siRNAs. As used herein, the term "small
interfering RNA" ("siRNA") (also referred to in the art as "short
interfering RNAs") refers to an RNA agent, preferably a
double-stranded agent, of about 10-50 nucleotides in length (the
term "nucleotides" including nucleotide analogs), preferably
between about 15-25 nucleotides in length, more preferably about
17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, the
strands optionally having overhanging ends comprising, for example
1, 2 or 3 overhanging nucleotides (or nucleotide analogs), which is
capable of directing or mediating RNA interference.
Naturally-occurring siRNAs are generated from longer dsRNA
molecules (e.g., >25 nucleotides in length) by a cell's RNAi
machinery (e.g., Dicer or a homolog thereof). The smaller RNA
segments then mediate the degradation of the target mRNA. Kits for
synthesis of RNAi are commercially available from, e.g. New England
Biolabsor Ambion. In one embodiment one or more of the chemistries
described above for use in antisense RNA can be employed in
molecules that mediate RNAi.
[0119] Alternatively, compound that promotes RNAi can be expressed
in a cell, e.g., a cell in a subject, to inhibit expression of
XBP-1 or a molecule in a biological pathway involving XBP-1. In
contrast to siRNAs, shRNAs mimic the natural precursors of micro
RNAs (miRNAs) and enter at the top of the gene silencing pathway.
For this reason, shRNAs are believed to mediate gene silencing more
efficiently by being fed through the entire natural gene silencing
pathway. The term "shRNA", as used herein, refers to an RNA agent
having a stem-loop structure, comprising a first and second region
of complementary sequence, the degree of complementarity and
orientation of the regions being sufficient such that base pairing
occurs between the regions, the first and second regions being
joined by a loop region, the loop resulting from a lack of base
pairing between nucleotides (or nucleotide analogs) within the loop
region. shRNAs may be substrates for the enzyme Dicer, and the
products of Dicer cleavage may participate in RNAi. shRNAs may be
derived from transcription of an endogenous gene encoding a shRNA,
or may be derived from transcription of an exogenous gene
introduced into a cell or organism on a vector, e.g., a plasmid
vector or a viral vector. An exogenous gene encoding an shRNA can
additionally be introduced into a cell or organism using other
methods known in the art, e.g., lipofection, nucleofection,
etc.
[0120] The requisite elements of a shRNA molecule include a first
portion and a second portion, having sufficient complementarity to
anneal or hybridize to form a duplex or double-stranded stem
portion. The two portions need not be fully or perfectly
complementary. The first and second "stem" portions are connected
by a portion having a sequence that has insufficient sequence
complementarity to anneal or hybridize to other portions of the
shRNA. This latter portion is referred to as a "loop" portion in
the shRNA molecule. The shRNA molecules are processed to generate
siRNAs. shRNAs can also include one or more bulges, i.e., extra
nucleotides that create a small nucleotide "loop" in a portion of
the stem, for example a one-, two- or three-nucleotide loop. The
stem portions can be the same length, or one portion can include an
overhang of, for example, 1-5 nucleotides.
[0121] In certain embodiments, shRNAs of the invention include the
sequences of a desired siRNA molecule described supra. In such
embodiments, shRNA precursors include in the duplex stem the 21-23
or so nucleotide sequences of the siRNA, desired to be produced in
vivo.
[0122] Another type of inhibitory compound that can be used to
inhibit the expression and/or activity of XBP-1 or a molecule in a
biological pathway involving XBP-1 (e.g., HIF.alpha.1 is an
intracellular antibody specific for said protein. The use of
intracellular antibodies to inhibit protein function in a cell is
known in the art (see e.g., Carlson, J. R., 1988. Mol. Cell. Biol.
8, 2638-2646; Biocca, S., et al. 1990. EMBO J. 9, 101-108; Werge,
T. M., et al. 1990. FEBS Letters 274, 193-198; Carlson, J. R.,
1993. Proc. Natl. Acad. Sci. USA 90, 7427-7428; Marasco, W. A., et
al. 1993. Proc. Natl. Acad. Sci. USA 90, 7889-7893; Biocca, S., et
al. 1994. Bio/Technology 12, 396-399; Chen, S. Y., et al. 1994.
Human Gene Therapy 5, 595-601; Duan, L., et al. 1994. Proc. Natl.
Acad. Sci. USA 91, 5075-5079; Chen, S. Y., et al. 1994. Proc. Natl.
Acad. Sci. USA 91, 5932-5936; Beerli, R. R., et al. 1994. J. Biol.
Chem. 269, 23931-23936; Beerli, R. R., et al. 1994. Biochem.
Biophys. Res. Commun. 204, 666-672; Mhashilkar, A. M., et al. 1995.
EMBO J. 14, 1542-1551; Richardson, J. H., et al. 1995. Proc. Natl.
Acad. Sci. USA 92, 3137-3141; PCT Publication No. WO 94/02610 by
Marasco et al.; and PCT Publication No. WO 95/03832 by Duan et
al.).
[0123] To inhibit protein activity using an intracellular antibody,
a recombinant expression vector is prepared which encodes the
antibody chains in a form such that, upon introduction of the
vector into a cell, the antibody chains are expressed as a
functional antibody in an intracellular compartment of the cell.
For inhibition of transcription factor activity according to the
methods of the invention (e.g., inhibition of HIF.alpha.1,
preferably an intracellular antibody that specifically binds the
protein is expressed within the nucleus of the cell. Nuclear
expression of an intracellular antibody can be accomplished by
removing from the antibody light and heavy chain genes those
nucleotide sequences that encode the N-terminal hydrophobic leader
sequences and adding nucleotide sequences encoding a nuclear
localization signal at either the N- or C-terminus of the light and
heavy chain genes (see e.g., Biocca, S., et al. 1990. EMBO J. 9,
101-108; Mhashilkar, A. M., et al. 1995. EMBO J. 14, 1542-1551). A
preferred nuclear localization signal to be used for nuclear
targeting of the intracellular antibody chains is the nuclear
localization signal of SV40 Large T antigen (see Biocca, S., et al.
1990. EMBO J. 9, 101-108; Mhashilkar, A. M., et al. 1995. EMBO J.
14, 1542-1551).
[0124] In another embodiment, an inhibitory compound of the
invention is a peptidic compound derived from the XBP-1 amino acid
sequence or the amino acid sequence of a molecule in a biologicalon
pathway involving XBP-1 (e.g., HIF.alpha.1). For example, in one
embodiment, the inhibitory compound comprises a portion of, e.g.,
XBP-1 or HIF.alpha.1 (or a mimetic thereof) that mediates
interaction of XBP-1, for example, with HIF1.alpha. such that
contact of XBP-1 or HIF1.alpha. with this peptidic compound
competitively inhibits the interaction of XBP-1 and
HIF1.alpha..
[0125] The peptidic compounds of the invention can be made
intracellularly in cells by introducing into the cells an
expression vector encoding the peptide. Such expression vectors can
be made by standard techniques using oligonucleotides that encode
the amino acid sequence of the peptidic compound. The peptide can
be expressed in intracellularly as a fusion with another protein or
peptide (e.g., a GST fusion). Alternative to recombinant synthesis
of the peptides in the cells, the peptides can be made by chemical
synthesis using standard peptide synthesis techniques. Synthesized
peptides can then be introduced into cells by a variety of means
known in the art for introducing peptides into cells (e.g.,
liposome and the like).
[0126] In addition, dominant negative proteins (e.g., of XBP-1 or
HIF1.alpha.) can be made which include XBP-1 or HIF1.alpha.
molecules (e.g., portions or variants thereof) that compete with
native (i.e., wild-type) molecules, but which do not have the same
biological activity. Such molecules effectively decrease, e.g.,
XBP-1 or HIF1.alpha. activity in a cell.
[0127] Other inhibitory agents that can be used to specifically
inhibit the activity of an XBP-1 or a molecule in a biological
pathway involving XBP-1 are chemical compounds that directly
inhibit expression, processing, post-translational modification,
and/or activity of, e.g., an XBP-1 (or HIF1.alpha.) or inhibit the
interaction between, e.g., XBP-1 and HIF1.alpha.. Such compounds
can be identified using screening assays that select for such
compounds, as described in detail above as well as using other art
recognized techniques.
[0128] In exemplary embodiments, one or more of the above-described
inhibitory compounds is formulated according to standard
pharmaceutical protocols to produce a pharmaceutical composition
for therapeutic use. A pharmaceutical composition of the invention
is formulated to be compatible with its intended route of
administration.
[0129] V. Prognostic Uses
[0130] Triple negative breast cancers comprise a very heterogeneous
group of cancers. There is conflicting information over prognosis
for the various subtypes but it is believed that, at least for more
aggressive subtypes, present method of prognosis are poor. It is
characterized by distinct molecular, histological and clinical
features including a particularly unfavorable prognosis despite
increased sensitivity to standard cytotoxic chemotherapy
regimens.
[0131] The present invention is based, at least in part on the
discovery of a gene expression signature indicative of XBP pathway
activation that is associated with poor prognosis in patients with
TNBC. As used herein, the term "gene expression signature" refers
to a specific pattern of detectable signals indicative of gene
expression in a sample. In one embodiment, the detectable signals
are nucleic acid hybridization signals, for example, signals
generated by hybridization of mRNAs in the sample to mRNA nucleic
acid probes, e.g. probes having sequence complementarity to the
mRNAs. Exemplary detectable labels include, but are not limited to,
radioactive labels, fluorescent labels probes, colorometric labels,
biotin labels, etc. Probes and/or mRNAs can be immobilized, for
example, on a chip, membrane, slide, film, etc. In other
embodiments, hybridization can be accomplished with one or more
components in solution. In exemplary aspects of the invention, a
"gene expression signature" consists of a plurality of signals of
varied intensity, the pattern of which is reproducible when
detected in replicate samples. In preferred aspects of the
invention, a "gene expression signature" consists of a plurality of
signals of increased intensity, for example, genes exhibiting
increased expression in a TNBC sample or cell. In other aspects of
the invention, a "gene expression signature" consists of a
plurality of signals of decreased intensity, for example, genes
exhibiting decreased expression in a TNBC sample or cell. In still
other aspects of the invention, a "gene expression signature"
consists of a plurality of signals of increased and decreased
intensity, for example, genes exhibiting increased and decreased
expression in a TNBC sample or cell.
[0132] In exemplary embodiments of the invention, a "gene
expression signature" is detected in a test sample (e.g., a
biological sample from a patient suspected of having or at risk for
developing TNBC, and compared to an appropriate control gene
expression signature profile (e.g., a signature from a known TNBC
sample or cell). In preferred embodiments, the "test sample" is a
sample isolated, obtained or derived from a subject, e.g., a human
subject. The term "subject" is intended to include living organisms
but preferred subjects are mammals, and in particular, humans. In
particularly preferred embodiments, the "test sample" is a sample
isolated, obtained or derived from a female subject, e.g., a female
human.
[0133] In some embodiments, the gene expression signature is
associated with a specific stage of TNBC. In some embodiments, the
gene expression signature features or consists essentially of mRNAs
that are coordinately regulated. These mRNAs may be coordinately
regulated, for example, by HIF1.alpha. transcriptional activity and
can comprise or consist of specific HIF1.alpha. targets, i.e.,
genes expressed as a result of HIF1.alpha. transcriptional
activity.
[0134] In preferred embodiments, a gene expression profiling test
is used to analyze the patterns of a plurality of genes, e.g.,
those set forth in Table 1 within a sample from a TNBC subject,
e.g., within a sample of cells from a breast tissue tumor in said
subject or from another sample of cancer cells from said subject to
help predict how likely it is that breast cancer, e.g., an
early-stage breast cancer will recur after initial treatment.
[0135] In exemplary embodiments, the invention features diagnostic
tests that quantify the likelihood of disease recurrence in
subjects, e.g., women subjects with triple-negative breast cancer
(TNBC). Such likelihood of disease recurrence is referred to herein
as "prognostic significance". In referred embodiments, the
diagnostic tests of the invention further assess the likely benefit
from certain types of cancer therapeutics, e.g., chemotherapy. Such
assessment is referred to herein as "predictive significance".
[0136] In exemplary aspects of the invention, the diagnostic tests
are designed or formatted to analyzes a panel genes within a sample
from a TNBC subject, e.g., cells or a tissue sample from a tumor of
said subject. From such an analysis, a practitioner or other health
professional (e.g., pathologist) can determine, for example,
prognostic significance and/or predictive significance. In
exemplary embodiments, the test provides for determination of a
"recurrence score". in exemplary embodiments, a recurrence score is
a numerical value, e.g., a number between 0 and 100, that
corresponds to a specific likelihood of breast cancer recurrence
within a certain time period after an initial diagnosis or
treatment. In some embodiments, the score corresponds to a
likelihood of recurrence within 5 years of the initial diagnosis or
treatment. In some embodiments, the score corresponds to a
likelihood of recurrence within 10 years of the initial diagnosis
or treatment. Based on such a score, a subject (e.g., a TNBC
patient) may be classified as low, intermediate or high risk for
recurrence. Such a classification may assume that said subject
follows a course of treatment including, for example, treatment
with anti-hormonal therapy, such as tamoxifen or aromatase
inhibitors (e.g., anastrozole), over the period of time following
diagnosis or treatment. Depending on the subject risk for
recurrence, treatment protocols may include anti-cancer drugs,
chemotherapy, treatment with anti-hormonal therapy, such as
tamoxifen or aromatase inhibitors, neoadjuvant hormonal therapy
(oncology) and the like.
[0137] In exemplary embodiments of the invention, the diagnostic
test is a noninvasive test that is performed on a small amount of
the tissue removed during the original surgery lumpectomy,
mastectomy, or core biopsy. In preferred embodiments, the tissue
sample (after the surgical procedure) is fixed (e.g.,
formalin-fixed) and embedded (e.g., paraffin-embedded) so as to be
preserved for further diagnostic testing. In other preferred
embodiments, the sample (specimen) is fresh tissue sample/specimen.
If using a fresh sample, the sample (from an unfixed tumor
specimen) can be placed in a preservative solution within a short
period of time, e.g., within an hour of surgery. Exemplary
preservatives include, but are not limited to, solutions containing
RNAse inhibitors.
[0138] In exemplary embodiments, a practitioner or other health
professional (e.g., pathologist) prepares the samples for testing,
(e.g., fixing, embedding, thin-sectioning) samples are analyzed,
e.g., in a laboratory or at a testing facility, for example, via
RT-PCR to determine expression of a plurality of genes, e.g.,
10-20, 20-30, 30-40 or more, from a gene signature of the
invention. In preferred embodiments, a panel of genes strongly
correlated with recurrence-free survival is features in a
diagnostic assay or kit of the invention. In exemplary embodiments
of the invention, the results of the featured diagnostic tests can
be integrated with other standard laboratory test results to help
practitioners and/or health care professionals formulate a
treatment plan based on the unique characteristics of the tumor or
cell sample.
[0139] Pluralities or panels of genes featured in the diagnostic
assays and/or kits of the invention can include cancer genes (those
correlated with recurrence) and can include, for example, reference
or control genes used to normalize the expression of the cancer
genes.
[0140] Various methodologies of the instant invention include step
that involves comparing a value, level, feature, characteristic,
property, etc. to a "suitable control", referred to interchangeably
herein as an "appropriate control". A "suitable control" or
"appropriate control" is any control or standard familiar to one of
ordinary skill in the art useful for comparison purposes. In one
embodiment, a "suitable control" or "appropriate control" is a
value or level, of one or more genes (or mRNAs of said genes, or
proteins expressed therefrom) as determined in a cell or sample
positive for TNBC, as described herein. In another embodiment, a
"suitable control" or "appropriate control" is a value or level, of
one or more genes (or mRNAs of said genes, or proteins expressed
therefrom) as determined in a cell or sample negative for TNBC,
e.g., that determined in a cell or organism, e.g., a control or
normal cell or organism, exhibiting, for example, normal traits. In
yet another embodiment, a "suitable control" or "appropriate
control" is a predefined value or level of one or more genes (or
mRNAs of said genes, or proteins expressed therefrom).
[0141] VI. Screening Assays
[0142] In certain aspects, the invention features methods for
identifying compounds useful in inhibiting the growth of TNBC
cells, such compounds having potential therapeutic use in the
treatment of TNBC. As described herein, the instant invention is
based, at least in part, on the discovery of a previously unknown
role for XPB1 is TNBC, such a role being linked to transcriptional
activity of HIF1.alpha.. Genome-wide mapping of the XBP1
transcriptional regulatory network revealed that XBP1 regulates the
hypoxia response through controlling HIF1.alpha. transcriptional
activity and the expression of HIF1.alpha. targets. Accordingly, in
exemplary aspects the invention features methods of identifying for
identifying compounds useful in inhibiting the growth of TNBC
cells, the methods featuring screening or assaying for compounds
that modulate, e.g., activate or increase, or inhibit or decrease,
the interaction of XBP1 and HIF 1.alpha., or biologically active
portions thereof. In exemplary aspects, the methods comprise:
providing an indicator composition comprising XBP1 and HIF1.alpha.,
or biologically active portions thereof; contacting the indicator
composition with each member of a library of test compounds; and
selecting from the library of test compounds a compound of interest
that decreases the interaction of XBP1 and HIF1.alpha., or
biologically active portions thereof, wherein the ability of a
compound to inhibit growth of TNBC cells is indicated by a decrease
in the interaction as compared to the amount of interaction in the
absence of the compound
[0143] As used herein, the term "contacting" (i.e., contacting a
cell e.g. a cell, with a compound) includes incubating the compound
and the cell together in vitro (e.g., adding the compound to cells
in culture) as well as administering the compound to a subject such
that the compound and cells of the subject are contacted in vivo.
The term "contacting" does not include exposure of cells to an
XBP-1 modulator that may occur naturally in a subject (i.e.,
exposure that may occur as a result of a natural physiological
process).
[0144] As used herein, the term "test compound" refers to a
compound that has not previously been identified as, or recognized
to be, a modulator of the activity being tested. The term "library
of test compounds" refers to a panel comprising a multiplicity of
test compounds.
[0145] As used herein, the term "indicator composition" refers to a
composition that includes a protein of interest (e.g., XBP-1 or a
molecule in a biological pathway involving XBP-1, e.g.,
HIF1.alpha.), for example, a cell that naturally expresses the
protein, a cell that has been engineered to express the protein by
introducing one or more of expression vectors encoding the
protein(s) into the cell, or a cell free composition that contains
the protein(s) (e.g., purified naturally-occurring protein or
recombinantly-engineered protein(s)).
[0146] As used herein, the term "cell" includes prokaryotic and
eukaryotic cells. In one embodiment, a cell of the invention is a
bacterial cell. In another embodiment, a cell of the invention is a
fungal cell, such as a yeast cell. In another embodiment, a cell of
the invention is a vertebrate cell, e.g., an avian or mammalian
cell. In a preferred embodiment, a cell of the invention is a
murine or human cell. As used herein, the term "engineered" (as in
an engineered cell) refers to a cell into which a nucleic acid
molecule e.g., encoding an XBP-1 protein (e.g., a spliced and/or
unspliced form of XBP-1) has been introduced.
[0147] As used herein, the term "cell free composition" refers to
an isolated composition, which does not contain intact cells.
Examples of cell free compositions include cell extracts and
compositions containing isolated proteins.
[0148] The ability of the test compound to modulate XBP-1 binding
to HIF1.alpha. can also be determined. Determining the ability of
the test compound to modulate XBP-binding to HIF1.alpha. can be
accomplished, for example, by coupling the HIF1.alpha. with a
radioisotope or enzymatic label such that binding of HIF1.alpha. to
XBP-1 can be determined by detecting the labeled HIF1.alpha. in a
complex. Alternatively, XBP-1 could be coupled with a radioisotope
or enzymatic label to monitor the ability of a test compound to
modulate XBP-1 binding to HIF1.alpha. in a complex. Determining the
ability of the test compound to bind to XBP-1 (or HIF1.alpha.) can
be accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to XBP-1 (or HIF1.alpha.) can be determined by detecting the
labeled compound in a complex. For example, targets can be labeled
with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively,
compounds can be labeled, e.g., with, for example, horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic
label detected by determination of conversion of an appropriate
substrate to product.
[0149] It is also within the scope of this invention to determine
the ability of a compound to interact with XBP-1 or HIF1.alpha.
without the labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a
compound with XBP-1 or HIF1.alpha. without the labeling of either
the compound or the XBP-1 or HIF1.alpha. (McConnell, H. M., et al.
1992. Science 257, 1906-1912). As used herein, a "microphysiometer"
(e.g., Cytosensor) is an analytical instrument that measures the
rate at which a cell acidifies its environment using a
light-addressable potentiometric sensor (LAPS). Changes in this
acidification rate can be used as an indicator of the interaction
between a compound and XBP-1 or HIF1.alpha..
[0150] The cells used in the instant assays can be eukaryotic or
prokaryotic in origin. For example, in one embodiment, the cell is
a bacterial cell. In another embodiment, the cell is a fungal cell,
e.g., a yeast cell. In another embodiment, the cell is a vertebrate
cell, e.g., an avian or a mammalian cell. In a preferred
embodiment, the cell is a human cell. The cells of the invention
can express endogenous XBP-1 or HIF1.alpha. or can be engineered to
do so. For example, a cell that has been engineered to express the
XBP-1 protein and/or HIF1.alpha. can be produced by introducing
into the cell an expression vector encoding the protein.
Recombinant expression vectors that can be used for expression of
XBP-1 or a HIF1.alpha..
[0151] In another embodiment, the indicator composition is a cell
free composition. XBP-1 or HIF1.alpha. expressed by recombinant
methods in a host cells or culture medium can be isolated from the
host cells, or cell culture medium using standard methods for
protein purification. For example, ion-exchange chromatography, gel
filtration chromatography, ultrafiltration, electrophoresis, and
immunoaffinity purification with antibodies can be used to produce
a purified or semi-purified protein that can be used in a cell free
composition. Alternatively, a lysate or an extract of cells
expressing the protein of interest can be prepared for use as
cell-free composition.
[0152] In one embodiment, the amount of binding of XBP-1 to
HIF1.alpha. in the presence of the test compound is greater than
the amount of binding of XBP-1 binding to HIF1.alpha. in the
absence of the test compound, in which case the test compound is
identified as a compound that enhances binding of XBP-1 to
HIF1.alpha.. In another embodiment, the amount of binding of the
XBP-1 to HIF1.alpha. in the presence of the test compound is less
than the amount of binding of the XBP-1 to HIF1.alpha. in the
absence of the test compound, in which case the test compound is
identified as a compound that inhibits binding of XBP-1 to HIF
1.alpha..
[0153] Binding of the test compound to XBP-1 or HIF.alpha.1 can be
determined either directly or indirectly as described above.
Determining the ability of XBP-1 (or HIF1.alpha.) protein to bind
to a test compound can also be accomplished using a technology such
as real-time Biomolecular Interaction Analysis (BIA) (Sjolander,
S., et al. 1991. Anal. Chem. 63, 2338-2345; Szabo, A., et al. 1995.
Curr. Opin. Struct. Biol. 5, 99-705). As used herein, "BIA" is a
technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the optical phenomenon of surface plasmon resonance (SPR) can be
used as an indication of real-time reactions between biological
molecules.
[0154] In the methods of the invention for identifying test
compounds that modulate an interaction between XBP-1 protein and
HIF1.alpha., the complete XBP-1 (or e.g HIF1.alpha.) protein can be
used in the method, or, alternatively, only portions of the protein
can be used. In one embodiment of the above assay methods of the
present invention, it may be desirable to immobilize either XBP-1
(or HIF 1.alpha.) for example, to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, or
to accommodate automation of the assay. Binding of a test compound
to a XBP-1 with HIF1.alpha. in the presence and absence of a test
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided in which a domain that allows one or both
of the proteins to be bound to a matrix is added to one or more of
the molecules. For example, glutathione-S-transferase fusion
proteins or glutathione-S-transferase/target fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are
then combined with the test compound or the test compound and
either the non-adsorbed target protein or XBP-1 (or HIF1.alpha.)
protein, and the mixture incubated under conditions conducive to
complex formation (e.g., at physiological conditions for salt and
pH). Following incubation, the beads or microtitre plate wells are
washed to remove any unbound components, the matrix is immobilized
in the case of beads, and complex formation is determined either
directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of binding or activity determined using standard
techniques.
[0155] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either an XBP-1 protein or HIF1.alpha. can be immobilized utilizing
conjugation of biotin and streptavidin. Biotinylated protein or
target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies which are reactive
with protein but which do not interfere with binding of the
proteins can be derivatized to the wells of the plate, and unbound
XBP-1 or HIF1.alpha. protein is trapped in the wells by antibody
conjugation. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with XBP-1
or HIF1.alpha., as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the XBP-1 or
HIF1.alpha..
[0156] Another aspect of the invention pertains to kits for
carrying out the screening assays, modulatory methods or diagnostic
assays of the invention.
[0157] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents, and published patent applications cited
throughout this application, as well as the figures and the
sequence listing, are hereby incorporated by reference.
EXAMPLES
Example 1
The UPR is Activated in Human Breast Cancer Patients
[0158] To determine whether the UPR is activated in breast cancer,
we used immunohistochemistry (IHC) to examine the phosphorylation
of PERK, a marker of UPR activation, in human primary breast tumor
samples. By staining breast cancer tissue microarrays (TMA)
containing 66 normal breast tissue samples and 40 tumor tissue
samples, we found that PERK was preferentially phosphorylated in
breast tumors, but not in normal breast tissue (FIG. 1A, 1B),
suggesting that activation of the UPR occurs specifically in
tumors. Next, the same TMA were stained with antibodies
specifically recognizing phosphorylation of eukaryotic
translational initiation factor 2.alpha. (eIF2.alpha.), another
marker of UPR activation. Similarly, eIF2.alpha. was phosphorylated
in malignant breast tumors but not normal breast tissue (FIG. 1C,
1D). Thus, the UPR is preferentially activated in breast
tumors.
Example 2
XBP1 is Required for Transformation of Immortalized Mammary
Epithelial Cells
[0159] The IRE1-XBP1 axis of the UPR shows robust conservation from
yeast to metazoans, including humans. To investigate the role of
XBP1 in cellular transformation, we used MCF10A immortalized
mammary epithelial cells that express ER-Src, a fusion of the Src
kinase oncoprotein (v-Src) and the ligand binding domain of the
estrogen receptor. Treatment of these cells with tamoxifen (TAM)
for 36 hr results in neoplastic transformation, including the
ability to form colonies in soft agar, increased motility and
invasive ability, and tumor formation upon injection into nude mice
(Iliopoulos, D., et al. 2009. Cell 139, 693-706). Knockdown of XBP1
expression with a highly effective shRNA (Figure S1) blocked the
neoplastic transformation of MCF10A ER-Src cells (FIG. 2A).
Furthermore, XBP1 silencing reduced the invasiveness and the
ability of MCF10A ER-Src cells to form colonies in soft agar and
tumors in immunodeficient mice (FIG. 2B-D). We tested the ability
of enforced XBP1 expression to transform MCF10A cells by
overexpression of the XBP1 spliced form (XBP1s) in MCF10A ER-Src
cells in the absence of tamoxifen. XBP1 overexpression was
sufficient to induce transformation in the absence of tamoxifen
(FIG. 2E). Furthermore, XBP1s overexpression increased colony
formation in a soft agar assay (FIG. 2F). Collectively, these
results demonstrate that XBP1 is both necessary and sufficient for
the transformation of mammary epithelial cells.
Example 3
XBP1 Inhibition Blocks Breast Cancer Cell Growth and Invasiveness
Both Ex Vivo and In Vivo; XBP1 Silencing Blocks Triple Negative
Breast Cancer Progression
[0160] To further characterize the function of XBP1 in breast
cancer, we first determined the activation status of XBP1 in
different breast cancer cell lines. Breast cancers can be
classified as luminal or basal-like, depending on their expression
of different cytokeratins (Perou, C. M., et al. 2000. Nature 406,
747-752; Vargo-Gogola, T., et al. 2007. Cancer 7, 659-672).
Unexpectedly, XBP1 was preferentially spliced and activated in
basal-like breast cancer cells (FIG. 3A), which harbor a
transcriptome similar to that of triple negative breast cancer
(TNBC), a subtype of breast cancer that is extremely aggressive and
difficult to target due to the lack of expression of the estrogen
(ER), progesterone (PR) and human epidermal growth factor 2 (HER2)
receptors (Foulkes, W. D., et al. 2010. N Engl J Med 363,
1938-1948). In particular, while XBP1 expression was readily
detected in both luminal and basal-like breast cancer cells, the
level of its spliced (activated) form was higher in the latter cell
type (FIG. 1A), which comprises primarily TNBC (Perou, C. M., et
al. 2000. Nature 406, 747-52; Vargo-Gogola, T., et al. 2007. Nat
Rev Cancer 7, 659-72; Herschkowitz, J. I., et al. 2007. Genome Biol
8, R76). Furthermore, silencing XBP1 expression decreased the
ability of different breast cancer cell lines to form colonies in
soft agar (FIG. 3B).
[0161] TNBC is a highly aggressive subtype of breast cancer
characterized by the absence of estrogen receptor (ER),
progesterone receptor (PR) and human epidermal growth factor 2
(HER2) expression (Foulkes, W. D., et al. 2010. N Engl J Med 363,
1938-48). We next demonstrated that silencing of XBP1 significantly
impaired soft agar colony formation (FIG. 1B) and invasiveness
(FIG. 1C) of multiple TNBC cell lines (MDA-MB-231, MBD-MB-468,
HBL-100, MDA-MB-436, MDA-MB-157), suggesting a potential role of
XBP1 in the regulation of anchorage-independent growth and
invasiveness of TNBC.
[0162] Interestingly, knockdown of XBP1 was more effective in
suppressing the proliferation of basal-like (MDA-MB-231,
MBD-MB-468, HBL-100, MDA-MB-157, MDA-MB-435, MDA-MB-436, SUM-159)
than luminal (MCF7, BT-474, SKBR3, T47D, MDA-MB-361) breast cancer
cell lines, consistent with the preferential splicing of XBP1 in
basal-like cells. Similarly, knockdown of XBP1 decreased the
invasiveness of breast cancer cell lines, a phenotype that was more
dramatic in basal-like lines (FIG. 3C). These data suggest that
XBP1 regulates the growth and invasiveness of breast cancer cells,
especially basal-like breast cancer cells.
[0163] To assess the function of XBP1 in vivo, we established an
orthotopic xenograft mouse model with inducible expression of shRNA
against XBP1. In particular, we infected MDA-MB-231 cells, a TNBC
cell line, with lentiviruses encoding XBP1 shRNAs under the control
of a doxycycline-inducible promoter. Cells infected with a
lentivirus encoding a scrambled LacZ shRNA served as a control.
Doxycycline treatment of cells infected with the XBP1 shRNA
lentivirus led to an 85% reduction in XBP1 mRNA levels compared to
cells grown in the absence of doxycycline (FIG. 3D).
[0164] Next, these MDA-MB-231 cells infected with the shRNA
lentiviruses were further infected with a retrovirus encoding
luciferase. After injection with retroviruses, these cells were
implanted (injected) orthotopically in the mammary glands of
NOD/SCID/IL2R.gamma.-/- mice. The kinetics of tumor growth were
monitored with bioluminescent imaging. At > two weeks after
implantation (19 days), prior to induction of the XBP1 shRNA, XBP1
shRNA and control tumors exhibited similar luciferase signals (FIG.
3E). These mice were then fed chow containing doxycycline to induce
the XBP1 shRNA and serially monitored using bioluminescence. After
4 weeks of XBP1 depletion a significant inhibition of tumor growth
was observed (FIGS. 3E and 3F). XBP1 was efficiently silenced in
the tumor (FIG. 3I). While tumors expressing control shRNA (n=8)
began to metastasize to the lungs 9 weeks after transplantation, no
metastasis was observed in the XBP1 shRNA xenograft tumors (n=8)
(FIG. 3E). To rule out off-target effects of the XBP1 shRNA, the
same assays were conducted with another inducible XBP1 shRNA
construct targeting a different region of XBP1 (FIG. 3J), which
yielded similar results (FIG. 3K). To exclude the possibility of
cell line specific effects, subcutaneous xenograft experiments were
performed using two other TNBC cell lines: MDA-MB-436 and HBL-100
cells. As expected, XBP1 silencing significantly repressed the
formation of MDA-MB-436 and HBL-100 TNBC-derived tumors (FIG. 3L).
Importantly, we examined the functional relevance of XBP1 in
primary human breast tumor cells. We inhibited XBP1 by siRNA in a
patient-derived TNBC xenograft model (BCM-2147). Silencing of XBP1
in this model significantly decreased tumor incidence (FIG. 3G) and
suppressed tumor growth (FIG. 3H), further supporting the role of
XBP1 in TNBC. Collectively, these results demonstrate that loss of
XBP1 suppresses the growth and metastasis (tumorigenicity and
progression) of human triple negative breast tumors.
Example 4
XBP1 is Required to Sustain Tumor Initiating Cell (TIC)
Self-Renewal; XBP1 is Required for Tumor Initiating Cells
[0165] Previous studies have shown that basal-like breast cancer
cells are more aggressive than luminal cells due to increased
numbers of a stem cell-like CD44.sup.high/CD24.sup.low
subpopulation, termed tumor initiating cells (TICs) (Al-Hajj, M.,
et al. 2003. Proc Natl Acad Sci USA 100, 3983-3988; Mani, S. A., et
al. 2008. Cell 133, 704-715). To interrogate the effect of XBP1 on
TICs, we used a model of breast epithelial cells (MCF10A) carrying
an inducible Src oncogene (ER-Src), in which the Src kinase
oncoprotein (v-Src) was fused with the ligand binding domain of the
estrogen receptor (Iliopoulos, D., et al. 2009. Cell 139, 693-706).
Recently, it has been shown that during transformation of MCF10A
ER-Src cells, there is formation of a CD44.sup.high/CD24.sup.low
population with TIC characteristics (Iliopoulos et al., 2011). In
particular, Treatment of these cells with tamoxifen (TAM) for 24-36
hr results in neoplastic transformation and the gain of a
CD44high/CD24low population with tumor-initiating property
(Iliopoulos, D., et al. 2011. Proc Natl Acad Sci USA 108,
1397-402). In transformed MCF10A ER-Src cells, knockdown of XBP1
blocked the formation of the CD44.sup.high/CD24.sup.low ER-Src TIC
population (reducing the CD44.sup.high/CD24.sup.low TIC
fraction)(FIG. 4B and Figures H-I). In this system, XBP1 was more
highly spliced in TICs (CD44.sup.high/CD24.sup.low) relative to
non-TICs (NTICs) (FIG. 4A). XBP1 silencing also suppressed the
ability of transformed MCF10A ER-Src cells to form mammospheres
(FIG. 4C), an assay used to assess the self-renewal of breast TICs
(Dontu, G., et al. 2003. Genes Dev 17, 1253-1270). These phenotypes
were not due to a direct effect of XBP1 on cell viability (FIGS.
4J-K).
[0166] To test if expression of XBP1 was sufficient to induce TIC
properties in NTICs, XBP1s was overexpressed in
CD44.sup.low/CD24.sup.high NTICs derived from MCF10A ER-Src cells.
This induced the formation of a population with a TIC-like
CD44.sup.high/CD24.sup.low surface phenotype and enhanced
mammosphere forming ability.
[0167] Tumorigenicity in a murine host is the gold standard for
evaluating the stem cell-like properties of TICs (Clarke, M. F., et
al. (2006). Cancer Res 66, 9339-9344). To further investigate if
the TICs induced by XBP1s expression in NTICs also display TIC
properties in a murine tumor formation assay, NTICs or NTICs with
enforced expression of XBP1s (XBP1s-NTIC) were injected into
NOD/SCID mice at a range of dilutions. Remarkably, as few as 100
XBP1s-NTICs cells were able to generate a tumor, whereas control
NTICs failed to form tumors at any dilution. Thus, under these
conditions, XBP1s is sufficient for the induction of functional
breast TICs.
[0168] In a limiting dilution experiment, TAM-treated MCF10A-ER-Src
cells bearing control shRNA were able to initiate tumors when as
few as 1.times.10.sup.4 or 1.times.10.sup.5 cells were implanted.
However, XBP1-depleted cells showed complete loss of tumor-seeding
ability even when 1.times.10.sup.6 cells were xenografted (FIG.
4D).
[0169] In addition to MCF10A ER-Src cells, we examined the effects
of XBP1 inhibition in TICs derived from breast cancer cell lines.
XBP1 inhibition suppressed the growth of mammospheres derived from
MDA-MB-231, MDA-MB-468 and MDA-MB-436 cells (FIG. 4F).
[0170] To evaluate the functional relevance of XBP1 in human cancer
patients, we sorted the CD44.sup.high/CD24.sup.low subpopulation
directly from human TNBC patient samples, and confirmed XBP1
splicing to be elevated in this fraction compared to the
CD44low/CD24high cells (FIG. 4E). Infection of the
CD44.sup.high/CD24.sup.low cells with lentivirus expressing XBP1
shRNA, inhibited the formation of mammospheres derived from a
number of patient-derived TNBC tissues (FIG. 4F). Conversely,
overexpression of XBP1s in NTICs (CD44.sup.high/CD24.sup.low)
sorted from primary human TNBC (or derived from breast cancer cell
lines) transformed them into TICs as based on surface phenotype.
Remarkably, these XBP1s-induced TICs are able to form tumors in
immunodeficient mice at very low dilutions (as low as 10
xenografted cells) whereas none of the control parental NTICs were
tumorigenic (FIG. 4G).
[0171] Collectively, these data establish a critical and unexpected
role of XBP1 in TICs, likely contributing to its function in
promoting triple-negative breast cancer
Example 5
XBP1 Silencing Increases Sensitivity and Reduces Resistance to
Chemotherapy; Inhibition of XBP1 Suppresses Tumor Relapse
[0172] Chemotherapy is the only systemic therapy currently used
clinically to treat TNBC. However, patients with TNBC have the
highest rate of relapse within 1-3 years despite the use of
adjuvant chemotherapy (Lehmann, B. D., et al. 2011. J Clin Invest
121, 2750-67). Moreover, TICs are resistant to chemotherapy and are
believed to be responsible for tumor relapse after chemotherapy
(Dean, M., et al., 2005. Nat Rev Cancer 5, 275-284). Given that
XBP1 appears to induce TIC differentiation, the role of XBP1 in
mediating the relapse of the MDA-MB-231 xenograft tumor after
chemotherapy was evaluated. It was believed that this approach
would yield further insights into the function of XBP1 in TNBC.
Treatment of MDA-MB-231 xenograft tumors with doxorubicin (i.p.)
every 5 days, from day 15 until day 30, suppressed tumor growth
(FIG. 4M). Relapse from treatment occurred on day 60, i.e., Tumor
relapse after treatment was detected from day 60 onwards.
Strikingly, combinatorial treatment with doxorubicin and XBP1 shRNA
not only blocked tumor growth but also inhibited tumor relapse
(FIG. 4N).
[0173] The presence of tumor initiating cells (TICs), characterized
by the cell surface phenotype CD44high/CD24low and the expression
of ALDH1 (Ginestier, C., et al. 2007. Cell Stem Cell 1, 555-67),
are thought to play a role in chemotherapy resistance and tumor
relapse after systemic adjuvant therapy (Dean, M., et al. 2005. Nat
Rev Cancer 5, 275-84; Al-Hajj, M., et al. 2003. Proc Natl Acad Sci
USA 100, 3983-8; Creighton, C. J., et al. 2009. Proc Natl Acad Sci
USA 106, 13820-5; Li, X., et al. 2008. J Natl Cancer Inst 100,
672-9). In order to test whether suppression of tumor relapse (this
increased sensitivity to chemotherapy) is due to an effect of XBP1
on TICs, we examined mammosphere-forming ability of cells (the
number of mammospheres) derived from the treated tumors (day 20).
Mammosphere assays are used to assess the activity of breast TICs
in vitro (Dontu, G., et al. 2003. Genes Dev 17, 1253-70).
Consistent with the previously observed enrichment of TIC following
chemotherapy (Creighton, C. J., et al. 2009. Proc Natl Acad Sci USA
106, 13820-5), mammosphere formation was increased in cells derived
from doxorubicin treated tumors (FIG. 4L). Intriguingly, tumors
treated with doxorubicin in combination with XBP1 knockdown
demonstrated substantially suppressed mammosphere growth (FIG. 4M),
suggesting that XBP1 silencing blunted chemotherapy-induced
expansion of the TIC pool. Thus, the combination of chemotherapy
and XBP1 knockdown suppresses breast tumor growth and prolongs
remission in breast xenografts.
[0174] Collectively, these data demonstrate that XBP1 is required
to sustain TIC self-renewal in breast cancer.
Example 6
XBP1 Interacts with HIF1.alpha. and Co-Occupies the Promoters of
HIF1.alpha. Targets; HIF1.alpha. is a Co-Regulator of XBP1 in
TNBC
[0175] Given the importance of XBP1 in the breast cancer models
above and to further understand how XBP1 contributes to TNBC, we
sought to identify transcriptional networks regulated by XBP1 and
to dissect the underlying mechanism by mapping the physiological
targets of XBP1s using ChIP coupled with high-throughput sequencing
(ChIP-seq). Tumor cells are exposed to hypoxia and glucose
deprivation, and these factors are appreciated to have a large
impact on tumor pathophysiology (Semenza, G. L. 2003. Nat Rev
Cancer 3, 721-32). XBP1s was highly expressed in MDA-MB-231 cells
by exposure to the physiological stressors (FIG. 5J) To examine if
these stressors of cellular physiology might induce XBP1 activation
via splicing, MDA-MB-231 cells were grown in hypoxic and glucose
deprivation conditions for 24 h. Exposure to hypoxia and glucose
deprivation induced splicing of XBP1, and this resulted in a
corresponding increase in the signal intensity detected in ChIP-seq
experiments. Using a ChIP-seq approach (using a polyclonal antibody
specifically recognizing the XBP1s protein), we identified a total
of 6317 high-confidence XBP1 binding sites in MDA-MB-231 cells.
13.9% of the binding sites mapped to promoters, and 73.6% were
found at distal intergenic and intronic regions (FIG. 5K). Notably,
the overlap of the genes bound by XBP1 in MDA-MB-231 cells versus
those bound in plasma cells or pancreatic beta cells was small
(Acosta-Alvear, D., et al. 2007. Mol Cell 27, 53-66). Therefore,
our study revealed a unique repertoire of XBP1 binding sites
specific for TNBCs. As expected, XBP1 extensively bound to genes
involved in the UPR pathway, such as DNAJB9, HSPA5, and EDEM. By
performing microarray and gene set enrichment analysis (GSEA) of
genes differentially expressed upon XBP1 depletion in MDA-MB-231
cells, we found that the UPR pathway was among the most enriched
categories, with significant enrichment of genes involved in ER
stress and UPR pathways indicating that XBP1 directly regulates the
UPR in TNBC cells.
[0176] To determine the in vivo sequence specificity of XBP1, we
derived the consensus sequence motifs by using a motif-discovery
algorithm MDScan (Liu, X. S., et al. 2002. Nat Biotechnol 20,
835-839). Notably, the predominant motif found was a perfect match
to the XBP1 consensus site GC/ACACGT (FIG. 5L), confirming the
validity of the ChIP-seq dataset. Remarkably, a HIF1.alpha. binding
motif showed statistically significant enrichment in our dataset
(enrichment of the HIF1.alpha. binding motif in the XBP1 sites
(p=1.0.times.10.sup.-30)) (FIG. 5A), suggesting potential
cooperation between HIF1.alpha. and XBP1, e.g., that HIF1.alpha.
frequently co-localizes to the same transcriptional regulatory
elements as XBP1. HIF1.alpha. is a ubiquitously expressed, O.sub.2
dependent subunit of Hypoxia Induced Factor (HIF1), known to play
essential roles in TNBC and in breast TICs self-renewal (Schwab, L.
P., et al. 2012. Breast Cancer Res 14, R6). The enrichment of the
HIF1.alpha. motif in the XBP1 ChIP-seq dataset raised the
possibility that XBP1 and HIF1.alpha. might interact in the same
transcriptional complex.
[0177] To assess this possibility, Flag-tagged HIF1.alpha. was
co-expressed with XBP1s in 293T cells cultured under hypoxia.
Treatment of cells with the proteasome inhibitor MG132 for 16 hours
was necessary to inhibit the basal turnover of HIF1.alpha..
Extracts were harvested and immunoprecipitated with M2 FLAG
antibody, and HIF1.alpha. was found to co-precipitate with XBP1s
(FIG. 5B). This interaction could also be observed with endogenous
proteins in the context of two TNBC cell lines. MDA-MB-231 and
Hs578T cells were treated with tunicamycin (TM), a potent
pharmacologic ER-stress inducer that triggers robust XBP1 splicing.
Nuclear extracts were harvested, and immunoprecipitation using an
anti-HIF1.alpha. antibody demonstrated the co-precipitation of XBP1
(FIGS. 5C and 5M). Thus, endogenous XBP1 interacts with HIF1.alpha.
in the nucleus.
[0178] To extend these results, we next asked whether XBP1 binds
together with HIF1.alpha. specifically at the site of HIF1.alpha.
target genes. Direct ChIP-qPCR was performed to examine the
co-occupancy XBP1 and HIF .alpha. at several well known HIF1.alpha.
direct targets including VEGFA, PDK1, DDIT4, JMJD1A and JMJD2C
(Xia, X, et al. 2009. Proc Natl Acad Sci USA 106, 4260-4265). As
shown in FIGS. 5D-F, and FIG. 5N, both XBP1 and HIF1.alpha. bind to
the promoters of VEGFA, PDK1, DDIT4, JMJD1A and JMJD2C under
hypoxic conditions, whereas control GST ChIP did not show any
enrichment. Next, we ascertained the functional contribution of
XBP1 to the regulation of HIF1.alpha. targets. As the physiologic
response to tissue hypoxia is initiated by the binding of the HIF-1
transcription factor to the hypoxia response element (HRE)
(Semenza, G. L., 2001. Cell 107, 1-3), a luciferase construct
containing three copies of HRE (FIG. 5G) was co-transfected
together with a construct encoding XBP1s into MDA-MB-231 cells.
XBP1s was able to transactivate the HRE reporter in a dose
dependent manner, whereas the empty vector had no effect (FIG. 5H).
Conversely, depletion of XBP1 by two independent shRNA constructs
dramatically reduced HRE activity under hypoxic conditions (FIG.
5I). Taken together, these data demonstrate that XBP1s interacts
with HIF1.alpha. and in turn the two collaborate to regulate the
promoters of HIF1.alpha. targets.
Example 7
XBP1 Regulates the Response to Hypoxia (the Hypoxia Response
Pathway)
[0179] Next, we profiled the differential transcriptome regulated
by XBP1 silencing in MDA-MB-231 cells using gene expression
microarray analysis. In particular, to identify the transcriptional
programs regulated by XBP1, we perturbed XBP1 expression in
MDA-MB-231 cells by shRNA and examined the effects on gene
expression by microarray analysis under the same conditions as the
above ChIP-seq assay. Gene set enrichment analysis (GSEA)
identified significant enrichment of genes in the hypoxia response
pathway (FIG. 6A, B). To verify the regulation of the hypoxia
response by XBP1, we exposed cells to hypoxia, and demonstrated
that depletion of XBP1 resulted in downregulation of HIF1.alpha.
targets VEGFA, PDK1, GLUT1 and DDIT4 expression (FIG. 6C, Figure
S4). This result indicates that XBP1 regulates the expression of
HIF1.alpha. targets under hypoxic conditions. Performing the same
experiment in another TNBC cell line, HS578T, yielded similar
results (FIG. 6D, FIG. 6H). Thus, XBP1 is an essential mediator of
the hypoxic response via its key function in regulating the
expression of HIF1.alpha. target genes.
[0180] To further understand the mechanism by which XBP1 regulates
HIF1.alpha. transcriptional pathways, we first examined the
correlation between XBP1 and HIF1.alpha. at genome-wide level. As
shown in FIG. 6E, a high level of XBP1 occupancy was associated
with increased occurrence of the HIF1.alpha. motif in TNBC
(p<1.times.10-5), suggesting a requirement of XBP1 for
HIF1.alpha. occupancy. Next, we depleted XBP1 and examined the
occupancy of HIF1.alpha. at HIF1.alpha.-XBP1 co-bound sites near
well-established HIF1.alpha. targets. MDA-MB-231 cells infected
with control shRNA or XBP1 shRNA were treated for 24 h under
hypoxic conditions, and the extracts were subjected to ChIP. As
expected, XBP1 knockdown reduced the occupancy of XBP1 on co-bound
sites (FIG. 6I). HIF1.alpha. levels were not altered by XBP1
depletion (FIG. 6J). XBP1 depletion substantially attenuated
HIF1.alpha. occupancy at the targets (FIG. 6F), suggesting that the
recruitment of HIF 1.alpha. is dependent on XBP1
[0181] To further understand the relationship between XBP1,
HIF1.alpha. and the basal transcription machinery, we examined the
recruitment of RNA polymerase II at the promoters of HIF1.alpha.
target genes. In particular, we carried out ChIP against RNA
polymerase II. Consistent with the reduction in HIF1.alpha. target
transcripts after XBP1 depletion, the binding of RNA polymerase II
to the XBP1-HIF1.alpha. co-bound sites was also significantly
reduced in the absence of XBP1 (FIG. 6G). As a control, RNA
polymerase II binding to .beta.-actin, which is not occupied by
XBP1, was not altered (FIG. 6G). Collectively, these data suggest
that XBP1 regulates HIF1.alpha. transcriptional activity by
controlling the binding of HIF1.alpha. to its targets and by the
recruitment of RNA polymerase II.
Example 8
XBP1Activation is Associated with Human Breast Cancer Prognosis
[0182] Through integrated analysis of XBP1 ChIP-seq data and gene
expression profiles, we identified a plurality of genes that are
directly bound and up-regulated by XBP1. This gene set was defined
as the XBP1 signature (FIG. 7A). The gene signature is also defines
by the genes set forth in Table 1.
TABLE-US-00001 TABLE 1 XBP1 gene signature Refseq Gene Symbol RP
value FDR NM_005080 XBP1 0.000308166 0.046 NM_001079539 XBP1
0.000616333 0.033 NM_173354 SIK1 0.007660895 0.026 NM_001177 ARL1
0.007856733 0.0225 NM_015021 ZNF292 0.010608268 0.0192 NM_001113182
BRD2 0.018319709 0.017 NM_005104 BRD2 0.020101806 0.016444444
NM_024116 TAF1D 0.021276877 0.014727273 NM_005321 HIST1H1E
0.026452097 0.013733333 NM_134470 IL1RAP 0.026969403 0.01425
NM_177444 PPFIBP1 0.031355838 0.014380952 NM_144949 SOCSS
0.031921637 0.014818182 NM_014011 SOCSS 0.032025593 0.014956522
NM_014840 NUAK1 0.032058195 0.015166667 NM_003410 ZFX 0.032864181
0.015851852 NM_012421 RLF 0.035372081 0.017483871 NM_002610 PDK1
0.036732609 0.018571429 NM_001259 CDK6 0.037469791 0.018666667
NM_001134368 SLC6A6 0.037723647 0.018918919 NM_003670 BHLHE40
0.038232511 0.018894737 NM_006265 RAD21 0.039985705 0.0195
NM_012330 MYST4 0.041773318 0.020095238 NM_004792 PPIG 0.041827844
0.020232558 NM_006699 MAN1A2 0.042287349 0.020347826 NM_006427
SIVA1 0.043459113 0.020857143 NM_001145306 CDK6 0.046056189
0.022346154 NM_021709 SIVA1 0.046086078 0.022566038 NR_027856 CLK1
0.047788652 0.023758621 NR_027855 CLK1 0.048191354 0.024305085
NM_004071 CLK1 0.048592674 0.024833333 NM_001162407 CLK1 0.04931818
0.025419355 NM_001135581 SLC1A4 0.050712807 0.026338462 NM_003286
TOP1 0.051189956 0.026848485 NM_018463 ITFG2 0.05599817 0.028027778
NM_020791 TAOK1 0.056306819 0.028273973 NM_004642 CDK2AP1
0.058411965 0.028973684 NM_004354 CCNG2 0.059493678 0.029777778
NM_006810 PDIA5 0.059980932 0.030292683 NM_003038 SLC1A4
0.060388037 0.030952381 NM_033026 PCLO 0.060740842 0.031035294
NM_001031723 DNAJB14 0.063887884 0.032593407 NM_022044 SDF2L1
0.068126387 0.034 NM_012328 DNAJB9 0.06931484 0.034632653 NM_018386
PCID2 0.070132067 0.035030303 NM_001127203 PCID2 0.07045563 0.03532
NM_052834 WDR7 0.07101882 0.035960784 NM_015285 WDR7 0.071327044
0.036368932 NM_003432 ZNF131 0.072904644 0.037364486 NM_018725
IL17RB 0.073397321 0.038558559 NM_014629 ARHGEF10 0.076846594
0.040537815 NM_005834 TIMM17B 0.078254854 0.041289256 NM_001127202
PCID2 0.078373692 0.04157377 NM_178812 MTDH 0.078864716 0.042080645
NM_015565 RNF160 0.079551116 0.042384 NM_173214 NFAT5 0.079829972
0.042692913 NM_138714 NFAT5 0.080330418 0.043410853 NM_138713 NFAT5
0.080828942 0.044333333 NM_020182 PMEPA1 0.080955732 0.044820896
NM_006599 NFAT5 0.081325577 0.045066667 NM_001113178 NFAT5
0.081820358 0.046246377 NM_001307 CLDN7 0.08389827 0.046628571
NM_206866 BACH1 0.085201186 0.046822695 NM_001006622 WDR33
0.085351526 0.047070423 NM_021913 AXL 0.085668045 0.047496503
NM_001080512 BICC1 0.086429554 0.047708333 NM_014607 UBXN4
0.086642454 0.048246575 NM_001699 AXL 0.086705455 0.048489796
NM_001186 BACH1 0.087932789 0.050313725 NM_001706 BCL6 0.089419251
0.051261146 NM_001042370 TROVE2 0.089715599 0.051594937 NM_005734
HIPK3 0.09027961 0.0521875 NM_001048200 HIPK3 0.091696467
0.054060606 NM_004641 MLLT10 0.095742597 0.057737143 NM_020354
ENTPD7 0.096224931 0.058034091 NM_001009569 MLLT10 0.097206843
0.058905028 NM_004600 TROVE2 0.097239566 0.059233333 NM_001042369
TROVE2 0.097349374 0.059436464 NM_015659 RSL1D1 0.097841743
0.059747253 NM_032991 CASP3 0.098056014 0.060174863 NM_004346 CASP3
0.098873885 0.0605 NM_002360 MAFK 0.100285797 0.061659574 NM_013409
FST 0.100786907 0.061978836 NM_033300 LRP8 0.102030543 0.062492147
NM_003376 VEGFA 0.102146358 0.06307772 NM_022066 UBE2O 0.103051603
0.063897959 NM_017522 LRP8 0.103179158 0.064304569 NM_004083 DDIT3
0.104217214 0.06504 NM_004631 LRP8 0.104316037 0.065792079
NM_001001925 MTUS1 0.105039098 0.066868293 NM_199170 PMEPA1
0.105246837 0.06763285 NM_032711 MAFG 0.10539111 0.068210526
NM_001018054 LRP8 0.105441559 0.068580952 NM_199169 PMEPA1
0.105647608 0.069549296 NM_001001924 MTUS1 0.106466031 0.070608295
NM_033668 ITGB1 0.107856482 0.071909502 NM_001025368 VEGFA
0.108110645 0.072198198 NM_001025367 VEGFA 0.109049116 0.07275
NM_005067 SIAH2 0.109127465 0.072915556 NM_199171 PMEPA1
0.109425982 0.073274336 NM_001025366 VEGFA 0.109980443 0.073929825
NM_006287 TFPI 0.112000579 0.07525 NM_018433 KDM3A 0.112719
0.075476395 NM_001455 FOXO3 0.113000887 0.075794872 NM_001146688
KDM3A 0.113091614 0.076153191 NM_025090 USP36 0.113105469
0.076559322 NM_012224 NEK1 0.113246019 0.077268908 NM_002359 MAFG
0.113434126 0.077548117 NM_001033756 VEGFA 0.114262021 0.078248963
NM_201559 FOXO3 0.115000173 0.079853659 NM_004850 ROCK2 0.11677596
0.08116 NM_177951 PPM1A 0.117567808 0.081698413 NM_015640 SERBP1
0.117780863 0.082086957 NM_001018069 SERBP1 0.118092488 0.082433071
NM_001018068 SERBP1 0.118404096 0.083276265 NM_001018067 SERBP1
0.118715685 0.08355814 NM_015497 TMEM87A 0.118930277 0.084030769
NM_001025369 VEGFA 0.119400069 0.084557252 NM_001973 ELK4
0.120484396 0.085222642 NM_022828 YTHDC2 0.121842701 0.087516484
NM_016578 RSF1 0.121898417 0.087744526 NM_206909 PSD3 0.122170784
0.08792 NM_006466 POLR3F 0.123368602 0.088527076 NM_012334 MYO1O
0.123689567 0.088834532 NM_014945 ABLIM3 0.123956467 0.089039427
NM_015046 SETX 0.127055781 0.091531469 NM_174907 PPP4R2 0.127746035
0.092090278 NM_006350 FST 0.128346778 0.092914089 NM_005135 SLC12A6
0.128533103 0.093130137 NM_005649 ZNF354A 0.128561915 0.093372014
NM_024949 WWC2 0.129706945 0.09427027 NM_031899 GORASP1 0.130596765
0.095006711 NM_138927 SON 0.132138364 0.097980456 NM_001143886
PPP1R12A 0.133036549 0.099647436
[0183] In exemplary embodiments, subset of the genes listed in
Table 1 can be selected to constitute a more simple gene signature.
For example, a subset of genes, e.g., 10-20, 20-30 or more genes
from Table 1 (or, for example, 5%, 10%, 15% 20% or more of the
genes in Table 1) can be selected having a high degree of
expression or representation in the gene signature. Alternatively,
a subset of genes, e.g., 10-20, 20-30 or more genes from Table 1
(or, for example, 5%, 10%, 15% 20% or more of the genes in Table 1)
can be selected having a low degree of expression or representation
in the gene signature.
[0184] Differentially expressed genes (DEGs) can be selected based
on low false discovery rate (FDR) (e.g., FDR for p-values from
t-test.) For example, genes with a RP value of <0.1, <0.09,
<0.08, <0.07, <0.06, <0.05, <0.04 or <0.02 can be
selected as DEGs. Alternatively, genes with a FDR<0.05,
<0.04, <0.3, or <0.2 can be selected as DEGs.
Alternatively, or in combination, DEGs can be selected based on
rank product (RP) value A lower absolute value for RP indicates a
higher degree of differential expression. The genes in Table 1 were
ranked in descending order of the absolute RP value.
[0185] RP ranking can characterize up-regulated genes and
down-regulated genes under one class. To obtain one RP value per
gene for comparison within results (or for comparison with ranking
according to other methods), a lower value can be defined as a net
value for a gene. A small net value for RP is therefore evidence of
differential expression. (See e.g., Kadota K et al. (2009).
Algorithm Mol. Biol. 4:7.)
[0186] To investigate the correlation of the XBP1 gene signature
with patient relapse-free survival, we performed survival analysis
using an aggregate breast cancer dataset that contains the gene
expression profile and the survival information for 109 TNBC
patient samples from 21 datasets (Lehmann, B. D., et al. 2011. J
Clin Invest 121, 2750-2767). Of the plurality of genes in the XBP1
signature, a subset of genes were represented on the TNBC
microarray datasets (FIG. 7A, Table 1).
[0187] As shown in FIG. 7B, the activation of the XBP1 pathway, as
represented by the higher expression of the XBP1 signature,
correlates with shorter relapse-free survival (Log-rank test,
p=0.00768). These findings were confirmed in an independent
validation cohort of 193 TNBC patients (FIG. 7C, Log-rank test,
p=6.3.times.10.sup.-6).
[0188] We have identified both the UPR and the hypoxia response as
XBP1 dependent pathways in TNBC. Interestingly, growing evidence
indicates that increased expression of HIF1.alpha. and HIF1.alpha.
targets, such as CA9 and GLUT1, are associated with worse clinical
outcome in basal-like human breast tumors (Bos, R., et al. 2003.
Cancer 97, 1573-1581; Hussein, Y. R., et al. 2011. Transl Oncol 4,
321-327; Semenza, G. L., 2010. Oncogene 29, 625-634; Tan, E. Y., et
al. 2009. Br J Cancer 100, 405-411), consistent with the
association of XBP1 with TNBC. To understand the clinical relevance
of these two XBP1-regulated pathways in TNBC. we examined mRNA
expression levels of multiple UPR markers in TICs and NTICs derived
from five human TNBC patients. This analysis (survival analysis)
revealed up-regulation of these marker genes in TICs relative to
NTICs, indicative of an association of the UPR pathway with TICs
and TNBC. Intriguingly, we also found that an elevated expression
of the UPR gene signature in TNBC was associated with decreased
relapse free survival (Log-rank test, p=0.00911) (FIG. 7D).
[0189] Collectively these data demonstrate that activation of XBP1
in TNBC patients is associated with poor clinical outcome.
Discussion
[0190] Patients with TNBC have a relatively poorer prognosis and
are more likely to recur and develop metastatic disease than other
breast cancer subtypes (Foulkes, W. D., et al. 2010. N Engl J Med
363, 1938-48; Lehmann, B. D., et al. 2011. J Clin Invest 121,
2750-67). The genes linked to TNBC are not well understood and
thus, unlike other breast cancer subtypes, effective targeted
therapies have not yet been identified for TNBC (Foulkes, W. D., et
al. 2010. N Engl J Med 363, 1938-48). Here, by manipulating the
expression of XBP1, the key component of the most evolutionarily
conserved branch of the UPR, in a panel of breast cancer cell lines
and in the patient-derived xenograft model, a key function for XBP1
in TNBC was discovered. XBP1 was activated in TNBC cells, and
silencing of XBP1 was very effective in suppressing the
tumorigenicity and progression of TNBCs. In addition to its
essential role in TNBC, it is expected that XBP1 may also affect
other subtypes of human breast cancer. TNBC typically contains a
higher proportion of tumor-initiating cells (TICs) (Blick, T., et
al. 2010. J Mammary Gland Biol Neoplasia 15, 235-52; Ricardo, S.,
et al. 2011. J Clin Pathol 64, 937-46). Relative to NTICs, TICs are
resistant to chemotherapy, and contribute to a significantly higher
incidence of recurrence and distant metastasis (Smalley, M., et al.
2003. Nat Rev Cancer 3, 832-44; Stingl, J., et al. 2007. Nat Rev
Cancer 7, 791-9). Progress in targeting this subpopulation with
novel therapeutics continues to be hampered by our incomplete
knowledge of the molecular pathways contributing to TIC identity.
It is thus demonstrated herein that XBP1 is a novel regulator for
breast TICs.
[0191] These studies are the first to demonstrate that compromising
the ER stress response significantly impairs the TIC population. It
is speculated that TICs residing in the stem cell niche require
robust UPR activation to cope with external stress. Hence TICs rely
on XBP1 activation and their function is compromised in its
absence. The increased activation of XBP1 in TICs is intriguing and
provides potentially novel strategies to target this subpopulation
of cancer cells. Hypoxia is known to promote aggressive tumor
phenotypes and HIF1.alpha. was recently demonstrated to be
essential for TNBC and breast TICs (Schwab, L. P., et al. 2012.
Breast Cancer Res 14, R6; Conley, S. J., et al. 2012. Proc Natl
Acad Sci USA 109, 2784-9; Montagner, M., et al. 2012. Nature 487,
380-4). Increased HIF1.alpha.a levels are also associated with
increased metastasis and decreased survival in patients with TNBC
(Semenza, G. L., 2010. Oncogene 29, 625-34; Bos, R., et al. 2003.
Cancer 97, 1573-81). The data presented herein reveal that XBP1
acts in TNBC through regulating the HIF1.alpha. transcriptional
program. HIF1.alpha. requires XBP1 to sustain downstream target
expression. Hypoxia is a physiological inducer of the UPR in cancer
(Wouters, B. G., et al. 2008. Nat Rev Cancer 8, 851-64). In the
studies, it was found that XBP1 functions in a positive feedback
loop to sustain the hypoxia response via regulating HIF1.alpha.
transcriptional activity. This feed-forward circuit ensures maximum
HIF activity and an efficient adaptive response to the cytotoxic
microenvironment of solid tumors. HIF activity is tightly
controlled during tumor progression, through translational and
post-translational regulation of HIF1.alpha., but relatively less
is known about how HIF1.alpha. transcriptional activity is
controlled (Kaelin, W. G., Jr., et al. 2008. Mol Cell 30, 393-402).
These studies reveal a novel function for XBP1 as a HIF1.alpha.
transcriptional cofactor. Herein is proposed a model in which these
two critical pathways, the UPR and the hypoxia response, are
physically interconnected and act together to mount an appropriate
adaptive response to perpetuate cancer cells in the hostile tumor
microenvironment. These data highlight the importance of XBP1 in
TNBC progression and recurrence. Activation of the XBP1 pathway is
correlated with poor patient survival in human TNBC patients, hence
inhibition of this pathway may offer novel treatment strategies for
this aggressive subtype of breast cancer. The use of UPR inhibitors
in combination with standard chemotherapy may greatly enhance the
effectiveness of anti-tumor therapies.
Experimental Procedures
[0192] Detailed protocols for all experimental procedures are
provided below.
Cell Culture and Treatments
[0193] The non-transformed breast cell line MCF10A cells contains
ER-Src, an integrated fusion of the v-Src oncoprotein, and the
ligand-binding domain of estrogen receptor (ER) (Iliopoulos, D., et
al., 2009. Cell 139, 693-706). These cells were grown in DMEM/F12
medium supplemented with 5% donor horse serum (Invitrogen), 20
ng/ml epidermal growth factor (EGF) (R&D systems), 10 ug/ml
insulin (Sigma), 100 ug/ml hydrocortisone (Sigma), 100 ng/ml
cholera toxin (Sigma), 50 units/ml pen/step (Gibco), with the
addition of puromycin (Sigma). Src induction and cellular
transformation was achieved by treatment with 1 uM 4-OH tamoxifen
(TAM), typically for 36 h as described previously (Iliopoulos, D.,
et al. 2009. Cell 139, 693-706; Iliopoulos, D., et al. 2010. Mol
Cell 39, 761-72).
[0194] All breast cancer cells were cultured according to Neve, R.
M., et al. 2006. Cancer Cell 10, 515-27. Following retroviral or
lentiviral infection, cells were maintained in the presence of
puromycin (2 ug/ml) (Sigma). For all hypoxia experiments, cells
were maintained in an anaerobic chamber (Coy laboratory) with 0.1%
O.sub.2. For glucose deprivation experiments, cells were maintained
in DMEM without glucose medium (Gibco) with 10% FBS (Gibco) and 50
units/ml of penicillin/streptomycin.
Orthotopic Tumor Growth Assays
[0195] Six week old female NOD/SCID/IL2R.gamma.-/- mice (Taconic)
were used for xenograft studies. Approximately 1.5.times.10.sup.6
viable tumor cells were resuspended in 40 ul growth factor reduced
Matrigel (BD Biosciences) and injected orthotopically into mammary
gland four as previously described (Zhang, Q., et al. 2009. Cancer
Cell 16, 413-424). Mice were supplied with chow containing 6 g
doxycycline/kg (Bioserv) for treatment. For bioluminescent
detection and quantification of cancer cells, mice were given a
single i.p. injection of a mixture of luciferin (50 mg/kg),
ketamine (150 mg/kg), and xylazine (12 mg/kg) in sterile water.
Five minutes later, mice were placed in a light tight chamber
equipped with a charge coupled device IVIS imaging camera
(Xenogen). Photons were collected for a period of 1-60 s, and
images were obtained by using LIVING IMAGE 2.60.1 software
(Xenogen) and quantified using IGOR Pro 4.09 A image analysis
software (WaveMatrics). The imaging intensity was normalized to the
luminescence signal of each individual mouse taken before the
Doxycycline chow treatment. The average luminescence ratio of
treatment group (LacZ or XBP1 shRNA) was plotted over the course of
doxycycline chow treatment. Results are presented as
mean.+-.standard error of the mean (SEM).
Sorting of TICs and NTICs (General)
[0196] To separate TICs from NTICs, flow cytometric cell sorting
was performed on single-cell suspensions that were stained with
CD44 antibody (FITC-conjugated) and with CD24 antibody
(PE-conjugated) (BD Biosciences) for 30 min. As used throughout,
TICs are defined by the minority CD44.sup.high/CD24.sup.low
population, whereas NTICs are defined by the majority
CD44.sup.low/CD24.sup.high.
Purification of TICs and NTICs from Patients with TNBC
(Detailed)
[0197] Five human invasive triple negative ductal carcinoma tissues
(stage III) were used in our TIC experiments (Iliopoulos, D., et
al. 2011. Proc Natl Acad Sci USA 108, 1397-402). Immunomagnetic
purification of TICs and NTICs was performed according to
Shipitsin, M., et al. 2007. Cancer Cell 11, 259-73. Briefly, the
breast tissues were minced into small pieces (1 mm) using a sterile
razor blade. The tissues were digested with 2 mg/ml collagenase I
(C0130, Sigma) and 2 mg/ml hyalurimidase (H3506, Sigma) in 370C for
3 h. Cells were filtered, washed with PBS and followed by Percoll
gradient centrifugation. The first purification step was to remove
the immune cells by immunomagnetic purification using an equal mix
of CD45 (leukocytes), CD15 (granulocytes), CD14 (monocytes) and
CD19 (B cells) Dynabeads (Invitrogen). The second purification step
was to isolate fibroblasts from the cell population by using CD10
beads for magnetic purification. The third step was to isolate the
endothelial cells by using an "endothelial cocktail" of beads (CD31
BD Pharmingen cat no. 555444, CD146 P1H12 MCAM BD Pharmingen cat
no. 550314, CD105 Abcam cat no. Ab2529, Cadherin 5 Immunotech cat
no. 1597, and CD34 BD Pharmingen cat no. 555820). In the final step
the CD44high cells were purified from the remaining cell population
using CD44 beads.
[0198] These cells were sorted for CD44high/CD24low (TIC) cells,
CD24high cells were also purified using CD24 beads. These cells
were sorted for CD44low/CD24high (NTICs) cells. These TIC and NTIC
populations were sorted again with CD44 antibody (FITC-conjugated)
(555478, BD Biosciences) and CD24 antibody (PE-conjugated) (555428,
BD Biosciences) in order to increase their purity (>99.2% in all
cases).
Mammosphere Formation Assay
[0199] Mammospheres were generated by placing cell lines in
suspension (1,000 cells/ml) in serum-free DMEM/F12 media,
supplemented with B27 (1:50, Invitrogen), 0.4% BSA, 20 ng/mL EGF,
and 4 .mu.g/ml insulin. After 6 days of incubation, mammospheres
were typically >75 mM in size with 97% bearing the
CD44.sup.high/CD24.sup.low phenotype. For serial passaging, 6-day
old mammospheres were harvested using a 70 um cell strainer,
whereupon they were dissociated to single cells with trypsin and
then re-grown in suspension for 6 days.
ChIP and ChIP-seq
[0200] ChIP assays were carried out as described previously (Chen,
X., et al. 2008. Cell 133, 1106-1117). Briefly, cells were
crosslinked with 1% formaldehyde for 10 min at room temperature,
and formaldehyde was then inactivated by the addition of 125 mM
glycine. Chromatin extracts containing DNA fragments with an
average size of 500 bp were immunoprecipitated by using the
antibodies described below. All ChIP experiments were repeated at
least three times.
[0201] ChIP was performed with XBP1 antibody (Biolegend, 619502);
HIF1.alpha. antibody (Abcam, ab2185), RNA Polymerase II antibody
(Millipore, 05-623) or GST antibody (Santa Cruz, sc-33613). The
primers used in FIG. 6 are listed in Table 2.
TABLE-US-00002 SUPPLEMENTARY TABLE 2 ChIP primer sequence Gene
Forward JMJD1A 1 TGTTCCTTCAGGTTCAATAGAATTTTTCCC (SEQ ID NO:) JMJD1A
2 CATCATTCATTATGGCCTTCAACTACTTTA (SEQ ID NO:) JMJD1A 3
CTTTCCTGTGAGATTCTTCCGCCA (SEQ ID NO:) JMJD1A 4
GGGTCCGGGAGGCTGTGCGTGTCTTGTGAG (SEQ ID NO:) JMJD1A 5
TCCCACACCGACGTTACCAAGAAGGATCTG (SEQ ID NO:) JMJD2C 1
AACTTCAAGGGGAATCTATGTATTGTTCAT (SEQ ID NO:) JMJD2C 2
TCCCGTTAGCCTTAGCTCAATTAATCACAT (SEQ ID NO:) JMJD2C 3
TCCTTCTACGCGAGTATCTTTCCC (SEQ ID NO:) JMJD2C 4
GATTATCGCTTGCTTTCTTACCTTGCTGGC (SEQ ID NO:) VEGFA
TCTTCGAGAGTGAGGACGTGTGT (SEQ ID NO:) PDK1 CGCCCTGTCCTTGAGCC (SEQ ID
NO:) DDIT4 CTAGAGCTCGCGGTCTGGTCTGGTCT (SEQ ID NO:) NDRG1
AACACGTGAGCTAAGCTGTCCGA (SEQ ID NO:) BETA-ACTIN
GGGACTATTTGGGGGTGTCT (SEQ ID NO:) Control TGAGGGTTCATCAAGCTGGTGTCT
(SEQ ID NO:) Reference JMJD1A 1 Reverse JMJD1A 2
TGGCCTATCCTAAGGTGACGCTATGA (SEQ ID NO:) JMJD1A 3
GAAGAAAGGCGTGGAGTTACTGGATA (SEQ ID NO:) Xia et al., 2009 JMJD1A 4
CCGCGAAATCGGTTATCAACTTTGGG (SEQ ID NO:) JMJD1A 5
CGGCGCTTTCACCTTTCTCTCCCCTCT (SEQ ID NO:) JMJD2C 1
ACTCGGCTCTATACAACCATTCCAAA (SEQ ID NO:) JMJD2C 2
CTACTAGAAAATCAACTGGACTCATGGCAC (SEQ ID NO:) JMJD2C 3
CTGGGTCCCTTGTGGCGTTTTCTCTA (SEQ ID NO:) Xia et al., 2009 JMJD2C 4
GTCACGTGGGCTTACAAACAGCTT (SEQ ID NO:) VEGFA
ACTGTATTACCAAGTTTGCGGGATACTGTA (SEQ ID NO:) Lee et al., 2009 PDK1
AAGGCGGAGAGCCGGAC (SEQ ID NO:) Lee et al., 2009 DDIT4
CGGTATGGAGCGTCCCCT (SEQ ID NO:) NDRG1 GGCGAAGAGGAGGTGGACGACGACGAG
(SEQ ID NO:)AAG Xia et al., 2009 BETA-ACTIN
ATGGAGGCAGAAGGAACATGTGAG (SEQ ID NO:) Gromak et al., 2006 Control
TCCCATAGGTGAAGGCAAAG (SEQ ID NO:) Xia et al., 2009
[0202] The ChIP-seq library was prepared using ChIP-Seq DNA Sample
Prep Kit (Illumina) according to the manufacturer's instructions.
XBP1 ChIP-seq peaks were identified using MACS package (Zhang, Y.,
et al. 2008. Genome Biol 9, R137) with a p-value cutoff of
1.times.10.sup.-7.
Tumor Initiation Assay Using Patient-Derived Tumors
[0203] Tumorgraft line BCM-2147 was derived by transplantation of a
fresh patient breast tumor biopsy (ER-PR-HER2-) into the cleared
mammary gland fat pad of immune-compromised SCID/Beige mice and
retained the patient biomarker status and morphology across
multiple transplant generations in mice. To overcome the challenge
of limited cell viability by dissociation of solid tumors, 10 mg
tumor pieces containing 1.3.times.10.sup.5 cells were transplanted
with basal membrane extract (Trevigen, Gaithersburg, Md.). The cell
number was calculated as average cell yield 1.3.times.10.sup.7
cells/gram.times.0.01 gram=1.3.times.10.sup.5 cells. For sustained
siRNA release in the first two weeks following transplantation,
porous silicon particles loaded with siRNA (scrambled control or
XBP1 siRNA) packaged in nanoliposomes were injected into the tumor
tissue with basal membrane extract at the time of transplantation.
Scrambled sequence [5' CGAAGUGUGUGUGUGUGGCdTdT 3']; XBP1 siRNA
sequence [5' CACCCUGAAUUCAUUGUCUdTdT 3']. Two weeks
post-transplantation, nanoliposomes containing siRNA (15 mg per
mouse) were injected I.V. twice weekly for 8 weeks. Mice were
monitored thrice weekly for tumor development, and tumors were
calipered and recorded using LABCAT Tumor Analysis and Tracking
System v6.4 (Innovative Programming Associates, Inc., Princeton,
N.J.). Tumor incidence is reported at 10 weeks
post-transplantation.
Invasion Assay
[0204] We performed invasion assays according to 49. Invasion of
the matrigel was conducted by using standardized conditions with BD
BioCoat growth factor reduced MATRIGEL invasion chambers
(PharMingen). Assays were conducted according to manufacturer's
protocol, by using 5% horse serum (GIBCO) and 20 ng/ml EGF (R&D
Systems) as chemoattractants.
Colony Formation Assay
[0205] 1.times.105 breast cancer cells were mixed 4:1 (v/v) with
2.0% agarose in growth medium for a final concentration of 0.4%
agarose. The cell mixture was plated on top of a solidified layer
of 0.8% agarose in growth medium. Cells were fed every 6 to 7 days
with growth medium containing 0.4% agarose. The number of colonies
was counted after 20 days. The experiment was repeated three times
and the statistical significance was calculated using Student's t
test.
Subcutaneous Xenograft Experiments
[0206] MCF10A ER-Src TAM-treated (36 h) cells or MDA-MB-436 or
HBL-100 breast cancer cells were injected subcutaneously in the
right flank of athymic nude mice (Charles River Laboratories).
Tumor growth was monitored every five days and tumor volumes were
calculated by the equation V (mm.sup.3)=a.times.b.sup.2/2, where a
is the largest diameter and b is the perpendicular diameter. When
the tumors reached a size of .about.100 mm.sup.3 (15 days) mice
were randomly distributed into 3 groups (5 mice/group). The first
group was used as control (non-treated), the second group was
intratumorally treated with shCtrl and the third group was
intratumorally treated with shXBP1. For each injection 10 ug of
shRNA was mixed with 2 ul of vivo-jetPEI (polyethylenimine) reagent
(cat. no 201-50G, PolyPlus Transfection SA) in a final volume of
100 ul. These treatments were repeated every five days for 4 cycles
(days 15, 20, 25, 30). In addition, in vivo dilution
xenotransplantation assays were performed in
NOD/SCID/IL2R.gamma.-/- mice. Mice were evaluated on a weekly basis
for tumor formation. All mice were maintained in accordance with
Dana-Farber Cancer Institute Animal Care and Use Committee
procedures and guidelines.
Gene Expression Microarray Analysis
[0207] MDA-MB-231 cells infected with control shRNA or XBP1 shRNA
lentiviruses grown in glucose free medium were treated in 0.1%
O.sub.2 in a hypoxia chamber for 24 h. Total RNA was extracted by
using RNeasy mini kit with on column DNase digestion (QIAGEN).
Biotin labeled cRNA was prepared from 1 ug of total RNA,
fragmented, and hybridized to Affymetrix human U133 plus 2.0
expression array. All Gene expression microarray data were
normalized and summarized using RMA (Irizarry, R. A., et al. 2003.
Nucleic Acids Res 31, e15). The differentially expressed genes were
identified using Limma (Smyth, G. K., et al. 2003. Methods Mol Biol
224, 111-36) (q.ltoreq.10%, fold change .gtoreq.1.5).
Motif Analysis
[0208] Flanking sequences around the summits (.+-.300 bp) of the
top 1,000 XBP1 binding sites were extracted and the repetitive
regions in these flanking sequences were masked. The consensus
sequence motifs were derived using Seqpos (Lupien, M., et al. 2008.
Cell 132, 958-70).
XBP1 Signature Generation
[0209] The XBP1 signature was generated by integrative analysis of
ChIP-seq and differential expression data using the method as
previously described (Tang, Q., et al. 2011. Cancer Res 71,
6940-7). Briefly, we first calculated the regulatory potential for
a given gene, Sg, as the sum of the nearby binding sites weighted
by the distance from each site to the TSS of the gene:
S.sub.g=.SIGMA..sub.i=1.sup.ke.sup.-(0.5+4.DELTA..sup.i.sup.)
where k is the number of binding sites within 100 kb of gene g and
.DELTA.i is the distance between site i and the TSS of gene g
normalized to 100 kb (e.g., 0.5 for a 50 kb distance). We then
applied the Breitling's rank product method (Breitling, R., et al.
2004. FEBS Lett 573, 83-92; Klisch, T. J., et al. 2011. Proc Natl
Acad Sci USA 108, 3288-93) to combine regulatory potentials with
differential expression t-values to rank all genes based on the
probability that they were XBP1 targets. Only genes with at least
one binding site within 100 kb from its TSS and a differential
expression t-value above the 75th percentile were considered (Tang,
Q., et al. 2011. Cancer Res 71, 6940-7). The FDR of XBP1 target
prediction was estimated by permutation (Breitling, R., et al.
2004. FEBS Lett 573, 83-92). At a FDR cutoff of 10% and
differential expression fold-change cutoff of 1.5, we obtained 119
up-regulated genes (HUGO gene symbol) as direct targets of
XBP1.
Survival Analysis (General)
[0210] Principle component analysis (PCA) was applied to patient
expression profiles of genes of interest and separated the samples
into 2 groups based on the median value of the first component.
Kaplan-Meier survival analysis was used to assess the significance
of survival difference. In cases where XBP1 signature genes were
the relevant gene set, a correlation value was calculated between
the relevant gene expression indexes of each patient and those of
the MDA-MB-231 cell line, and the correlations of the 2 groups were
compared and the significance of difference was assessed by
t-test.
Survival Analysis (Detailed)
[0211] We performed survival analysis using an aggregated
compendium of gene expression profiles of 383 TNBC samples from 21
breast cancer datasets (Rody, A., et al. 2011. Breast Cancer Res
13, R97). Of the 119 XBP1 signature genes, 91 genes had
corresponding probes in this dataset. To avoid potential
confounding factors such as heterogeneity among the samples, we
randomly split all 383 TNBC samples into two datasets with similar
size (190 and 193 cases) and evaluated the correlation of the XBP1
gene signature with relapse free survival using these two datasets
respectively. We separated patients into two subgroups: one with
higher and the other with lower expression of XBP1 signature. The
subgroup classification was performed as described previously
(Marotta, L. L., et al. 2011. J Clin Invest 121, 2723-35). Patients
were considered to have higher XBP1 signature if they had average
expression values of all the genes in the XBP1 signature above the
60th percentile (Marotta, L. L., et al. 2011. J Clin Invest 121,
2723-35). Kaplan-Meier survival analysis was performed and log-rank
test was used to assess the statistical significance of survival
difference between these 2 groups. A similar analysis was performed
for the HIF pathway signature (VEGFA, PDK1, DDIT4, SLC2A1, KDM3A,
NDRG1, PFKFB3, PIK3CA, RORB, CREBBP, PIK3CB and EGLN1).
[0212] Virus Production and Infection
[0213] The Phoenix packaging cell line was used for the generation
of ecotropic retroviruses and all retroviral infections were
carried out as described previously (Martinon, F., et al. 2010. Nat
Immunol 11, 411-8). The 293T packaging cell line was used for
lentiviral amplification and all lentiviral infections were carried
out as previously described (Martinon, F., et al. 2010. Nat Immunol
11, 411-8). In brief, viruses were collected 48 and 72 hr after
transfection, filtered, and used for infecting cells in the
presence of 8 mg/ml polybrene prior to drug selection with
puromycin (2 .mu.g/ml). shRNA constructs were generated by The
Broad Institute. Targeting of GFP mRNA with shRNA served as a
control. Optimal targeting sequences identified for human XBP1 were
5'-GACCCAGTCATGTTCTTCAAA-3', and 5'-GAACAGCAAGTGGTAGATTTA-3',
respectively. Knockdown efficiency was assessed by real-time PCR
for XBP1.
[0214] Luciferase Assay
[0215] For FIG. 5H, MDA-MB-231 cells were co-transfected with
3.times.HRE luciferase (3.times.HRE-Luc) plasmid (Yan, Q., et al.
2007. Mol Cell Biol 27, 2092-102) and XBP1s overexpression
construct (Kaser, A., et al. 2008. Cell 134, 743-56) or control
vector by using Lipofectamine 2000 (Invitrogen). A Renilla
luciferase plasmid (pRL-CMV from Promega) was co-transfected as an
internal control. Cells were harvested 36 hr after transfection,
and the luciferase activities of the cell lysates were measured by
using the Dual-luciferase Reporter Assay System (Promega). For FIG.
5I, MDA-MB-231 cells were co-transfected with 3.times.HRE-Luc and
two inducible XBP1 shRNA construct (in pLKO-Tet-On vector) or
control shRNA construct by using Lipofectamine 2000 (Invitrogen).
Cells were treated with doxycycline for 48 h and hypoxia for 24 h
before the luciferase activities of the cell lysates were
measured.
[0216] Statistical Analysis
[0217] The significance of differences between treatment groups
were identified with a Student's t-test. P values of less than 0.05
were considered statistically significant.
[0218] Coimmunoprecipitation
[0219] Transfected cells were lysed in cell lysis buffer (50 mM
Tris HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 10%
glycerol with protease inhibitor cocktail) for 1 hour. M2 beads
(Sigma) were incubated with the whole cell extracts at 4.degree. C.
for overnight. The beads were washed with cell lysis buffer four
times. Finally, the beads were boiled in 2.times. sample buffer for
10 minutes. The eluents were analyzed by Western blot. Nuclear
extracts were used to perform the endogenous co-IP as described
previously (Xu, J., et al. 2010. Genes Dev 24, 783-98). Briefly, 5
mg of nuclear extracts were incubated with 5 ug of anti-HIF1.alpha.
antibody (Novus Biologicals, NB 100-479) at 4.degree. C. for
overnight. The protein complexes were precipitated by addition of
protein A agarose beads (Roche) with incubation for 4 hr at
4.degree. C. The beads were washed four times and boiled for 5 min
in 2.times. sample buffer.
[0220] Real-Time PCR Analysis
[0221] 1 ug of RNA sample was reverse-transcribed to form cDNA,
which was subjected to SYBR Green based real-time PCR analysis.
Primers used for .beta.-actin forward: 5'-CCTGTACGCCAACACAGTGC-3'
and reverse 5'-ATACTCCTGCTT GCTGATCC-3'; for VEGFA forward
5'-CACACAGGATGGCTTGAAGA-3' and reverse 5'-AGGGCAGAATCATCACGAAG-3';
for PDK1 forward 5'-GGAGGTCTCAACACGAGGTC-3' and reverse
5'-GTTCATGTCACGCTGGGTAA-3'; for GLUT1 forward
5'-TGGACCCATGTCTGGTTGTA-3' and reverse 5'-ATGGAGCCCAGCAGCAA-3'; for
JMJD1A forward 5'-TCAGGTGACTTTCGTTCAGC-3' and reverse
5'-CACCGACGTTACCAAGAAGG-3'; for DDIT4 forward
5'-CATCAGGTTGGCACACAAGT-3' and reverse 5'-CCTGGAGAGCTCGGACTG-3';
for MCT4 forward 5'-TACATGTAGACGTGGGTCGC-3' and reverse 5'
CTGCAGTTCGAGGTGCTCAT-3'; for XBP1 splicing forward
5'-CCTGGTTGCTGAAGAGGAGG-3' and reverse 5'-CCATGGGGAGATGTTCTGGAG-3';
for XBP1 total forward 5'-AGGAGTTAAGACAGCGCTTGGGGATGGAT-3' and
reverse 5'-CTGAATCTGAAGAGTCAATACCGCCAGAAT-3'.
[0222] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0223] This invention is further illustrated by the following
examples which should not be construed as limiting.
EQUIVALENTS
[0224] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *