EP2391372A1

EP2391372A1 - Methods for treating estrogen receptor positive cancer by x-box binding protein 1 inhibition

Info

Publication number: EP2391372A1
Application number: EP10736464A
Authority: EP
Inventors: David Kiewlich
Original assignee: Bayer Healthcare LLC
Current assignee: Bayer Healthcare LLC
Priority date: 2009-01-30
Filing date: 2010-01-29
Publication date: 2011-12-07
Also published as: WO2010088498A1; EP2391372A4; CA2749947A1

Abstract

Provided is a method of inhibiting proliferation of estrogen receptor alpha positive tumor cells comprising the inhibition of X-box binding protein-1 (Xbp1), including inhibiting estrogen-induced proliferation.

Description

METHODS FOR TREATING ESTROGEN RECEPTOR POSITIVE CANCER BY X-BOX BINDING PROTEIN 1 INHIBITION

REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority to U.S. Provisional Application No. 61/148,518, filed 30 January 2009 which is hereby incorporated by reference.

FIELD

[002] The present application relates to X-box Binding Protein 1 , Xbp1 , and methods for treating estrogen receptor alpha (ERa) positive cancer by the inhibition of Xbp1 , particularly breast cancer.

REFERENCE TO SEQUENCE LISTING

[003] This application incorporates by reference the attached sequence listing in both paper and electronic copy in .txt format, created January 19, 2009. Applicant further certifies that the content contained in the paper and electronic copies are identical.

BACKGROUND

[004] Estrogen is essential for the growth and survival of the ductal epithelial cells of the breast, as well as most primary breast cancers. Estrogen binds to the estrogen receptor (ER), a member of the superfamily of nuclear hormone receptors that act as transcription factors. While there are two distinct estrogen receptors, alpha (ERa) and beta (ERβ), only ERa is believed to play a major role in primary breast cancer. Upon binding to estrogen, the dimerized receptor binds to specific DNA sequences called estrogen response elements (EREs) and modulates transcription. [005] Gene expression profiling studies of breast tumors established groups of genes that were co-expressed with ERa and included the transcription factor Xbp1 . The high expression of Xbp1 in ERa positive cancers was confirmed by immunohistochemistry of primary breast cancer specimens. Xbp1 is a basic leucine zipper-containing transcription factor that is a key mediator of a cell program called the unfolded protein response (UPR). The UPR is a survival response that is activated when there is an overload of unfolded or misfolded proteins in the endoplasmic reticulum (EnR). Although the detection machinery is not fully understood, the UPR can be activated by hypoxia or nutrient starvation, common conditions in solid tumors, or through the action of drugs, such as tunicamycin (Tm). Tm blocks the glycosylation of proteins, which, in turn, interferes with protein folding. Once the UPR is initiated, a 26 bp fragment from the Xbp1 mRNA is removed generating a translationally frameshifted protein designated Xbp1 S. [006] A potential explanation for the co-expression of Xbp1 and ERa has been determined, where over-expression of Xbp1 and ERa led to estrogen independent expression of an ERE- driven reporter gene. The increase in reporter gene transcription was still sensitive to anti- estrogens. This study also showed that Xbp1 and ERa proteins physically interact. Subsquent research has shown Xbp1 acts to modify and relax chromatin, although it was not clear if this was the reason for the increased ERa transcriptional activity of the reporter gene. More recently, over- expression of Xbp1 allowed MCF7 cells to continue cell division in the presence of tamoxifen (OHT), a selective estrogen receptor modulator.

[007] While these studies have shown that over-expression of Xbp1 increases the activity of ERa, it has not been determined if Xbp1 is required for ERa driven growth or transcription.

DESCRIPTION OF THE DRAWINGS

[008] Figure 1. Optimization of Xbp1 si RNA knockdown in MCF7 cells, a. MCF7 Cells were transfected with a panel of siRNAs targeting Xbp1 mRNA using RNAiMAX lipid. Taqman analysis was performed to determine expression level of Xbp1 mRNA and results were normalized to human, large ribosomal protein mRNA (human PO) and graphed with the level of expression in untreated cells set at 1 . Inset: Western blotting shows that siRNA 391 , 392, 474 and 447 all result in reduction of Xbp1 S protein following induction by 10ug/ml Tunicamycin for 4 hours. Equal loading was determined by anti-actin monoclonal antibody (lower band), b. MCF7 cells were transfected with lipid alone (Lipid), negative control siRNA (NEGsi) negative contol siRNA pool (NEGP), Dharmacon's Xbp1 targeting pool of siRNAs (X-Pool), or single Xbp1 targeting siRNAs (391 , 392, 447 or 474) with either RNAiMAX lipid (RNAi)or Hyperfect lipid (Hyper) for 1 , 2 or 3 days. Xbp1 mRNA expression level was determined by Taqman analysis, normalized to human PO and graphed with the level of expression in lipid treated only cells set at 1 for each time point and lipid type. Error bars represent 1 standard deviation.

[009] Figure 2. Xbp1 Knockdown Prevents Estrogen Induced MCF7 Cell Growth. MCF7 cells were transfected with siRNAs (Xbp1 siRNA-391 , -392), targeting Xbp1 or negative control siRNA (Neg siRNA), or Hyperfect lipid alone (Lipid) and starved for estrogens for 2 days. Cells were then incubated with 10nM E2 or 10nM E2 and 1 uM ICI 182,780 (1 uM ICI) as a control for ERa dependent growth. Growth was monitored with WST over 7 days. Cell number was normalized at day 1 and data is graphed as fold change. Error bars are 1 standard deviation.

[010] Figure 3. Xbp1 Knockdown Prevents Estrogen Induced Induction of ERa Regulated Genes. MCF7 with just medium (No Treat), Hyperfect lipid only (Lipid), or transfected with negative control siRNA (Neg siRNA), negative control siRNA pool (Neg Pool), siRNAs targeting Xbp1 (Xbp1 siRNA-391 , -392) or Dharmacon siRNA pool targeting Xbp1 (X-Pool) starved for estrogens for 2 days, as in Figure 2, then cells were stimulated with 1 nM E2 for either 4 or 24 hours. RNA expression levels were determined by Taqman, normalized with human PO and graphed relative to the DMSO induced, media only cells set at 1 . a. Relative expression of Xbp1 mRNA (XBP), b. Cyclin D1 mRNA (CyclinDI ), c. insulin-like growth factor binding protein 4 mRNA (IGFBP4), d. Treefoil factor 1 mRNA (TFF1 ). Error bars represent 1 standard deviation.

[011 ] Figure 4. Xbp1 Knockdown Does Not Affect Expression of ERa mRNA. MCF7 with just medium (No Treat), Hyperfect lipid only (Lipid), or transfected with negative control siRNA (Neg siRNA), negative control siRNA pool (Neg Pool), siRNAs targeting Xbp1 (Xbp1 siRNA-391 , - 392) or Dharmacon siRNA pool targeting Xbp1 (X-Pool) starved for estrogens for 2 days, as in Figure 3, then cells were stimulated with 1 nM E2 for either 4 or 24 hours. ERa mRNA expression levels were determined by Taqman, normalized with human PO and graphed relative to the DMSO induced, media only cells set at 1 . Error bars represent 1 standard deviation.

SUMMARY

[012] Provided is a method for modulating proliferation of cancer cells comprising contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa). In some embodiments, proliferation is inhibited.

[013] Also provided is a method of treating cancer comprising administering a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cancer is an estrogen receptor alpha positive cancer.

[014] In some embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, hepatocarcinoma and B-cell lymphoma. Thus, in some embodiments, the cancer cells are selected from the group consisting of breast cells, ovarian cells, endometrium cells, liver cells and B-cell lymphocytes.

[015] Additionally, provided is a method for inhibiting estrogen receptor alpha tumor cells from utilizing estrogen as a growth factor comprising contacting a human X-box binding protein-1 (Xbp1 ) inhibitor with the estrogen receptor alpha tumor cell, whereby estrogen-induced proliferation of the estrogen receptor alpha tumor cells is inhibited.

[016] Further, provided is a method of inhibiting estrogen-induced proliferation of breast tumor cells comprising contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa).

[017] In some embodiments, the Xbp1 inhibitor is an antisense nucleic acid molecule, an anti- Xbp1 antibody, antibody mimetic, a siRNA oligonucleotide, an aptamer, a soluble recombinant Xbp1 protein fragment, a small molecule, a peptide or a peptide mimetic. In some embodiments, the siRNA oligonucleotide is one of SEQ ID NO: 2-5.

DETAILED DESCRIPTION

[018] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[019] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an inhibitor" is a reference to one or more inhibitors and includes equivalents thereof known to those skilled in the art, and so forth.

[020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[021] All publications and patents mentioned herein are hereby incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

X-Box Binding Protein-1 and Breast Cancer

[022] X-Box Binding protein 1 (Xbp1 , cDNA sequence provided in SEQ ID NO:1 ) is a basic leucine zipper-containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum. Most primary breast cancers are ERa positive and are dependent on ERa transcriptional activity for growth and survival. Xbp1 is shown to be highly expressed in ERa positive breast cancer cell lines and clinical isolates.

[023] In normal ERa positive ductal cells of the breast, estrogen stimulation does not induce growth. However, most ERa positive cancers, which are believed to be derived from ERa positive ductal cells, rely on estrogen for growth and survival. The majority of ERa regulated genes have been determined from analysis of cancer cell lines, and for breast cancer. In this background, there is high expression of Xbp1 that is not seen in the limited studies of normal, ERa positive breast cells.

[024] MCF7 cells, which are a model of estrogen receptor alpha (ERa) positive breast cancer, express high levels of Xbp1 and are dependent on estrogen for growth and ERa regulated transcription. Further overexpression of Xbp1 in ERa positive breast cancer cells, including MCF7, leads to estrogen independent ERa regulated transcription and anti-estrogen resistance, suggesting that Xbp1 expression influences ERa regulated transcription. However, it is yet to be shown whether ERa requires Xbp1 as a cofactor for transcription.

X-Box Binding Protein-1 Inhibition

[025] MCF7 cells are unable to grow in response to estrogen and ERa regulated gene expression is inhibited when Xbp1 mRNA is knocked down (see Figs. 1 and 2). These results, together with the high expression of Xbp1 seen in ERa positive breast cancers, but not in normal mammary ductal cells, suggests that high levels of Xbp1 enable ERa tumor cells to use estrogen as a growth factor. Thus, high levels of Xbp1 expression could represent an early transformation step leading to ERa positive breast cancer, and could explain why a normal ERa positive breast ductal cell line has not been cultured. Thus, it is suggested that the interaction of Xbp1 and ERa results in fundamental changes in the repertoire of genes positively regulated by estrogen. In particular, our results suggest that high expression of Xbp1 enables ERa positive breast cells to use estrogen as a growth factor.

[026] Thus, estrogen regulated growth of an ERa positive breast cancer model cell line is dependent on the presence of Xbp1 . In the absence of Xbp1 , the cells seem to be unable to respond to estrogen stimulation. This potentially explains why normal, ERa positive breast cells that have low levels of Xbp1 expression do not grow in response to estrogen stimulation and why, thus far, these cells have never been cultured as a primary culture or cell line. Therefore, the upregulation of Xbp1 could represent an early, critical transformation step in the carcinogenesis of the breast allowing ERa positive breast cells to use estrogen as a growth factor.

[027] The impact of inhibition of Xbp1 expresssion, including siRNA knockdown of Xbp1 mRNA, on estrogen-regulated growth and ERa transcriptional activity was studied. It is found that knockdown of Xbp1 mRNA renders a model cell line for ERa positive breast cancer unable to grow in response to estrogen and inhibits the estrogen regulated ERa transcriptional activation of a number of ERa regulated genes.

Methods of Use

[028] As used herein, various terms are defined below.

[029] The term "treatment" includes any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject.

[030] The phrase "therapeutically effective" means the amount of agent administered that will achieve the goal of improvement in a disease, condition, and/or disorder severity, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment. [031] The term "pharmaceutically acceptable" means that the subject item is appropriate for use in a pharmaceutical product.

[032] Accordingly, one embodiment of this invention provides for a method of treating cancer comprising administering a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cancer is an estrogen receptor alpha positive cancer.

[033] Typically, estrogen receptor alpha is expressed in breast cancers. However, other cancers are also estrogen receptor alpha positive. For example, in some embodiments, the estrogen receptor alpha positive cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, hepatocarcinoma and B-cell lymphoma. Thus, in some embodiments, the cancer cells are selected from the group consisting of breast cells, ovarian cells, endometrium cells, liver cells and B-cell lymphocytes.

[034] The term "combination therapy" or "co-therapy" means the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder. Such administration encompasses co-administration of two or more therapeutic agents in a substantially simultaneous manner or administration of each type of therapeutic agent in a sequential manner.

[035] Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a Xbp1 inhibitor or variant thereof and one or more additional therapeutic agents, as well as administration of Xbp1 inhibitor or variant thereof and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, Xbp1 inhibitor or variant thereof and a therapeutic agent may be administered to the patient together in a single dosage composition or each agent may be administered in separate dosage formulations.

[036] Where separate dosage formulations are used, the Xbp1 inhibitor or variant thereof and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).

[037] Also provided is a method for modulating proliferation of breast cancer cells, wherein the method comprises contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa). In some embodiments, proliferation is inhibited.

[038] Additionally, provided is a method for inhibiting estrogen receptor alpha tumor cells from utilizing estrogen as a growth factor comprising contacting a human X-box binding protein-1 (Xbp1 ) inhibitor with the estrogen receptor alpha tumor cell, whereby estrogen-induced proliferation of the estrogen receptor alpha tumor cells is inhibited.

[039] Further, provided is a method of inhibiting estrogen-induced proliferation of breast tumor cells comprising contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa). [040] In some embodiments, the Xbp1 inhibitor is an antisense nucleic acid molecule, an anti- Xbp1 antibody, an antibody mimetic, a siRNA oligonucleotide, an aptamer, a soluble recombinant Xbp1 protein fragment, a small molecule, a peptide or a peptide mimetic.

[041] In some embodiments, the Xbp1 inhibitor is used to block expression of Xbp1 . In other embodiments, the Xbp1 inhibitor is to block Xbp1 activity. For example, since it is known that Xbp1 and ERa physically interact, either directly or within a complex, then inhibition may be preventing the interaction of Xbp1 with ERa. An inhibitor, such as a small molecule, antibody, or antibody mimetic, could therefore inhibit that interaction and may block Xbp1 's influence on the ERa transcriptional activity.

[042] In some embodiments, the siRNA oligonucleotide is one of those listed in Table I.

TABLE I. Xbp1 siRNA oligonucleotides

Pharmaceutical compositions

[043] The Xbp1 inhibitor or a variant thereof as described herein may be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be non-pyrogenic. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers may be employed including, but not limited to saline, glycine, or the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and the like. The concentration of Xbp1 inhibitor or variant thereof in such pharmaceutical formulation may vary widely, and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration.

[044] The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which may be used pharmaceutically. Pharmaceutical compositions of the invention may be administered by subcutaneous means.

[045] Formulations suitable for subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20^th edition, 2000).

Determination of a Therapeutically Effective Dose

[046] The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to the amount of an agent that may be used to effectively treat a disease (e.g., breast cancer) compared with the efficacy that is evident in the absence of the therapeutically effective dose.

[047] The therapeutically effective dose may be estimated initially in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.

[048] The exact dosage may be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration may be adjusted to provide sufficient levels of the agent or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.

EXAMPLES

[049] It will be apparent to those skilled in the art that the examples and embodiments described herein are by way of illustration and not of limitation, and that other examples may be used without departing from the spirit and scope of the present invention, as set forth in the claims.

Example 1. Materials and Methods

[050] All experiments were performed at least 3 times and error bars represent one standard deviation. Paired Student's T-Test were used to determine p-values.

Reagents and antibodies: [051] Anti-Xbp1 S polyclonal antibody was purchased from BioLegend (San Diego, CA). Anti- Rabbit HRP labeled antibody was purchased from Pierce Biotechnology (now Thermo Scientific, Rockford, IL). 17-β-estradiol (E2) and tunicamycin (Tm) were purchased from Sigma-Aldrich (St. Louis, MO). IC 1-182,780 (ICI), was purchased from Tocris Biosciences (Ellisville, MO), stock solutions of 1 mM were prepared in DMSO. siRNAs 1 1 1391 (Xbp1 391 , sequence: 5'- gctggaacagcaagtggtagattta-3', SEQ ID NO: 2) and 1 1 1392 (Xbp1 392, 5'- ccttgtagttgagaaccaggagtta-3', SEQ ID NO: 3) (Invitrogen Carlsbad, CA) and 5447 (Xbp1 447, 5'- ggctcgaatgagtgagctg-3', SEQ ID NO: 4), 139474 (Xbp1 474, 5'-gggtggtagttttccctaa-3', SEQ ID NO: 5) and non-targeting siRNA (Neg siRNA) (Applied Biosciences, Foster City, CA), were prepared in manufacture's buffer at 20 μM and stored at -80 °C. RNAiMAX was purchased from Invitrogen. HiPerfect was purchased from Qiagen (Valencia, CA).

Cell Lines and Tissue Culture:

[052] Breast cancer cell line MCF7 was obtained from the ATCC. Cells were cultured in growth medium consisting of MEM Alpha (Gibco/lnvitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (ATCC Maryland, MD), 10 ml/L non-essential amino-acids, 1 mM Na Pyruvate, 10 mM HEPES, 2 mM Glutamax-1 and 0.5 mg/ml insulin (Gibco/lnvitrogen). The trypsin replacement TripLE Express solution (Gibco/lnvitrogen) was used to passage the cells.

[053] For estrogen-induction experiments, cells were cultured in NPR medium consisting of phenol red-free MEM Alpha (Gibco/lnvitrogen), supplemented with 5% dextran/activated charcoal stripped fetal bovine serum (HyClone, now Thermo Fisher, Logan, UT), 10 ml/L non-essential amino-acids, 1 mM Na Pyruvate, 10 mM HEPES and 2 mM Glutamax-1 (Gibco/lnvitrogen). siRNA knockdown:

[054] MCF7 cells were seeded at 10⁶ cells in a 10 cm plate. siRNAs (non-targeting or targeting Xbp1 ) at a final concentration of 5 nM were mixed with RNAiMAX or HiPerfect in OptiMEM (Invitrogen Carlsbad, CA) or lipid alone in OptiMEM (10% final concentration) then added to cells in NPR medium (90% final concentration) for 2 days. Those samples used for the western blot inset in Figure 1 a were transfected in growth media and the UPR triggered with 10 μg/ml Tunicamycin for 4 hours.

E2 Dependent Growth Study:

[055] MCF7 cells were transfected as above, then plated at 5 x 10³ per well in quadruplicate in 96 well plates in NPR medium. Twenty-four (24) hours after plating, one plate was analyzed with WST according to manufacturer's instructions and all samples were normalized to 1 . All additional plates were treated with 10 nM E2 in NPR medium, or 10 nM E2 with 1 uM ICI 182,780 as a control for inhibition of ERa regulated growth, and one plate was analyzed by WST at each time point. Medium was replaced every 2-3 days.

E2 Dependent Gene Expression: [056] MCF7 cells were transfected as above and cells were plated at a density of 2 x 10⁵ cells/well of a 6-well tissue culture treated plate (Corning, Lowell, MA) in NPR medium before E2 stimulation. MCF7 cells were stimulated with 1 nM final concentration E2 for 4 or 24 hours and RNA was purified wth the AIIPrep DNA/RNA/Protein Mini Kit from Qiagen according to the manufacturer's instructions except total RNA was eluted in H2O supplemented with the RNAsecure reagent (Applied Biosciences, Foster City, CA). RNA concentrations were determined on an Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences, Now GE Healthcare, Pittsburgh, PA) and had an OD 260/280 of at least 1 .6. RNA integrity was determined by denaturing gel electrophoresis.

Real Time PCR:

[057] Total RNA was reverse transcribed with Superscript III (Invitrogen) according to the manufacture's instructions. Briefly, 2-3 μg of RNA was added to a mix of dNTPs (Invitrogen), random pentadecamers (15N, Operon, Huntsville, AL), RNasin Plus (Promega, Fitchburg, Wl) and heated to 65 °C for 5 min, then rapidly cooled in an ice water bath for 5 minutes. To this was added the Superscript III first strand buffer, DTT and Superscript III enzyme. This was incubated at 25 O for 15 min, 50 ⁰C for 90 min and then 75 ⁰C for 15 min. The cDNA mix was diluted 50 fold and used immediately or stored at -80 °C until needed.

[058] Real time PCR (Taqman) was performed on a Stratagene 3000P Taqman machine (Stratagene, Now Agilent Technologies, La JoIIa, CA) using the Taqman universal buffer and inventoried Taqman gene assays (Applied Biosystems, Foster City, CA) as per the manufacturer's recommended conditions. Samples were normalized using the human large ribosomal protein (human PO) Taqman assay (Applied Biosystems).

Western Blotting:

[059] Western blotting was preformed according to (20). Briefly, 30 μg of each cell lysate was separated in a 4-12% NuPAGE gel (Invitrogen) and transferred with the iBLOT system (Invitrogen). Equal loading was verified by Ponceau S (Sigma-Aldrich) staining. Membranes were blocked with Protein Free blocker (Pierce Biotechnology) with 5% non-fat milk, 0.05% TWEEN®- 20 (polyoxyethylenesorbitan monolaurate) and 0.01 % SDS. Antibodies were diluted in the blocking buffer. Blots were rinsed with H2O then washed 3x with PBS with 0.05% TWEENΘ-20. HRP labeled anti-Rabbit secondary antibody, for the Biolegend anti-Xbp1 S antibody, was diluted in blocking buffer. Blots were washed as above. HRP was detected by SuperSignal Dura (Pierce Biotechnology) and BioMax MR film (Kodak, Rochester, NY).

Example 2. Validation of Xbp1 siRNA knockdown

[060] Since there have been very few reports of siRNA mediated Xbp1 knockdown, we tested a panel of siRNAs targeting Xbp1 on the model ERa positive breast cancer cell line MCF7 to determine those which would result in the most effective knockdown of Xbp1 mRNA. Various Xbp1 siRNAs were transfected into MCF7 cells. Taqman analysis showed that all tested siRNAs against Xbp1 resulted in substantial knockdown of Xbp1 (Figure 1 a). To determine that the RNA knockdown seen by the Taqman also resulted in reduction in protein, cells were transfected with the siRNAs as above and then challenged with Tm to generate Xbp1 S protein, which is more readily detectable than the Xbp1 unspliced (Xbp1 U) protein. The protein knockdown was analyzed by western blotting (Figure 1 a inset). Four siRNAs, (391 , 392, 447 and 474) that were the most effective in knocking down both Xbp1 mRNA and protein levels were chosen for future studies.

[061] To optimize siRNA transfection, MCF7 cells were transfected with the 4 chosen siRNAs using two different lipids, and the expression of Xbp1 mRNA was determined over three days. While the lipids alone or the negative siRNAs had no effect on Xbp1 mRNA expression, the 4 Xbp1 -targeting siRNAs all effectively reduced Xbp1 mRNA expression (Figure 1 b). Further, the knockdown of Xbp1 mRNA was considerably longer lasting when the siRNAs were transfected with the HyperFect lipid (>3 days) compared to the RNAiMAX lipid (~1 day, Figure 1 b). Since the knockdown was superior with the HyperFect lipid, all subsequent experiments used this lipid exclusively.

Example 3. Xbp1 Knockdown Inhibits Estrogen Dependent Growth of MCF7 Cells

[062] Since estrogen dependent growth is one hallmark of MCF7 cells, we first decided to test the impact of Xbp1 knockdown on growth. MCF7 cells were transfected with siRNAs targeting Xbp1 , a negative control siRNA, or lipid alone and starved for estrogens for 2 days. Cells were then incubated with 10 nM 17- β -estradiol (E)2 or 10 nM E2 and 1 μM ICI 182,780 (ICI) to block ERa dependent activity and monitored for growth for the next 7 days. Over the 7 days, the untreated control cells doubled approximately twice in response to E2 stimulation as did cells treated with the negative control siRNA, or with lipid alone (Figure 2). However, cells transfected with siRNAs targeting Xbp1 , as well as the cells treated with ICI, failed to grow in response to E2.

Example 4. Xbp1 Knockdown Inhibits Expression of Several Estrogen Response Genes

[063] To verify the effect of Xbp1 knockdown on ERa driven transcription specifically, we looked at the expression of several well established estrogen regulated genes. MCF7 cells were transfected with siRNAs as per the above growth studies (Figure 2) but cells were stimulated with 1 nM E2 for only 4 or 24 hours. As expected, the lipid only and negative control siRNA did not affect induction of Xbp1 mRNA in response to estrogen, (Figure 3a). The siRNAs targeting Xbp1 did result in reduction of Xbp1 mRNA, and while there was some increase of Xbp1 mRNA in response to estrogen at 4 and 24 hours, that increase was small (Figure 3a). Since the expression of Xbp1 was effectively suppressed even with estrogen stimulation, several known estrogen- regulated genes were assayed to determine their expression levels. The estrogen induction of all three genes were significantly reduced when Xbp1 was knocked down, but unaffected by the lipid or negative control siRNA (Figure 3b-d). In results not shown, we have observed reduction of 3 additional estrogen regulated genes. One possible explanation for this effect could be that knockdown of Xbp1 could be affecting the expression of ERa. However, we do not see any impact on ERa mRNA expression levels by any of the tested conditions (Figure 4).

[064] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. 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.

Claims

1 . A method for modulating proliferation of cancer cells comprising contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa).

2. The method of claim 1 , wherein the Xbp1 inhibitor is selected from the group consisting of antisense nucleic acid molecules, anti-Xbp1 antibodies, antibody mimetics, siRNA oligonucleotides, aptamers, soluble recombinant Xbp1 protein fragments, small molecules, peptides and peptide mimetics.

3. The method of claim 3, wherein the siRNA oligonucleotides are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

4. The method of claim 1 , wherein the proliferation of the cancer cells is inhibited.

5. The method of claim 1 , wherein the cancer cells are selected from the group consisting of breast cells, ovarian cells, endometrium cells, liver cells and B-cell lymphocytes.

6. A method for treating cancer comprising administering a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cancer is an estrogen receptor alpha positive cancer.

7. The method of claim 6, wherein the Xbp1 inhibitor is selected from the group consisting of antisense nucleic acid molecules, anti-Xbp1 antibodies, antibody mimetics, siRNA oligonucleotides, aptamers, soluble recombinant Xbp1 protein fragments, small molecules, peptides and peptide mimetics.

8. The method of claim 7, wherein the siRNA oligonucleotides are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

9. The method of claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, hepatocarcinoma and B-cell lymphoma.

10. A method for inhibiting estrogen receptor alpha tumor cells from utilizing estrogen as a growth factor comprising contacting a human X-box binding protein-1 (Xbp1 ) inhibitor with the estrogen receptor alpha tumor cell, whereby estrogen-induced proliferation of the estrogen receptor alpha tumor cells is inhibited.

1 1 . The method of claim 10, wherein the Xbp1 inhibitor is selected from the group consisting of antisense nucleic acid molecules, anti-Xbp1 antibodies, antibody mimetics, siRNA oligonucleotides, aptamers, soluble recombinant Xbp1 protein fragments, small molecules, peptides and peptide mimetics.

12. The method of claim 1 1 , wherein the siRNA oligonucleotides are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

13. A method of inhibiting estrogen-induced proliferation of tumor cells comprising contacting the cell with a human X-box binding protein-1 (Xbp1 ) inhibitor, wherein the cells express estrogen receptor alpha (ERa).

14. The method of claim 13, wherein the Xbp1 inhibitor is selected from the group consisting of antisense nucleic acid molecules, anti-Xbp1 antibodies, antibody mimetics, siRNA oligonucleotides, aptamers, soluble recombinant Xbp1 protein fragments, small molecules, peptides and peptide mimetics.

15. The method of claim 14, wherein the siRNA oligonucleotides are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

16. The method of claim 13, wherein the tumor cells are from cancers selected from the group consisting of breast cells, ovarian cells, endometrium cells, liver cells and B-cell lymphocytes.