WO 2014/113820 PCT/US2014/012405 [001] Title [002] Estrogen Receptor Inhibitors [003] Cross-Reference To Related Applications 5 [004] This application claims priority to U.S. Provisional Patent Application No. 61/754,292, for Estrogen Receptor Alpha Inhibitors, to Shapiro, et al., filed on 18 January 2013, which is incorporated herein in its entirety. [005] Statement Regarding Federally Sponsored Research Or Development 10 [006] This invention was made with government support under contract number PHS R01 DK01 71909 awarded by the National Institutes of Health. The government has certain rights in the invention. [007] Backqround 15 [008] Estrogen Receptor in Human Disease [009] The hormone estrogen binds to a protein called the estrogen receptor (ER). The complex of estrogen and the estrogen receptor bind to specific DNA sequences in the cell nucleus causing or blocking the copying of the nearby DNA and stimulating or decreasing the production of the RNA blueprints that specify the 20 production of proteins that stimulate cell division and migration and cell death. The estrogen-ERa complex plays a role in the growth and spread (metastases) of many cancers and in endometriosis. Although the roles of estrogens and estrogen receptor are best understood in breast cancer, estrogens and estrogen receptor are known to play important roles in ovarian, uterine/endometrial, cervical, liver and lung cancer. In 25 several other cancers, including, colon pancreatic and brain, ERa is often present, but a direct role of estrogens has not been demonstrated. The important role of estrogens in breast cancer is illustrated by the widespread therapeutic use of aromatase inhibitors that block estrogen production and the selective estrogen receptor modulators tamoxifen and Faslodex/fulvestrant/ICI 182,780 (ICI: ICI 30 182,780 (Imperial Chemical Industries 182,780; also known as Faslodex and fulvestrant) that work by competing with estrogens for binding to estrogen receptor a (ERa). [010] While these therapies are extremely useful, tumors eventually develop resistance. Many of these resistant breast tumors still require ERa for growth but do 1 WO 2014/113820 PCT/US2014/012405 not require estrogens for growth. Still others no longer require ERa protein for growth, but still contain ERa. These two classes of resistant tumors do not respond to current therapies based on aromatase inhibitors that inhibit the synthesis of estrogens, or antagonists, such as tamoxifen or fulvestrant/Faslodex that prevent 5 estrogen binding to ERa. [011] Most epithelial ovarian cancer presents at an advanced stage (Stage 3 or 4). Although 30-70% of these tumors are ERa positive, endocrine therapy for ovarian cancer using agents developed to treat ERa* breast cancer is largely ineffective. Since endocrine therapy is ineffective, tumors that recur after surgery are 10 treated with combination chemotherapy, usually with paclitaxel and cisplatin. Although most tumors are initially responsive to combination chemotherapy using taxanes and platinum, after several cycles of treatment tumors recur as resistant ovarian cancer. Therapeutic options for these resistant tumors are poor and approximately two thirds of patients die within 5 years. 15 [012] It is widely accepted that by stimulating the growth of endometrial cells, estrogens, acting via ERa, play an important role in endometriosis. In this disease, endometrial cells, which normally line the uterus, detach from the uterus, attach at sites outside the uterus, including the ovaries, pelvic lining, bowel and rectum and proliferate in response to estrogen binding to ERa, leading to pain and infertility. 5 20 10% of premenopausal women in the United States have symptom of endometriosis. Current therapies for endometriosis aim at reducing estrogen levels. [013] Known Actions of Estrogen-ERa [014] At the molecular level, estrogens, such as 17-p-estradiol (also E2 or 25 E 2 ), bind to ERa and are known to act via two pathways. (i) The best-characterized action of E2-ERa complex is regulation of nuclear gene transcription. E2-ERa can increase or decrease expression of a gene. When bound to a potent estrogen, such a E2, ERa dimerizes and binds to DNA sequences called estrogen response elements (EREs) and closely related sequences. E2-ERa can also bind to ERE half 30 sites near SP1 and AP1 sites and be brought to DNA by tethering through other proteins bound at SP1 and AP1 sites. On DNA, E2-ERa exhibits a conformation that enables the recruitment of coactivators. The bound coactivators help assemble a multi-protein complex that facilitates both chromatin remodeling and formation of an active transcription complex. (ii) The E2-ERa complex can also rapidly activate 2 WO 2014/113820 PCT/US2014/012405 several plasma membrane-associated protein kinase-based signaling pathways. E2 ERa was not known to act at the endoplasmic reticulum to induce efflux of calcium from the lumen of the endoplasmic reticulum into the cytosol and activate the unfolded protein response. 5 [015] The Unfolded Protein Response [016] The main sensor system for response to cell stress is the endoplasmic reticulum sensor system, the unfolded protein response (UPR). The UPR is activated in response to diverse cell stresses including accumulation of unfolded protein, 10 altered redox potential, metabolic stress, and some drugs. The UPR consists of three main branches that together balance the synthesis of new proteins with the availability of chaperones and other proteins to help fold and transport proteins within cells. Moderate and transient activation of the UPR is protective, while extensive and sustained UPR activation induces cell death. In response to cell stress, the 15 transmembrane kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase) is activated by autophosphorylation. P-PERK phosphorylates eukaryotic initiation factor 2a (elF2a), resulting in inhibition of protein synthesis. The other arms of the UPR initiate with activation of the transcription factor ATF6a (activating transcription factor 6 a), leading to increased protein folding capacity and activation 20 of the splicing factor IRE1a (inositol-requiring protein 1a), which alternatively splices the transcription factor XBP1, resulting in production of active spliced (sp)-XBP1 and increased protein folding capacity. Activation of the UPR is usually transient, and the UPR is turned off by production of specific proteins that reverse activation of the PERK arm of the UPR and dephosphorylate elF2a, and by production of chaperone 25 proteins, such as BiP/GRP78 (binding immunoglobulin protein; also known as 78 kiloDalton glucose-regulated protein). [017] The inside, or lumen, of the endoplasmic reticulum (EnR) contains a high calcium concentration compared to the cytosol. Release of this Ca 2 * from the lumen into the cytosol activates the UPR. The increased cytosol Ca 2 * is a 30 proliferation signal, but very high Ca 2 + levels are toxic. Known UPR activators thapsigargin and ionomycin activate the UPR by depleting EnR Ca 2 *. [018] E2-ERa was not known to directly activate the UPR. Until now, hyperactivation of the UPR through use of an ERa inhibitor had not been described or proposed as a drug strategy. 3 WO 2014/113820 PCT/US2014/012405 [019] AMPK and Protein Synthesis [020] Living systems take in energy from their environment. Using catabolic (breakdown) processes, nutrients are broken down, and ATP is produced from ADP 5 and phosphate. Most cellular processes require energy and ATP and hydrolyze ATP to ADP + phosphate or AMP + pyrophosphate. The enzyme AMP kinase (AMPK; adenosine monophosphate kinase) plays a key role in maintaining the balance between production of ATP and use and breakdown of ATP. AMPK is a master regulator of energy balance, or homeostasis. AMPK is activated by metabolic 10 stresses that inhibit ATP production, such as hypoxia, or increase the use of ATP, such as exercise, by AMP and by several protein kinase systems. Phosphorylation activates AMPK. Activated AMPK is a protein kinase that phosphorylates diverse targets resulting in inhibition of pathways that use energy and stimulation of pathways that produce energy. Cell proliferation is one important process that uses 15 energy. [021] One energy utilizing pathway that can be inhibited by AMPK is production of new proteins. One important site for regulation of protein synthesis is elongation, the process by which additional amino acids are added to a growing polypeptide chain. The protein eukaryotic elongation factor 2 (eEF2) is active in its 20 unphosphorylated state and inactive in its phosphorylated state. eEF2 is phosphorylated by eEF2 kinase. eEF2 kinase is the only protein known to phosphorylate eEF2. A pathway leading to inhibition of protein synthesis by activation of AMPK leading to activation of eEF2 kinase has been identified: P-AMPKI(active) -- eEF2-KI(active)--P-eEF21(inactive). While BHPI (3,3,bis(4 25 hydroxyphenyl)-7-methyl-1,3,dihydro-2H-indol-2-one) activates AMPK in ERa containing cells and inactive phosphorylated eEF2 is produced, other pathways for BHPI activation of eEF2K (eukaryotic elongation factor 2 kinase) are not excluded. [022] Summary 30 [023] In certain embodiments, the present disclosure provides a method for the killing of an ERa-containing cell comprising: exposing the cell to an effect amount of BHPI, a derivative thereof, or a pharmaceutically acceptable salt thereof. In embodiments, the present disclosure provides, a method of inhibiting growth of an 4 WO 2014/113820 PCT/US2014/012405 ERa-containing cell comprising contacting said cell with an effective amount of BHPI, a derivative thereof, or a pharmaceutically acceptable salt thereof. [024] In another embodiment, the cell is a cancer cell. In another embodiment, the cancer cell is a human cancer cell. In another embodiment, the 5 cancer cell is in a human patient. In another embodiment, the cancer cell is one or more of ovarian, uterine/endometrial, cervical, lung and liver cancer. [025] In another embodiment, the present disclosure provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of BHPI, derivative thereof, or pharmaceutically acceptable salt thereof. In 10 another embodiment, the patient in need of treatment is a human patient. In another embodiment, the cancer is one or more of ovarian, uterine/endometrial, cervical, lung and liver cancer. [026] In another embodiment, the disclosure provides a method for the killing of an ERa-containing cell comprising: exposing the cell to an effect amount of the 15 compound of the formula: YX 20 X Structure A 25 or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CC13, -CHC12, -CBr3, -CHBr2; Y can be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, CC13, -CHC12, -CBr3, -CHBr2. In various embodiments the halogen can be one or more of fluorine, bromine, or chlorine. In one embodiment, the alkyl can be methyl. 30 [027] In another embodiment, the disclosure provides a method of inhibiting growth of an ERa-containing cell comprising contacting said cell with an effective amount the compound of the formula of Structure A or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CC13, -CHC12, -CBr3, -CHBr2; Y can be hydrogen or 5 WO 2014/113820 PCT/US2014/012405 hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CC13, -CHC12, CBr3, -CHBr2. In embodiments the halogen can be one or more of fluorine, bromine, or chlorine. In another embodiment, the alkyl can be methyl. [028] The disclosure relates at least in part to certain inhibitors and methods. 5 In another embodiment, the inhibitors are small molecule inhibitors that inhibit growth of cells. In particular embodiments, the cells are cancer cells. In embodiments, the inhibition is by targeting a novel estrogen receptor-dependent pathway. [029] In another embodiment, the present disclosure provides a composition comprising any feature described, either individually or in combination with any 10 feature, in any configuration. [030] In another embodiment, the disclosure provides a pharmaceutical formulation comprising a composition of the disclosure. In another embodiment, the disclosure provides a pharmaceutical formulation of a compound described herein. In another embodiment, the disclosure provides a method of synthesizing a 15 composition of the disclosure or a pharmaceutical formulation thereof. In another embodiment, a pharmaceutical formulation comprises one or more excipients, carriers, and/or other components as would be understood in the art is provided. Preferably, the components meet the standards of the National Formulary ("N"), United States Pharmacopoeia ("USP"), or Handbook of Pharmaceutical 20 Manufacturing Formulations. In another embodiment, an effective amount of a composition of the disclosure can be a therapeutically effective amount. [031] Compounds of this disclosure and compounds useful in the methods of this disclosure include those of the formula(s) described herein and pharmaceutically-acceptable salts and esters of those compounds. Salts include any 25 salts derived from the acids of the formulas herein which acceptable for use in human or veterinary applications. The term esters refers to hydrolysable esters of compounds including diphosphonate compounds of the formulas herein. Salts and esters of the compounds of the formulas disclosed herein can include those which have the same therapeutic or pharmaceutical (human or veterinary) general 30 properties as the compounds of the formulas herein. In another embodiment, a composition of the disclosure is a compound or salt or ester thereof suitable for pharmaceutical formulations. [032] Compounds disclosused can have prodrug forms. Prodrugs of the compounds of the disclosure are useful in embodiments including compositions and 6 WO 2014/113820 PCT/US2014/012405 methods. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or therapeutically active form of a compound of the disclosure is a prodrug. Various examples and forms of prodrugs are well known in the art. A prodrug, such as a pharmaceutically acceptable prodrug can represent prodrugs of 5 the compounds of the disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. Prodrugs of the disclosure can be rapidly transformed in vivo to a parent compound of a compound 10 described herein, for example, by hydrolysis in blood or by other cell, tissue, organ, or system processes. [033] In another embodiment, the disclosure contemplates pharmaceutically active compounds either chemically synthesized or formed by in vivo biotransformation to compounds of formulae described herein. 15 [034] In another embodiment, the disclosure provides a method for inhibiting growth of a cell comprising any method described, in any order, using any modality. [035] In other embodiments the disclosure provides a composition comprising any feature described, either individually or in combination with any feature, in any configuration. 20 [036] Further embodiments, forms, features, aspects, benefits, objects, and advantages of the present application shall become apparent from the detailed description and figures provided herewith. [037] Brief Description of the Fiqures 25 [038] Figure 1 is a schematic representation of the scheme for high throughput screening and characterization of "hits". [039] Figure 2 shows the structure and chemical name of the ERa inhibitor, BHPI (3,3,bis(4-hydroxyphenyl)-7-methyl-1,3,dihydro-2H-indol-2-one). [040] Figure 3 shows the results of a dose-response study of BHPI inhibition 30 of the ERE-luciferase reporter gene. [041] Figure 3A shows the results of a dose-response study of the effect of estrogen on expression of an estrogen response element-luciferase reporter. 7 WO 2014/113820 PCT/US2014/012405 [042] Figure 3B shows the results of a dose-response study of the effect of BHPI on expression of an estrogen response element-luciferase reporter and an androgen response element-luciferase reporter. [043] Figure 4 shows the effect of BHPI on expression of an estrogen 5 regulated gene in the presence of low and high concentrations of estrogen. [044] Figure 5 shows the structures of BHPI (Figure 5A) and of an inactive control compound (Figure 5B). [045] Figure 6 shows studies of BHPI interaction with ERa. [046] Figure 6A shows the effect of BHPI and a control compound on the 10 fluorescence emission spectrum of full-length ERa. [047] Figure 6B shows the effect of BHPI on protease sensitivity of ERa ligand binding domain (LBD) using proteinase K analyzed by SDS polyacrylamide gel electrophoresis. [048] Figure 6C shows the effect of BHPI on protease sensitivity of ERa 15 ligand binding domain (LBD) using chymotrypsin analyzed by SDS polyacrylamide gel electrophoresis. [049] Figure 7 shows qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of mRNAs in several cell lines. [050] Figure 7A-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa 20 mediated induction of pS2 mRNA in MCF-7 human breast cancer cells. [051] Figure 7A-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of GREB-1 mRNA in MCF-7 cells. [052] Figure 7A-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of SDF-1 mRNA in MCF-7 cells. 25 [053] Figure 7B-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of pS2 mRNA in T47D human breast cancer cells. [054] Figure 7B-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of GREB-1 mRNA in T47D cells. [055] Figure 7B-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa 30 mediated induction of SDF-1 mRNA in T47D cells. [056] Figure 7C-1 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of pS2 mRNA in BG-1 human ovarian cancer cells. [057] Figure 7C-2 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of GREB-1 mRNA in BG-1 cells. 8 WO 2014/113820 PCT/US2014/012405 [058] Figure 7C-3 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of SDF-1 mRNA in BG-1 cells. [059] Figure 8 is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated down-regulation of an mRNA. 5 [060] Figure 9A is a Western blot analysis of the effect of BHPI on ERa levels. [061] Figure 9B is a Western blot analysis of the effect of BHPI on ERa subcellular localization. [062] Figure 1 OA is a qRT-PCR analysis of the effect of BHPI on E2-ERa 10 mediated induction of pS2 mRNA in MCF-7 cells. [063] Figure 1 OB is a qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of GREB-1 mRNA s in MCF-7 cells. [064] Figure 10C is a chromatin immunoprecipitation (ChIP) study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to the estrogen 15 regulated pS2 gene. [065] Figure 1 OD is a chromatin immunoprecipitation (ChIP) study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to estrogen regulated GREB-1 genes. [066] Figure 11 is a qRT-PCR analysis of the effect of increased ERa 20 expression on BHPI inhibition of E2-ERa induction of an mRNA. [067] Figure 12 shows dose-response studies of the effect of BHPI on proliferation of ERa-positive and ERa-negative cancer cells. The cell lines used were: [068] Figure 12A-1 - ERa-positive MCF-7 human breast cancer cells. 25 [069] Figure 12A-2 - ERa-negative MDA-MB-231 human breast cancer cells. [070] Figure 12B-1 - ERa-positive BG-1 human ovarian cancer cells. [071] Figure 12B-2 - ERa-negative ES2 human ovarian cancer cells. [072] Figure 12C-1 - ERa-positive ECC-1 human endometrial cancer cells. [073] Figure 12C-2 - ERa-negative HeLa human cervical cancer cells. 30 [074] Figure 12D-1 - ERa-positive PC-3 human prostate cancer cells. [075] Figure 12D-2 - ERa-negative DU145 human prostate cancer cells. [076] Figure 13 shows MTS assays analyzing effects of different concentrations of BHPI on proliferation of ERa-positive and ERa-negative cancer 9 WO 2014/113820 PCT/US2014/012405 cells, showing that BHPI inhibits proliferation in diverse ERa-positive cancer cell lines and has no effect on cell growth in ERa-negative cell lines. The cell lines used were: [077] Figure 13A - ERa-positive MCF-7 human breast cancer cells. [078] Figure 13B - ERa-positive T47D human breast cancer cells. 5 [079] Figure 13C - ERa-positive kBluc-T47D human breast cancer cells. [080] Figure 13D - ERa-positive HCC1 500 human breast cancer cells. [081] Figure 13E - ERa-positive ZR-75-1 human breast cancer cells. [082] Figure 13F - ERa-positive BT-474 human breast cancer cells. [083] Figure 13G - ERa-positive MCF1 OAERIN9 human breast cancer cells. 10 [084] Figure 13H - ERa-positive MCF7ERaHA human breast cancer cells. [085] Figure 131 - ERa-negative MDA-MB-231 human breast cancer cells [086] Figure 13J - ERa-negative MCF 1 0A human breast cancer cells [087] Figure 13K - ERa-positive BG-1 human ovarian cancer cells. [088] Figure 13L - ERa-positive OVCAR-3 human ovarian cancer cells. 15 [089] Figure 13M - ERa-positive CAOV-3 human ovarian cancer cells. [090] Figure 13N - ERa-negative ES2 human ovarian cancer cells. [091] Figure 130 - ERa-negative IGROVE-1 human ovarian cancer cells. [092] Figure 13P - ERa-positive ECC-1 human endometrial cancer cells. [093] Figure 13Q - ERa-positive Ishikawka human endometrial cancer cells. 20 [094] Figure 13R - ERa-negative HeLa human cervical cancer cells. [095] Figure 13S - ERa-positive PC-3 human prostate cancer cells. [096] Figure 13T - ERa-negative DU145 human prostate cancer cells. [097] Figure 13U - ERa-negative 201 T human lung cancer cells. [098] Figure 13V - ERa-negative 273T human lung cancer cells. 25 [099] Figure 13W - ERa-negative H1793 human lung cancer cells. [0100] Figure 13X - ERa-negative A549 human lung cancer cells. [0101] Figure 13Y - ERa-negative HepG2 human hepatoma (liver) cancer cells. [0102] Figure 13Z - ERa-negative nonmalignant MEF Mouse embryo 30 fibroblasts. [0103] Figure 14A contains the structure of the chemical scaffolding of BHPI and related chemical structures. [0104] Figure 14B is a table listing preferred substitutions for compounds with the chemical structure shown in Figure 14A. 10 WO 2014/113820 PCT/US2014/012405 [0105] Figure 14C is a dose-response study showing the effect of BHPI on proliferation of ERa-positive T47D, human breast cancer cells. [0106] Figures 14D-L show dose-response studies comparing the effect of each of 9 compounds structurally related to BHPI on proliferation of ERa-positive 5 T47D, human breast cancer cells. [0107] Figure 15A shows the effect of BHPI and antiestrogens on EGF stimulated cell proliferation of T47D human breast cancer cells. [0108] Figure 15B shows the effect of BHPI and antiestrogens on EGF stimulated cell proliferation of BG-1 human ovarian cancer cells. 10 [0109] Figure 16 shows dose-response studies of the effect of BHPI on proliferation of ERa positive cancer cell lines resistant to current therapies. The cell lines used were: [0110] Figure 16A - BT-474 human breast cancer cells. [0111] Figure 16B - ZR-75-1 human breast cancer cells. 15 [0112] Figure 16C - Caov-3 human ovarian cancer cells. [0113] Figure 16D - OVCAR-3 human ovarian cancer cells. [0114] Figure 17 shows the effect of BHPI on anchorage-independent growth of ERa positive human cancer cells. [0115] Figure 17A is a photomicrograph of anchorage-independent growth of 20 ERa positive human cancer cells treated with DMSO vehicle. [0116] Figure 17B is a photomicrograph of anchorage -independent growth of ERa positive human cancer cells treated with E2. [0117] Figure 17C is a photomicrograph of anchorage-independent growth of ERa positive human cancer cells treated with E2 and BHPl. 25 [0118] Figure 17D shows the quantitation of colonies formed after treatment with vehicle, E2, and E2 and BHPl. [0119] Figure 18 shows the effect of BHPI on tumor size in a mouse xenograft model of estrogen-dependent cancer. [0120] Figure 19 shows the effect of BHPI on human breast tumors in a 30 mouse xenograft model showing tumor size (Figure 19A); tumor weight (Figure 19B); mouse body weight (Figure 19C); and mouse food intake (Figure 19D). [0121] Figure 20 is a Western blot analysis showing levels of ERa levels in different cell lines and the effect of BHPI incorporation of 35 S-methionine into protein in those cell lines. 11 WO 2014/113820 PCT/US2014/012405 [0122] Figure 21 A shows the effect of BHPI on incorporation of 35 S-methionine into protein in ERa-positive and ERa-negative cells. [0123] Figure 21 B is a comparison of the effects of BHPI and antiestrogens on incorporation of 35 S-methionine into protein in ERa-positive and ERa-negative cells. 5 [0124] Figure 22A shows the effect of BHPI on incorporation of 3 5 S-methionine into protein after knockdown of ERa. [0125] Figure 22B shows the effect of BHPI on incorporation of 35 S-methionine into protein after degradation of ERa with the antiestrogen IC. [0126] Figure 22C is a Western blot of ERa in cells treated with BHPI and the 10 antiestrogen IC. [0127] Figure 23A is a Western blot analysis showing levels of ERa in cells overexpressing ERa. [0128] Figure 23B is a dose-response study of the effect of increasing levels of ERa on BHPI inhibition of the incorporation of 35 S-methionine into protein. 15 [0129] Figure 24 is a comparison of the effect of BHPI and UPR activators on protein synthesis measured by incorporation of 35 S-methionine into protein. [0130] Figure 25 shows the effect of BHPI and the UPR activator thapsigargin on intracellular calcium measured using the calcium sensing dye Fluo-4. [0131] Figure 25A is a photomicrograph of the effect of a low concentration of 20 BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium. [0132] Figure 25B-1 is a photomicrograph of the effect of a high concentration of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium 25 [0133] Figure 25B-2 is a graphical representation of the effect of a high concentration of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium. [0134] Figure 25C-1 is a photomicrograph of the effect of the UPR activator thapsigargin (THG) on intracellular calcium in MCF-7 cells. 30 [0135] Figure 25C-2 is a graphical representation of the effect of the UPR activator thapsigargin (THG) on intracellular calcium in MCF-7 cells. [0136] Figure 26 is a comparison of the effect of BHPI on intracellular calcium levels in ERa-positive and ERa-negative cancer cells. 12 WO 2014/113820 PCT/US2014/012405 [0137] Figure 26A is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in BG-1 cells with and without extracellular calcium. [0138] Figure 26B-1 is a photomicrograph of the effect of a high concentration 5 of BHPI or thapsigargin (THG) on intracellular calcium in HeLa cells without extracellular calcium. [0139] Figure 26B-2 is a graphical representation of the effect of the BHPI and thapsigargin on intracellular calcium in HeLa cells. [0140] Figure 27A shows the effect of inhibitors of calcium channel opening on 10 intracellular calcium levels after BHPI treatment. [0141] Figure 27B shows the effect of inhibitors of calcium channel opening on protein synthesis measured by incorporation of 35 S-methionine into protein. [0142] Figure 28 is a model for activation of the three arms of the UPR. [0143] Figure 29A is a Western blot analysis showing the effect of BHPI on 15 phosphorylation and levels of PERK and elF2a. [0144] Figure 29B-1 is a Western blot analysis showing the effect of RNAi knockdown of PERK on phosphorylation and level of elF2a. [0145] Figure 29B-2 shows the effect of RNAi knockdown of PERK on protein synthesis measured by incorporation of 35 S-methionine into protein. 20 [0146] Figure 29C is a qRT-PCR analysis of the effect of RNAi knockdown on PERK mRNA levels. [0147] Figure 29D is a Western blot analysis showing the effect of RNAi knockdown on PERK on PERK protein levels. [0148] Figure 30A shows incorporation of 35 S-methionine into protein as a 25 function of time after addition of BHPl. [0149] Figure 30B contains Western blots of the effect of BHPI on phosphorylation and level of elF2a in different lines as a function of time after addition of BHPl. The cell lines used were MCF-7 cells in Figure 30B-1; BG-1 cells in Figure 30B-2; T47D cells in Figure 30B-3; and MCF-7 cells in Figure 30B-4. 30 [0150] Figure 30C is a Western blot analysis of the effect of BHPI on phosphorylation and level of elF2a in ERa-negative cancer cells. [0151] Figure 30D is a qRT-PCR analysis of mRNA levels of UPR-related mRNAs in MCF-7 ERa-positive cancer cells treated with BHPl. 13 WO 2014/113820 PCT/US2014/012405 [0152] Figure 30E is a qRT-PCR analysis of mRNA levels of UPR-related mRNAs in BG-1 ERa-positive cancer cells treated with BHPl. [0153] Figure 31 is a qRT-PCR analysis of spliced and unspliced UPR-related mRNAs in ERa-positive cancer cells treated with BHPl. 5 [0154] Figure 31A is a qRT-PCR analysis of unspliced XBP-1 mRNA in ERa positive cancer cells treated with BHPI and no estrogen. [0155] Figure 31 B is a qRT-PCR analysis PCR of spliced XBP-1 mRNA in ERa-positive cancer cells treated with BHPI and no estrogen. [0156] Figure 31C is a qRT-PCR analysis of unspliced XBP-1 mRNA in ERa 10 positive cancer cells treated with BHPI with and without estrogen. [0157] Figure 31 D is a qRT-PCR analysis of spliced XBP-1 mRNA in ERa positive cancer cells treated with BHPI with and without estrogen. [0158] Figure 32 is a Western blot analysis of the level of full length and spliced ATF6a in ERa-positive cancer cells treated with BHPI using MCF-7 cells in 15 Figure 32A and T47D cells in Figure 32B. [0159] Figure 33A is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2. [0160] Figure 33B is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2K. 20 [0161] Figure 33C is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated AMPK. [0162] Figure 33D-1 is a qRT-PCR analysis of p58 mRNA levels in BHPI treated cells. [0163] Figure 33D-2 is a Western blot analysis showing the effect of BHPI on 25 levels of BiP and p58'IP. [0164] Figure 34 is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2 in T47D ERa-positive cancer cells in Figure 34A and in HeLa ERa-negative cancer cells in Figure 34B. [0165] Figure 35A is a Western blot analysis showing the effect of THG 30 (thapsigargin) on phosphorylated and unphosphorylated elF2a. [0166] Figure 35B shows incorporation of 35 S-methionine into protein as a function of time in cells treated with THG. [0167] Figure 35C is a Western blot analysis showing the effect of TUN (tunicamycin) on phosphorylated and unphosphorylated elF2a. 14 WO 2014/113820 PCT/US2014/012405 [0168] Figure 35D is a qRT-PCR analysis of CHOP mRNA levels in TUN (tunicamycin) treated cells. [0169] Figure 35E is a Western blot analysis showing the effect of TUN (tunicamycin) on levels of BiP protein. 5 [0170] Figure 35F is a Western blot showing the effect of TUN (tunicamycin) on levels of p58'IP. [0171] Figure 36A-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in T47D breast cancer cells visualized using the dye Fluo 4. 10 [0172] Figure 36A-2 is a graphical representation of the effect of estrogen on intracellular calcium levels T47D breast cancer cells visualized using the dye Fluo-4. [0173] Figure 36B-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in PEO4 ovarian cancer cells visualized using the dye Fluo-4. 15 [0174] Figure 36B-2 is a graphical representation of the effect of estrogen on intracellular calcium levels PEO4 ovarian cancer cells visualized using the dye Fluo-4. [0175] Figure 37A-1 are photomicrographs showing the effect of estrogen on intracellular calcium levels in cells treated with calcium channel blockers visualized 20 using the dye Fluo-4. [0176] Figure 37A-2 is a graphical representation of the effect of estrogen on intracellular calcium levels in cells treated with calcium channel blockers visualized using the dye Fluo-4. [0177] Figure 37B is a Western blot analysis showing the effect of calcium 25 channel blockers and E2 on levels of phosphorylated and unphosphorylated elF2a. [0178] Figure 38A are photomicrographs showing the effect of RNAi knockdown of ERa on intracellular calcium levels visualized using the dye Fluo-4. [0179] Figure 38B is a graphical representation of the effect of RNAi knockdown of ERa on intracellular calcium levels visualized using the dye Fluo-4. 30 [0180] Figure 39A is a qRT-PCR analysis of the effect of E2 on the level of spliced XBP1 mRNA. [0181] Figure 39B is a qRT-PCR analysis of the effect of E2 on the levels of SERP1 and ERDJ mRNAs. 15 WO 2014/113820 PCT/US2014/012405 [0182] Figure 39C is a qRT-PCR analysis of the effect of E2 and antiestrogens on the level of spliced XBP1 mRNA. [0183] Figure 39D is a qRT-PCR analysis of the effect of RNAi knockdown of ERa on the level of spliced XBP1 mRNA. 5 [0184] Figure 39E is a Western blot analysis of the effect of E2 and antiestrogens on the level of full-length and spliced ATF6a protein in T47D breast cancer cells. [0185] Figure 39F is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in BG-1 ovarian cancer cells. 10 [0186] Figure 39G is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in PEO4 ovarian cancer cells. [0187] Figure 39H is a qRT-PCR analysis of the effect of E2 on the level of BiP mRNA in ERa containing cancer cells. [0188] Figure 391 is a Western blot analysis of the effect of E2 on the level of 15 BiP protein in MCF-7 cells. [0189] Figure 39J is a qRT-PCR analysis of the effect of RNAi knockdown of ERa and E2 on the level of BiP mRNA. [0190] Figure 40A is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated PERK. 20 [0191] Figure 40B is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated elF2a. [0192] Figure 40C shows incorporation of 35 S-methionine into protein in cells treated with E2 and the antiestrogen ICI 182,780 (ICI). [0193] Figure 41 A shows the effect of the calcium chelator BAPTA-AM on E2 25 ERa stimulated cell proliferation. [0194] Figure 41 B shows the effect of calcium channel blockers on E2-ERa stimulated cell proliferation in in MCF-7 breast cancer cells. [0195] Figure 41C shows the effect of calcium channel blockers on E2-ERa stimulated cell proliferation in BG-1 ovarian cancer cells. 30 [0196] Figure 41 D shows the results of luciferase assays analyzing the effect of calcium channel blockers on E2-ERa stimulated expression of an ERE-luciferase reporter gene. 16 WO 2014/113820 PCT/US2014/012405 [0197] Figure 41 E is a qRT-PCR analysis of the effect of effect of calcium channel blockers on E2-ERa regulated expression of cellular pS2 and GREB1 mRNAs. [0198] Figure 42A is a qRT-PCR analysis showing the effects of E2-ERa over 5 time on mRNAs for each UPR arm in MCF-7 cells. [0199] Figure 42B shows the effect of estrogen on growth of MCF-7 tumors in athymic mice. [0200] Figure 42C is a qRT-PCR analysis showing the levels of GREB-1 and pS2 mRNAs in mouse tumors with and without E2. 10 [0201] Figure 42D is a qRT-PCR analysis showing the levels of UPR-related mRNAs in mouse tumors with and without E2. [0202] Figure 42E is an analysis of publically available patient microarray data showing levels of estrogen-regulated mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma 15 of the breast, and invasive ductal carcinoma tissue. [0203] Figure 42F is an analysis of publically available patient microarray data showing levels of UPR-related mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast, and invasive ductal carcinoma tissue. 20 [0204] Figure 43 is a model for E2-ERa regulation of the UPR. [0205] Figure 44 shows the effect of prior activation of the UPR by E2 and by TUN on subsequent cell proliferation in cells later treated with TUN. [0206] Figure 45 is a table showing the genes that comprise the UPR gene index used in bioinformatics studies. 25 [0207] Figure 46 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts. [0208] Figure 46A is a bioinformatic analysis of data from two microarray chips (U133A in Figure 46A-1 and U133B in Figure 46A-2) showing Kaplan-Meier survival plots comparing time of relapse-free survival in breast cancer patients 30 expressing high and low levels of UPR index genes. [0209] Figure 46B is a bioinformatic analysis of data from two microarray chips (U133A) and (U133B) showing time to relapse in 277 breast cancer patients, hazard ratio, and p-Values for individual components of the UPR gene index. 17 WO 2014/113820 PCT/US2014/012405 [0210] Figure 46C is a bioinformatic analysis of data from microarray chips using the UPR gene signature alone (univariate analysis) comparing time to relapse in breast cancer patients using the UPR gene signature and current prognostic markers (multivariate analysis). 5 [0211] Figure 46D is a bioinformatic analysis of microarray data showing time to relapse in 474 breast cancer patients, hazard ratio, and p-Value for individual components of the UPR gene index. Microarray analysis was performed prior to initiation of tamoxifen therapy. [0212] Figure 46E is a bioinformatic analysis of microarray data from two 10 microarray chips (U133A) and (U133B) showing time to relapse in 236 breast cancer patients; shown are hazard ratio and p-Values for individual components of the UPR gene index. [0213] Figure 47 is a bioinformatic analysis of microarray data from ERa positive breast cancer patients comparing expression of classical estrogen-regulated 15 genes and UPR index components. [0214] Figure 48 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts. [0215] Figure 48A shows Kaplan-Meier plots of time of relapse-free survival for patients grouped by level of expression (low, medium and high) of the UPR gene 20 index using bioinformatic analysis of microarray data. [0216] Figure 48B shows Kaplan-Meier plots of time of overall survival for patients grouped by level of expression of the UPR gene index using bioinformatic analysis of microarray data. [0217] Figure 48C is a bioinformatic analysis of data from microarray chips 25 using the UPR gene signature alone (univariate analysis) comparing time of relapse free survival and overall survival in breast cancer patients using the UPR gene signature and current prognostic markers (multivariate analysis). [0218] Figure 49 is a bioinformatic analysis of publically available microarray data from ovarian cancer patients with early stage and highly malignant tumors. 30 [0219] Figure 50 shows a Kaplan-Meier plot of time of relapse-free survival in ovarian cancer patients grouped by high and low expression of UPR genes using publically available microarray data. 18 WO 2014/113820 PCT/US2014/012405 [0220] Detailed Description of the Preferred Embodiments [0221] A small molecule inhibitor (compound) that blocks ERa action is described. In certain embodiments, the compound can have the following structure: 5 10 Structure A 15 Structure A or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CC13, -CHC12, CBr3, -CHBr2; Y can be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CC13, -CHC12, -CBr3, -CHBr2. In embodiments the halogen can be one or more of fluorine, bromine, or chlorine. In another 20 embodiment, the alkyl can be methyl. [0222] In another embodiment, the small molecule inhibitor of Structure A is BHPI, 3,3,bis(4-hydroxyphenyl)-7-methyl-1,3,dihydro-2H-indol-2-one (Figure 14A), or a derivative thereof, or a salt thereof, or a pharmaceutical formulation thereof. Unlike the current drugs tamoxifen, Fulvestrant and Raloxifen, the small molecule inhibitor 25 does not work by competing with estrogens for binding to ERa. This small molecule inhibits protein synthesis in cancer cells that contain ERa. Independent of its ability to inhibit protein synthesis, the small molecule potently inhibits estrogen-ERa mediated gene expression. The compound works by distorting the previously unknown ability of estrogen-ERa to activate the unfolded protein response (UPR). In 30 the presence of the compound and ERa, this normal action of estrogen-ERa is distorted, and the arm of the unfolded protein response pathway that results in inhibition of protein synthesis is strongly activated. Since the ERa positive cancer cells cannot make new proteins and cannot regulate gene expression in response to estrogen that might help them shut down this arm of the UPR pathway, they don't 19 WO 2014/113820 PCT/US2014/012405 grow and ultimately die. In cells that lack ERa, the compound has no effect on protein synthesis or on cell proliferation - even at levels hundreds of times higher than those that block growth of ERa positive cancer cells. In a mouse xenograft study, the compound produced rapid regression of large pre-existing tumors with no 5 evidence of systemic toxicity. Because this compound targets a previously not described interaction of estrogen-ERa with components of the unfolded protein response, it represents a new therapeutic target. The compound is effective in ERa containing breast, ovarian, and endometrial cancer cells even when the cells do not require estrogen for growth and are resistant to the currently used drugs, tamoxifen 10 and fulvestrant, and it works in multidrug resistant cell lines. This compound has a novel mode of action fundamentally distinct from current small molecule therapeutics that target ERa. [0223] Through high throughput screening, compounds of the class 3,3-bis(4 hydroxyphenyl)-1,3-dihydro-2H-indol-2-one (also known as: 3,3-bis(4 15 hydroxyphenyl)-2-oxindoles) were identified as compounds that stop the growth of estrogen-ERa-containing cancer cells by inhibiting estrogen-ERa-regulated gene expression and mRNA production, and by a novel mechanism; they inhibit the synthesis of new proteins in cancer cells that contain estrogen receptor a (ERa). Since estrogen-ERa regulated gene expression and synthesis of new proteins are 20 each required for cells to proceed through the cell cycle, the compounds rapidly and completely stop the growth of the ERa-containing cancer cells, and the cells eventually die. As exemplified by BHPI, these compounds have an exceptionally attractive set of properties that make them excellent candidates for targeting ERa positive cancers and other diseases such as endometriosis. 25 [0224] These are a new class of non-competitive inhibitors of ERa action that block both the well-known classical pathway of estrogen-ERa regulated gene expression and a newly identified pathway that links ERa action to the unfolded protein response, leading to ERa-dependent inhibition of protein synthesis. The combination of inhibition of estrogen-ERaa-regulated gene expression and inhibition 30 of protein synthesis stops cell growth, and the cells eventually die. The compound produces rapid and dramatic tumor regression and shrinkage in a xenograft model and may shrink lesions in endometriosis as it is cytotoxic to tumors, not simply cytostatic. 20 WO 2014/113820 PCT/US2014/012405 [0225] At a concentration of 100 nM, the compound, BHPI, effectively and rapidly inhibits estrogen-ERa regulated gene expression and protein synthesis. BHPI is effective in numerous ERa-containing breast, ovarian, and endometrial cancer cells tested. At 100 nM, BHPI inhibits estrogen-ERa regulated gene expression, 5 protein synthesis, and cell proliferation in ERa-containing cancer cell lines. In contrast, at 10,000 nM (a 100 fold higher concentration), BHPI has no detectable effect on growth in all tested cell lines that do not contain ERa. This is a much larger therapeutic window than most existing drugs and other ERa inhibitors. [0226] By inhibiting ERa action, BHPI targets the entire spectrum of 10 pathologies associated with ERa. These include, but are not limited to, breast, ovarian, endometrial, liver cancer, and endometriosis. [0227] Inhibiting estrogen-ERa regulated gene expression alone blocks cell proliferation but does not usually kill cells. Simultaneous ERa-dependent inhibition of protein synthesis ultimately leads to cell death. BHPI does not kill non-cancerous 15 ERa-containing cells. Both ERa and the other proteins in the UPR that link ERa to protein synthesis are often overproduced in many cancers relative to normal cells that contain ERa. In addition, cancer cells that are resistant to tamoxifen and other current antiestrogens overexpress this arm of the UPR and are actually more susceptible to killing by BHPI than non-resistant cancer cells that contain ERa. This 20 makes the cancer cells selective targets for the compound._BHPI is effective in widely used breast cancer models resistant to the major ER inhibitors used clinically to treat cancer, such as tamoxifen and fulvestrant. It is also effective in a widely used multidrug resistant ovarian cancer cell line resistant to adriamycin/doxorubicin, cisplatin, taxol and other standard chemotherapy agents. BHPI is fully effective in 25 several ERa-containing cell lines in which estrogen does not stimulate cell growth. [0228] BHPI is fairly low molecular weight and is simple to synthesize. Compounds in this family are orally available. As exemplified by the compound BHPI, these are non-competitive independent inhibitors of both the ability of ERa to regulate gene expression and of the UPR. These compounds are potent inhibitors of 30 cell proliferation in ERa-containing cells. [0229] The BHPI family is unrelated to known inhibitors of ERa. BHPI does not act by competing with estrogens for binding in the ligand-binding pocket of ERa. Thus, it is completely different both in structure and site of action from current Selective Estrogen Receptor Modulators (SERMS) which include, but are not limited 21 WO 2014/113820 PCT/US2014/012405 to, tamoxifen, Falsodex/fulvestrant/ICI 182,780 and raloxifene. A few non competitive ER inhibitors have been described. Pyrimidines, guanyl hydrazones and amphipathic benzenes have been reported as potential inhibitors of the binding of coactivators to ERa. These compounds are structurally distinct from the BHPI family 5 of compounds and have not been shown to act as specific inhibitors of ERa dependent growth of cancer cells. [0230] BHPI and its family members are inhibitors of both known and novel actions of ERa. That inhibition has important implications for treatment of ERa dependent human pathologies and represents a novel method of use for BHPI and 10 structurally related compounds. [0231] BHPI and the related small molecules described in this application represent a new class of therapeutic agents for estrogen receptor a-dependent ovarian, endometrial/uterine, and breast cancers, and for endometriosis. BHPI is effective in in ERa-containing cancer cells that are resistant to current therapeutics 15 that target ERa and that are resistant to widely used chemotherapy agents including adriamycin/doxorubicin, cisplatin, and taxol. There exists a large set of cancers that are difficult or impossible to treat with current therapies for which these compounds exhibit great potential. In potency, efficacy, specificity, toxicity, and ease of production, these compounds exhibit an extremely favorable profile. This group of 20 compounds targets both the action of ERa-regulation on gene expression and the linkage between ERa and the unfolded protein response that controls the rate of synthesis of new proteins. [0232] Estrogens action of binding to estrogen receptor a (ERa) plays a key role in the growth and metastases of cancers of the reproductive system including 25 breast, ovarian, uterine/endometrial. Liver cancers are also fueled by estrogens binding to ERa. Although existing therapies that focus on small molecules that inhibit the synthesis of estrogens or on competing with estrogens for binding to ERa are initially effective, the tumors eventually become resistant. This is due to the inability of existing therapies to completely inhibit tumor growth resulting in outgrowth of 30 genetic variants that no longer require estrogen or ERa for growth. Many of these resistant tumors contain ERa, but they no longer need it to grow and are therefore resistant to current therapies. BHPI and related compunds have a fundamentally different mode of action that makes this class of compounds a far more versatile drug. BHPI targets the interaction of ERa with the UPR and with a system that 22 WO 2014/113820 PCT/US2014/012405 regulates elongation and independently inhibits ERa-mediated gene expression. The key proteins in the pathways it targets that lead to inhibition of protein synthesis are overexpressed in many cancers. BHPI works when ERa is present, it does not require that estrogen be present, and it works in cancer cells that do NOT require 5 ERa to grow. BHPI blocks cell growth and works in breast cancer cells that are resistant to tamoxifen and fulvestrant, the current mainstream ERa-targeting therapies. It is also effective in ovarian cancer cells that are resistant to common chemotherapy agents including doxorubicin, cisplatin and taxol. BHPI is effective in cell lines derived from most types of reproductive cancers including breast, ovarian, 10 and endometrial. BHPI has potential effectiveness in a wide range of advanced cancers that are not effectively targeted by current therapies. [0233] Because BHPI completely inhibits protein synthesis, it both blocks cell growth and eventually kills the cells. Current agents targeting ERa (tamoxifen, fulvestrant) prevent ERa from working, but usually do not kill the cancer cells. BHPI 15 induces rapid regression of large pre-existing ERa positive cancers in a mouse xenograft model. [0234] BHPI and structural analogues of this family have the following structural elements. 1 an oxindole ring, which is an indoline ring derivative containing a carbonyl at the 2-position of the nitrogen ring; and two o-phenol (4-hydroxy phenol) 20 rings emerging from the 3-position of the nitrogen ring. The aggregate of these components yields the compound, 3,3-diphenyloxindole. [0235] In one embodiment, a class of compounds useful for killing ERa positive cells is provided, the complounds having the formula: Y 25 X 30 Structure A 23 WO 2014/113820 PCT/US2014/012405 of Structure A or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or CF3, -CHF2, -CC13, -CHC12, CBr3, -CHBr2; Y can be hydrogen or hydroxyl; and Z can be hydrogen, alkyl, halogen, or -CF3, -CHF2, -CC13, -CHC12, -CBr3, -CHBr2. 5 [0236] In another embodiment, the disclosure provides a method of inhibiting growth of an ERa-containing cell comprising contacting said cell with an effective amount the compound of Structure A or a pharmaceutically acceptable salt thereof wherein, each independently, X can be hydrogen, alkyl, halogen, or -CF 3 , -CHF 2 , CC13, -CHC1 2 , -CBr 3 , -CHBr 2 ; Y can be hydrogen or hydroxyl; and Z can be hydrogen, 10 alkyl, halogen, or -CF 3 , -CHF 2 , -CC13, -CHC1 2 , -CBr 3 , -CHBr 2 . In embodiments the halogen can be one or more of fluorine, bromine, or chlorine. In another embodiment, the alkyl can be methyl. [0237] Compounds of the general structure of Structure A are useful in the treatment of diseases related to the function of estrogen receptor a or diseases in 15 cells containing estrogen receptor a. These include but are not limited to cancer of the breast, ovary, uterus and cervix, liver, colon, lung, and endometriosis. [0238] Any type of estrogen receptor a containing cell may be treated, including but not limited, to cells of the breast, ovary, uterine endometrium, cervix, liver, colon, lung and prostate. In one embodiment, a compound is used for 20 treatment of cells in which estrogen binding to estrogen receptor a stimulates growth of the cells. In another embodiment, a compound is used to treat cells in which the presence of estrogen receptor is sufficient to stimulate growth of the cells. In another embodiment, a compound is used to treat cells that contain estrogen receptor and in which estrogen and estrogen receptor do not themselves stimulate growth of the 25 cells. In each of these embodiments, the disclosed compound acts through estrogen receptor a to inhibit the ability of estrogen, bound to estrogen receptor a, to increase or decrease the expression of specific genes, and the compound acting through estrogen receptor a activates the unfolded protein response pathway. [0239] In one embodiment, a compound of Structure A acting through 30 estrogen receptor a activates the unfolded protein response pathway. Activation of one arm of this pathway inhibits synthesis of new protein. The compound may also activate the AMPK pathway and causes inhibition of elongation to further inhibit protein synthesis. This long-term inhibition of protein synthesis leads to cessation of cell growth and death of many target cells. Examples of cells in which this pathway 24 WO 2014/113820 PCT/US2014/012405 of estrogen receptor a action can be used therapeutically to inhibit cell growth include, but are not limited to, breast cancer, ovarian cancer, uterine endometrial cancer, uterine endometrial cells in the disease endometriosis, cervical cancer, liver cancer (hepatocellular carcinoma), colorectal cancer lung cancer, and prostate 5 cancer. Any type of cell containing estrogen receptor a may be treated including, but not limited to, breast, gynecological including ovarian, uterine endometrial, cervical, and vulval cells, liver, colon, lung, and prostate cells. [0240] Compounds different from the structural class of BHPI and related compounds may also act through estrogen receptor a to activate the PERK arm of 10 the unfolded protein response and inhibit protein synthesis and, therefore, reduce or inhibit growth and, in some cases, kill cells containing both estrogen receptor a and unfolded protein response components. These include, but are not limited to, breast cancer, ovarian cancer, uterine endometrial cancer, uterine endometrial cells in the disease endometriosis, cervical cancer, liver cancer (hepatocellular carcinoma), 15 colorectal cancer, lung cancer, and prostate cancer. [0241] In some embodiments, a compound not related to the class of compounds of Structure A is provided for targeting the estrogen receptor a to activate the PERK arm of the unfolded protein response and inhibit protein synthesis and, therefore, reduce or inhibit growth and, in some cases, kill cells containing both 20 estrogen receptor a and unfolded protein response components. [0242] Also provided is a compound of the general formula of Structure A as defined herein for use as a medicament, specifically, the use of a compound of the general formula of Structure A for the preparation of a medicament for the treatment of cancer, endometriosis, and other estrogen receptor related diseases in a 25 mammal. Such a medicament may be used in combination therapy with one or more other chemotherapeutic agents. [0243] Also provided is a method for treating a mammal either susceptible to cancer or endometriosis or suffering from cancer or endometriosis; the method comprising administering to the mammal a therapeutically effective quantity of the 30 compound of the general formula of Structure A. [0244] The disclosed compounds may be present as racemic mixtures or the individual isomers, such as diasteriomers or enantiomers. In various embodiments, the formulation ecompasses each and every possible enantiomer and diasteriomer as well as racemates and mixtures that may be enriched in one of the possible 25 WO 2014/113820 PCT/US2014/012405 steriosiomers. In another embodiment, forms in which the compound may be present as salt including pharmaceutically acceptable acidic and basic salts are provided. [0245] Compounds of the general formula of Structure A are suitably formulated as pharmaceuticals with a composition appropriate to the most suitable 5 route of administration. The route of administration may be any desirable route that leads to a concentration in the target tissue or blood that is therapeutically effective. Administration routes that may be applicable include but are not limited to, oral, subcutaneous, intravenous, parenteral, vaginal. The choice of route of administration depends on the physical and chemical properties of the compound in the 10 formulation, on the particular disease, and on the severity of the condition. [0246] The compound may represent any portion of the total in a pharmaceutical composition and will generally be in the range of 1-95% by weight of the total weight of the composition. The dosage form will be suitable to the method of administration. The composition may be in the form of powders, granules, emulsions, 15 suspensions, gels, ointments, creams injectables, sprays, and any other suitable form. [0247] The pharmaceutical compositions will follow accepted pharmaceutical practice. This will involve a pharmaceutically acceptable carrier and may involve composition with other agents. Pharmaceutically acceptable compositions may be 20 formulated to release the active compound immediately or nearly immediately after administration or to release the active over a predetermined time period. These compositions are referred to as timed release or controlled release formulations. Controlled release formulations involve formulations designed to produce a substantially constant concentration of the drug in the target tissue and/or in the 25 blood over an extended period of time. [0248] While the disclosed compounds may be combined with any drug, in one preferred embodiment, an effective compound is combined with the anticancer drug paclitaxel or with other taxanes. Paclitaxel activity is stimulated by calcium. BHPI and related active compounds increase intracellular calcium levels by opening 30 an endoplasmic reticulum calcium channel. Therefore BHPI is expected to increase the effectiveness of paclitaxel. [0249] In other embodiments, any compound that acts through estrogen receptor a to open a calcium channel is provided. In another embodiment, the compound is combined with paclitaxel and/or other taxanes to increase their activity 26 WO 2014/113820 PCT/US2014/012405 as anticancer drugs. BHPI and related compounds may be combined with paclitaxel and other taxanes to treat estrogen receptor a containing ovarian cancer and other cancers in which taxanes are used therapeutically. [0250] Therefore, with respect to the use and method of treatment, the 5 compound may be combined with paclitaxel and other taxanes, with other chemotherapeutic agents that inhibit estrogen synthesis including Letrozole, Anastrosole, Exemestane, with selective estrogen receptor modulators, tamoxifen, raloxifene, fulvestrant, or other therapeutic agents used to treat cancer, endometriosis, or other diseases in which estrogen receptor a is present. 10 [0251] In another embodiment, a composition of Formula A may be used in estrogen receptor alpha containing cells to open the endoplasmic reticulum calcium channel and release calcium into the cytoplasm of the cell. This includes opening the endoplasmic reticulum IP3R calcium channel, the ryanodine calcium channel, both the IP3R calcium channel and the ryanodine calcium channel and other endoplasmic 15 reticulum calcium channels. [0252] In another embodiment, the use of any compound that acts through estrogen receptor a to open the endoplasmic reticulum IP3R calcium channel, the ryanodine calcium channel, both the IP3R calcium channel and the ryanodine calcium channel, and other endoplasmic reticulum calcium channels is provided. 20 [0253] In another embodiment, a compound of Formula A may be used in estrogen receptor a containing cells to activate the PERK arm of the UPR resulting in inhibition of protein synthesis. [0254] In another embodiment, the use of any compound that acts through estrogen receptor a to activate the PERK arm of the UPR resulting in inhibition of 25 protein synthesis is provided. [0255] In another embodiment, the use of any compound that acts through estrogen receptor a to activate the metabolic sensor AMPK, resulting in inhibition of elongation by phosphorylation of eEF2 and inhibition of protein synthesis is provided. [0256] In another embodiment, the use of any compound in estrogen receptor 30 a containing cells to activate both the PERK arm of the UPR and AMPK, resulting in long-term inhibition of protein synthesis at two sites is provided. [0257] A method for identifying cancer patients in which therapy using BHPI or a compound of Structure A is likely to be most effective is provided. UPR is elevated in resistant ERa positive tumors (Figures 46-50). By performing a biopsy, evaluating 27 WO 2014/113820 PCT/US2014/012405 tumors using the UPR index and the level of estrogen receptor a, and determining those tumors with the highest levels of UPR index genes and ERa, it is possible to identify tumors most susceptible to treatment with BHPL. [0258] Breast cancers with very high levels of ERa are resistant to tamoxifen 5 therapy. Use of BHPI or a related compound is especially effective in inhibiting protein synthesis in cells that contain very high levels of ERa (Figure 23B). Thus, the use of BHPI as a cancer therapy is expected to be especially effective in the sub class of therapy-resistant cancers that express very high levels of ERa. [0259] A method of treating ERa containing cancers resistant to current 10 cancer therapies including antiestrogens, such as tamoxifen, aromatase inhibitors such as letrozole and taxanes such as paclitaxel is provided. Therapy-resistant ERa containing tumors overexpress the UPR index (Figures 46, 48-50). Thus, these tumors are especially susceptible to BHPI therapy. [0260] A method of inducing death of ERa containing breast cancer cells 15 resistant to current therapy is provided. Because therapy resistant cancer cells overexpress the genes of the UPR index, these ERa containing cells are especially sensitive to BHPL. They do not just stop growing; they rapidly die (Figures 16A and 16B). [0261] A method for treating ERa containing breast cancer cells whose growth 20 is stimulated by epidermal growth factor and epidermal growth factor receptors is provided. This includes, but is not limited to the Her2/NEU positive class of breast cancers and ovarian cancer cells (see Figure 15A and 15B). [0262] A method for treating ERa containing cancers that are resistant to therapy because they overexpress multidrug resistant resistance proteins, including, 25 but not limited to, multidrug resistance protein 1 (MDR1) is provided. OVCAR-3 cells overexpress MDR1 and are resistant to therapeutically relevant concentrations of at least eight anti-cancer drugs, but theyrespond to BHPI (Figure 16D). [0263] Method for treating ERa containing ovarian cancers that are resistant to antiestrogens (tamoxifen and ICI/Fulvestrant) and antiproliferative 30 chemotherapeutics including paclitaxel and other taxanes and/or cisplatin is provided (see Figure 16C). Caov-3 cells are resistant to ICI, the active form of tamoxifen (4 OHT), paclitaxel, and in some cases cisplatin, but they respond to treatment with BHPL. 28 WO 2014/113820 PCT/US2014/012405 [0264] A method for inhibiting growth of uterine fibroids in patients with endometriosis is provided. The fibroids that cause endometriosis use estrogen and ERa to stimulate growth and are expected to have their growth inhibited by BHPl. [0265] A method for determining whether a tumor is a candidate for therapy 5 with BHPI or a compound of Formula A is provided. The method comprises removing all or part of the tumor by biopsy or surgery; analyzing the tumor for ERa (usually done using an ELISA), extracting RNA from the tumor; performing microarray analysis to determine levels of UPR index genes; where elevated levels of UPR index genes indicate that the tumor is a good candidate for therapy with BHPI or a 10 compound of Formula A. As an alternative to, or in addition to microarray analysis of UPR index genes, live tumor cells may be inserted into an orthologous mouse model and directly testing the tumor for therapeutic response using BHPI or a compound of Formula A. Tumor size can be measured with calipers or using imaging. [0266] BHPI elicits three effects in ER a positive cells including inhibition of 15 estrogen ERa-regulated gene expression (which was a previously known action of ERa); activation of the UPR; and activation of AMPK. A screen for small molecules that carry out any two of these three effects identifies a unique small molecule that targets these pathways. Such a screen can be used to identify addition therapeutic compounds. 20 [0267] A screen for small molecules that activate the unfolded protein response only in ERa positive cells is provided. Typical cell lines would be ERa positive cells, such as MCF-7 or T47D cells, compared to ERa negative cells from the same tissue type, such as MDA-MB-231 cells. After treatment with a test compound, the readout would be either rapid inhibition of protein synthesis only in 25 the ERa positive cells, or activation of any of the arms of the UPR only in the ERa positive cells. These readouts can be formation of phospho-elF2 alpha, formation of spliced XBP-1, formation of spliced ATF6 alpha, and others readouts. These can be monitored using Western blots, ELISA, qRT-PCR for spliced XBP1, or other methods. 30 [0268] A kit for treating cancer with BHPI or a structurally related compound is provided. The kit includes an ELISA assay for determing whether ERa is present; materials for RNA extraction; and a microarray for identifying the level of UPR gene index expression. The microarray may be a commercially available microarray capable of testing for expression levels of many cell genes, or a custom microarray 29 WO 2014/113820 PCT/US2014/012405 for testing only genes in the UPR index. BHPI or a related compound, alone as the active ingredient, or combined with another drug as a medicine for oral delivery or subcutaneous injection would be provided. [0269] Use of the UPR index as a prognostic marker to identify tumors and 5 other ERa responsive conditions, such as endometriosis, that are good candidates for treatment with BHPI or a related compound is provided. [0270] Compounds of the general formula of Structure A are suitably formulated as pharmaceuticals with a composition appropriate to the most suitable route of administration. The route of administration may be any desirable route that 10 leads to a concentration in the target tissue or blood that is therapeutically effective. Administration routes that may be applicable include but are not limited to, oral, subcutaneous, intravenous, parenteral, vaginal. The choice of route of administration depends on the physical and chemical properties of the compound in the formulation, on the particular disease, and on the severity of the condition. 15 [0271] The compound may represent any portion of the total in a pharmaceutical composition and will generally be in the range of 1-95% by weight of the total weight of the composition. The dosage form will be suitable to the method of administration. The composition may be in the form of powders, granules, emulsions, suspensions, gels, ointments, creams injectables, sprays, and any other suitable 20 form. [0272] The pharmaceutical compositions will follow accepted pharmaceutical practice. This will involve a pharmaceutically acceptable carrier and may involve composition with other agents. Pharmaceutically acceptable compositions may be formulated to release the active compound immediately or nearly immediately after 25 administration or to release the active over a predetermined time period. These compositions are referred to as timed release or controlled release formulations. Controlled release formulations involve formulations designed to produce a substantially constant concentration of the drug in the target tissue and/or in the blood over an extended period of time. 30 [0273] Whenever a range is given, for example, a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. 30 WO 2014/113820 PCT/US2014/012405 [0274] In additional embodiments, the pharmaceutical composition is in unit dosage form by weight of the compound. In such embodiments each unit dosage typically consists of 1-100 mg administered daily. In various embodiments, dosages in the ranges of about 1 to about 100 mg administered daily; about 1 to about 10 mg 5 administered daily; about 10 to about 20 mg administered daily; about 20 to about 30 mg administered daily; about 30 to about 40 mg administered daily; about 40 to about 50 mg administered daily; about 50 to about 60 mg administered daily; about 60 to about 70 mg administered daily; about 70 to about 80 mg administered daily; about 80 to about 90 mg administered daily; about 90 to about 100 mg administered 10 daily; about 10 to about 90 mg administered daily; about 20 to about 80 mg administered daily; about 30 to about 70 mg administered daily; and about 40 to about 60 mg administered daily are provided. [0275] The compound is generally administered in the range of 0.01-2 mg per kg body weight daily. In various embodiments, daily dosages in the ranges of about 15 0.01 to about 2 mg per kg body weight; about 0.01 to about 0.2 mg per kg body weight;_about 0.2 to about 0.4 mg per kg body weight; about 0.4 to about 0.6 mg per kg body weight; about 0.6 to about 0.8 mg per kg body weight; about 0.8 to about 1.0 mg per kg body weight; about 1.2 to about 1.4 mg per kg body weight; about 1.4 to about 1.6 mg per kg body weight; about 1.6 to about 1.8 mg per kg body weight; 20 about 1.8 to about 2 mg per kg body weight; about 0.2 to about 1.8 mg per kg body weight; about 0.4 to about 1.6 mg per kg body weight; about 0.6 to about 1.4 mg per kg body weight; about 0.8 to about 1.2 mg per kg body weight are provided. [0276] For injection, the dosage of the compound to prevent or treat diseases is typically about 1 to about 100 mg dose administered daily. For oral adminiistration 25 the compound may be administered once or twice daily at a dose of about 1 to about 100 mg. [0277] In additional embodiments, the pharmaceutical composition is in unit dosage form by weight of the compound. In such embodiments each unit dosage typically consists of 1-100 mg administered daily. In various embodiments, dosages 30 in the ranges of about 1 to about 100 mg administered daily; about 1 to about 10 mg administered daily; about 10 to about 20 mg administered daily; about 20 to about 30 mg administered daily; about 30 to about 40 mg administered daily; about 40 to about 50 mg administered daily; about 50 to about 60 mg administered daily; about 60 to about 70 mg administered daily; about 70 to about 80 mg administered daily; 31 WO 2014/113820 PCT/US2014/012405 about 80 to about 90 mg administered daily; about 90 to about 100 mg administered daily; about 10 to about 90 mg administered daily; about 20 to about 80 mg administered daily; about 30 to about 70 mg administered daily; and about 40 to about 60 mg administered daily are provided. 5 [0278] The compound is generally administered in the range of 0.01-2 mg per kg body weight daily. In various embodiments, daily dosages in the ranges of about 0.01 to about 2 mg per kg body weight; about 0.01 to about 0.2 mg per kg body weight;_about 0.2 to about 0.4 mg per kg body weight; about 0.4 to about 0.6 mg per kg body weight; about 0.6 to about 0.8 mg per kg body weight; about 0.8 to about 1.0 10 mg per kg body weight; about 1.2 to about 1.4 mg per kg body weight; about 1.4 to about 1.6 mg per kg body weight; about 1.6 to about 1.8 mg per kg body weight; about 1.8 to about 2 mg per kg body weight; about 0.2 to about 1.8 mg per kg body weight; about 0.4 to about 1.6 mg per kg body weight; about 0.6 to about 1.4 mg per kg body weight; about 0.8 to about 1.2 mg per kg body weight are provided. 15 [0279] For injection, the dosage of the compound to prevent or treat diseases is typically about 1 to about 100 mg dose administered daily. For oral adminiistration the compound may be administered once or twice daily at a dose of about 1 to about 100 mg. [0280] The following formulations illustrate representative pharmaceutical 20 dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X'): 25 (i) Tablet 1 mq/tablet 'Compound X 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 30 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 Total 300.0 35 32 WO 2014/113820 PCT/US2014/012405 (ii) Tablet 2 mq/tablet 'Compound X 20.0 Microcrystalline cellulose 410.0 Starch 50.0 5 Sodium starch glycolate 15.0 Magnesium stearate 5.0 Total 500.0 (iii) Capsule mq/capsule 10 'Compound X 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 15 Total 600.0 (iv) Iniection 1 (1 mq/mL) mq/mL 'Compound X (free acid form) 1.0 Dibasic sodium phosphate 12.0 20 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL 25 (v) Iniection 2 (10 mq/mL) mq/mL 'Compound X (free acid form) 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 30 Polyethylene glycol 400 200.0 0.1 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL 35 (vi) Aerosol mg/can 'Compound X 20 Oleic acid 10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000 40 Dichlorotetrafluoroethane 5,000 (vii) Topical Gel 1 wt.% 'Compound X 5% Carbomer 934 1.25% 45 Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben 0.2% Purified water q.s. to 1 0Og 50 33 WO 2014/113820 PCT/US2014/012405 (viii) Topical Gel 2 wt.% 'Compound X 5% Methylcellulose 2% Methyl paraben 0.2% 5 Propyl paraben 0.02% Purified water q.s. to 1 0Og (ix) Topical Ointment wt.% 'Compound X 5% 10 Propylene glycol 1% Anhydrous ointment base 40% Polysorbate 80 2% Methyl paraben 0.2% Purified water q.s. to 1 0Og 15 (x) Topical Cream 1 wt.% 'Compound X 5% White bees wax 10% Liquid paraffin 30% 20 Benzyl alcohol 5% Purified water q.s. to 1 0Og (xi) Topical Cream 2 wt.% 'Compound X 5% 25 Stearic acid 10% Glyceryl monostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropyl palmitate 2 % 30 Methyl Paraben 0.2% Purified water q.s. to 1 0Og [0281] These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above 35 pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable 40 equivalents and proportions may be varied, according to the desired properties of the dosage form of interest. [0282] Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 30 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length 34 WO 2014/113820 PCT/US2014/012405 alkyl groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-30 carbon atoms. Cyclic alkyl groups include those having one or more rings. Cyclic alkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 1 0-member carbon ring and 5 particularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbon rings in cyclic alkyl groups can also carry alkyl groups. Cyclic alkyl groups can include bicyclic and tricyclic alkyl groups. Alkyl groups are optionally substituted. Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted. Specific alkyl groups include methyl, ethyl, n-propyl, iso 10 propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted. Substituted alkyl groups include fully halogenated or semihalogenated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or 15 iodine atoms. Substituted alkyl groups include fully fluorinated or semifluorinated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms. An alkoxy group is an alkyl group linked to oxygen and can be represented by the formula R-O. [0283] Aryl groups include groups having one or more 5- or 6-member 20 aromatic or heteroaromatic rings. Aryl groups can contain one or more fused aromatic rings. Heteroaromatic rings can include one or more N, 0, or S atoms in the ring. Heteroaromatic rings can include those with one, two or three N, those with one or two 0, and those with one or two S, or combinations of one or two or three N, 0 or S. Aryl groups are optionally substituted. Substituted aryl groups include among 25 others those which are substituted with alkyl or alkenyl groups, which groups in turn can be optionally substituted. Specific aryl groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups, all of which are optionally substituted. Substituted aryl groups include fully halogenated or semihalogenated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more 30 fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms. Substituted aryl groups include fully fluorinated or semifluorinated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms. [0284] Arylalkyl groups are alkyl groups substituted with one or more aryl groups wherein the alkyl groups optionally carry additional substituents and the aryl 35 WO 2014/113820 PCT/US2014/012405 groups are optionally substituted. Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups. Alkylaryl groups are alternatively described as aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. 5 Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl. Substituted arylalkyl groups include fully halogenated or semihalogenated arylalkyl groups, such as arylalkyl groups having one or more alkyl and/or aryl having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms. 10 [0285] Optional substitution of any alkyl and aryl groups includes substitution with one or more of the following substituents: halogens, -CN, -COOR, -OR, COR, OCOOR, CON(R)2 , -OCON(R)2, -N(R)2, -N02, -SR, -S02R, -S02N(R)2 or -SOR groups. Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are 15 optionally substituted. Optional substitution of alkenyl groups includes substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted. Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted. 20 [0286] Optional substituents for alkyl and aryl groups include among others: COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which are optionally substituted;-COR where R is a hydrogen, or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups 25 all of which groups are optionally substituted; -CON(R)2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds; -OCON(R)2 where each R, independently of each other R, is a 30 hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds; -N(R)2 where each R, independently of each other R, is a hydrogen, or an alkyl group, acyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or 36 WO 2014/113820 PCT/US2014/012405 phenyl or acetyl groups all of which are optionally substituted; or R and R can form a ring which may contain one or more double bonds; -SR, -SO2R,or -SOR where R is an alkyl group or an aryl groups and more specifically where R is methyl, ethyl, propyl, butyl, phenyl groups all of which are optionally substituted; for -SR, R can be 5 hydrogen; -OCOOR where R is an alkyl group or an aryl groups; -SO2N(R)2 where R is a hydrogen, an alkyl group, or an aryl group and R and R can form a ring; -OR where R=H, alkyl, aryl, or acyl; for example, R can be an acyl yielding OCOR* where R* is a hydrogen or an alkyl group or an aryl group and more specifically where R* is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are 10 optionally substituted. [0287] Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups. Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di , tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4 15 halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenyl groups, 3- or 4 alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo substituted naphthalene groups. More specifically, substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, 20 particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups, and methoxyphenyl groups, particularly 4 methoxyphenyl groups. [0288] As to any of the above groups which contain one or more substituents, it is understood, that such groups do not contain any substitution or substitution 25 patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this disclosure include all stereochemical isomers arising from the substitution of these compounds. [0289] When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a 30 formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination. Additionally, unless otherwise specified, all isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure. For example, it will be understood that any one or more hydrogens in a molecule disclosed can be replaced 37 WO 2014/113820 PCT/US2014/012405 with deuterium or tritium. Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known 5 that one of ordinary skill in the art can name the same compounds differently. [0290] The disclosed compounds may be present as racemic mixtures or the individual isomers, such as diasteriomers or enantiomers. In various embodiments, the formulation ecompasses each and every possible enantiomer and diasteriomer as well as racemates and mixtures that may be enriched in one of the possible 10 steriosiomers. In another embodiment, forms in which the compound may be present as salt including pharmaceutically acceptable acidic and basic salts are provided. [0291] Many of the molecules disclosed herein contain one or more ionizable groups [groups from which a proton can be removed (e.g., -COOH) or added (e.g., amines) or which can be quaternized (e.g., amines)]. All possible ionic forms of such 15 molecules and salts thereof are intended to be included individually in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art can select from among a wide variety of available counterions those that are appropriate for preparation of salts of this disclosure for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt 20 may result in increased or decreased solubility of that salt. [0292] Examples [0293] General Procedures 25 [0294] The following procedures were used in the Examples described below. [0295] Cell Culture and Reagents [0296] MCF-7 (MCF-7: Michigan Cancer Foundation-7), T47D, T47D-kBluc, HCC-1 500 (HCC-1 500: human carcinoma cells-1 500), ZR-75-1, MCF1 0A (MCF 1 0A: 30 Michigan Cancer Foundation 1 0A), MDA MB-231, CaOV-3 (CAOV-3: Cancer Ovarian-3), OVCAR-3 (OVCAR-3: Ovarian Carcinoma 3), IGROV-1, ES2, ECC-1 (ECC-1: endometrial carcinoma cells-1), HeLa, PC-3 (PC-3: prostate cancer cells-3), DU145, H1793, A549, MEF (MEF: mouse embryo fibroblast) and HepG2 (HepG2: hepatoma G2) cells were obtained from the ATCC. Dr. E. Wilson provided HeLa 38 WO 2014/113820 PCT/US2014/012405 AR1 3 cells, Dr. K. Korach provided BG-1 cells, Dr. B. H. Park provided MCF1 OAERIN9 cells (MCF1 OAERIN9: Michigan Cancer Foundation 1 0A estrogen receptor in (positive) 9), Dr. R. Schiff provided BT-474 cells, and Dr. E. Alarid provided MCF7ERaHA cells (MCF7ERaHA: MCF-7 estrogen receptor a hemaggluttinin). Prior to experiments, to 5 deplete cells of estrogens in the serum and medium, ERa positive cell lines were maintained for 4 days in medium supplemented with phenol red-free charcoal dextran (CD) treated serum. 17p-Estradiol (E2), 4-hydroxytamoxifen (4-OHT), 2 aminoethyl diphenylborinate (2-APB), and tunicamycin (TUN) were obtained from Sigma Aldrich. ICI 182,780 (ICI) was obtained from Tocris Biosciences. Ryanodine 10 (RyR) was obtained from Santa Cruz Biotechnology. The following antibodies were purchased from Cell Signaling: Phospho-elF2a (Ser51) (#3398), elF2a (#5324), Phospho-PERK (#3179), PERK (#5683), and BiP (#3177). Additional antibodies used were ATF6a (Imgenex, CA), p-Actin (Sigma, MO), and a-Tubulin (Sigma, MO). 15 [0297] Cell Proliferation Assays [0298] Cells proliferation assays were carried out as previously described in Andruska, N., et al., Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERalpha-induced proliferation of breast cancer cells. J Biomol Screen. 2012; 17(7):921-32. For each cell line, cell number was calculated from a 20 standard curve of the number of plated cells versus A490. [0299] Chemical Libraries and Screening The small molecule libraries screened were (1) the ~-150,000 small molecule Chembridge MicroFormat small molecule library; (2) the -10,000 small molecule 25 University of Illinois Marvel library developed by Drs. K. Putt and P. Hergenrother (Putt K.S., Hergenrother P.J., A nonradiometric, high-throughput assay for poly(ADP ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation. Anal Biochem. 2004;333(2):256-64); and the -2,000 small molecule NCI diversity set obtained from NIH (National Institutes of Health). High throughput screening for 30 small molecule inhibitors of endogenous E2-ERa induced expression of the stably transfected (ERE)3-luciferase reporter in T47D-KBluc cells was carried out using the assay described in Andruska N. et al., Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERalpha-induced proliferation of breast cancer cells. J Biomol Screen. 2012; 17(7):921-32. 39 WO 2014/113820 PCT/US2014/012405 [0300] Luciferase Assays [0301] Reporter gene assays were carried out, as previously described in Andruska, N., et al., Evaluation of a luciferase-based reporter assay as a screen for 5 inhibitors of estrogen-ERalpha-induced proliferation of breast cancer cells. J Biomol Screen. 2012; 17(7):921-32. Briefly, for the primary screen in 384-well plates, cells were harvested at a density of 1-million cells/ml in RPMI-1 640, plated at a density of 10,000 cells/well by pipetting 10 pl of cells into each well using a Matrix Wellmate dispenser. The final concentration of test compounds in each well was 7.14 pM. The 10 screening medium contained 0.1% (v/v) EtOH, 0.07% (v/v) DMSO (DMSO: dimethyl sulfoxide), and 10 nM E2. Plates were centrifuged for 2 minutes at 500 rpm, and incubated for 24 hours. For follow-on testing in 96-well plates, cells were switched to 10% CD-FBS (FBS: fetal bovine serum) for four days prior to experiments, and plated at a density of 50,000 cells/well in 96-well plates. The medium was replaced 15 the next day with medium containing the test compounds, with or without hormone, incubated for 24 hours and luciferase (luc: luciferase) assays were performed using Bright Glow reagent (Promega, WI). [0302] Quantitative Reverse Transcrilptase PCR (qRT-PCR) 20 [0303] RNA was extracted using a QiaShredder kit (Qiagen) for cell homogenization and purified with the RNAeasy mini-kit (Qiagen, CA). cDNA was prepared from 0.5 pg of RNA by reverse transcription using a DyNAmo cDNA synthesis kit (Finnzymes, Finland). Quantitative PCR assays were performed in triplicate, usually from each of 3 sets of cells. Reactions contained 10 ng of cDNA 25 and 50 nM forward and reverse primers in 15 pl and were carried out using Power SYBR Green PCR Mastermix (Applied Biosystems). The fold change in expression of each gene was calculated using the AACt method with the ribosomal protein 36B4 used as the internal control, as described in Kretzer, N.M., et al., A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer 30 cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem. 2010; 285(53):41863-73. 40 WO 2014/113820 PCT/US2014/012405 [0304] Chromatin lmmunolprecilpitation [0305] MCF-7 cells were depleted of estrogens by 3 days of culture in 5% CD FBS. Cells were pretreated with 1 pM BHPI or DMSO (0.1%) as a control for 105 minutes, and then were treated with either 10 nM E2 or an ethanol-vehicle control 5 (0.1%) for 45 minutes. ChIP was carried out as described in Cherian, M.T., et al., A competitive inhibitor that reduces recruitment of androgen receptor to androgen responsive genes. J Biol Chem. 2012; 287(28):23368-80. [0306] siRNA Transfections 10 [0307] siRNA (short interfering RNA) knockdowns were performed using ON TARGETplus SMARTpools, each containing a mixture of 4 siRNAs (Dharmacon, CO). Transfections were performed using DharmaFECT1 Transfection Reagent (Dharmacon, CO). To knockdown ERa, MCF1 OAERIN9 cells were treated for 16 hours with either human ERa SMARTpool (SMARTpool by Dharmacon) (ESR1) siRNA or 15 Non-targeting Control Pool siRNA. Cells were treated with transfection complex for 16 hours, and medium was replaced with DMEM/F1 2 (DMEM/F1 2: Dulbecco's Minimum Essential Medium/Hams Medium F1 2), supplemented with 2% CD-FBS. ERa knockdown at the mRNA and protein level was assessed every 24 hours following transfection. The effects of BHPI on protein synthesis following ERa 20 knockdown were assessed 3-days post-knockdown by treating cells with either 0.1% DMSO loading control or 100 nM BHPI for the indicated times, and protein synthesis was then assessed by measuring 35 S-methionine incorporation. To knockdown PERK, MCF-7 cells were maintained in MEM (MEM: minimal essential medium) containing 5% CD-FBS for 4 days prior to plating cells in serum-free MEM. Cells 25 were treated with ON-TARGETplus Human PERK (EIF2AK3) siRNA or ON TARGETplus Non-targeting Control Pool siRNA. Cells were treated with transfection complexes for 16 hours and medium was replaced with MEM, supplemented with 10% CD-calf serum. To assess PERK knockdown at the mRNA and protein level, mRNA and protein samples were collected every 24 hours post-transfection. Since 30 E2-ERa induces PERK (see Figure 5b), knockdown experiments were carried out in the absence of estrogen. The effects of BHPI on protein synthesis following PERK knockdown were assessed 3-days post-knockdown by treating cells with either 1% DMSO loading control or 250 nM BHPI for the indicated times and protein synthesis was then assessed by measuring 35 S-methionine incorporation. 41 WO 2014/113820 PCT/US2014/012405 [0308] Immunoblottinq [0309] Western blotting carried out as described in Andruska, N. et al., Evaluation of a luciferase-based reporter assay as a screen for inhibitors of 5 estrogen-ERalpha-induced proliferation of breast cancer cells. J Biomol Screen. 2012; 17(7):921-32. The following antibodies were used: ERa [6F1 1] antibody (Biocare Medical, CA), Phospho-elF2a (Ser51) (#3398; Cell Signaling Technology), elF2a (#5324; Cell Signaling Technologies, MA), Phospho-eEF2 (#2331; Cell Signaling Technology, MA), eEF2 (#2332; Cell Signaling Technology, MA), 10 Phospho-p44/42 MAPK (#4370; Cell Signaling Technology, MA), p44/42 MAPK (#4695; Cell Signaling Technology, MA), Phospho-PERK (#3179; Cell Signaling Technology, MA), PERK (#5683; Cell Signaling Technology, MA), ATF6a (Imgenex, CA), Phospho-AMPKa (#2535, Cell Signaling Technology, MA), AMPKa (adenosine monophosphate kinase a (subunit)) (#2603, Cell Signaling Technology, MA), 15 Phospho-AMPKp1 (AMPKp1: adenosine monophosphate kinase P1 (subunit)) (#4181, Cell Signaling Technology, MA), AMPKp1/2 (#4150, Cell Signaling Technology, MA), BiP (#3177, Cell Signaling Technology, MA), p 5 8 IPK (#2940, Cell Signaling Technology, MA), laminin A/C (Santa Cruz, CA), p-Actin (Sigma, MO), and a-Tubulin (Sigma, MO). Bound antibodies were detected using horseradish 20 peroxidase-conjugated secondary antibodies and chemiluminescent immunodetection with an ECL Detection Kit (GE Healthcare, NJ), and were visualized using a Phosphorlmager. [0310] Nuclear-cytolplasmic Distribution of ERa 25 [0311] MCF-7 cells were pre-treated with 1 pM BHPI or DMSO (0.1%) for 30 minutes, followed by 2 hours with or without E2. Nuclear and cytoplasmic extraction was carried out on -6 million cells/treatment using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoScientific). Lamin A/C and a-Tubulin, were used as nuclear and cytoplasmic markers, respectively. 30 [0312] Protein Synthesis [0313] Protein synthesis rates were evaluated by measuring incorporation of 35 5-methionine into newly synthesized protein. Cells were plated at a density of 10,000 cells/well in 96-well plates. Cells were incubated for 30 minutes with 3 pCi of 42 WO 2014/113820 PCT/US2014/012405 35 S-methionine (PerkinElmer, MA) per well at 37 0 C. Cells were washed two times with PBS, and lysed using 30 pL of RIPA buffer. Cell lysates were collected in microfuge tubes and clarified by centrifugation at 13,000 x g for 10 minutes at 4 0 C. Samples were normalized to total protein, and the appropriate volume of sample was 5 spotted onto Whatman 540 filter paper discs and immersed in cold 10% TCA (trichloroacetic acid). The filters were washed once in 10% TCA and 3 times in 5% TCA and air dried Trapped protein was then solubilized and the filters were counted. [0314] Calcium Imaging 10 [0315] Cytoplasmic Ca 2 , concentrations were measured using the calcium sensitive dye, Fluo-4 AM (Fluo-4: 2-{[2-(2-{5-[bis(carboxymethyl)amino]-2 methylphenoxy}ethoxy)-4-(2,7-difluoro-6-hydroxy-3-oxo-3H-xanthen-9 yl)phenyl](carboxymethyl)amino}acetic acid). The cells were grown on 35 mm fluorodish cell culture plates (Cat#FD35-1 00, World Precision Instruments) for two 15 days prior to imaging experiments. The cells were loaded with 5 pM Fluo-4 AM (Life Technologies, CA) in HEPES-based buffer (140 mM NaCl, 4.7 mM KCI, 1.13 mM MgCl 2 , 10 mM HEPES, 10 mM Glucose, pH = 7.4) for 30 minutes at 37 0 C before measurement of intracellular calcium. The cells were washed three times with HEPES buffer to remove extracellular Fluo-4-AM dye and incubated with either 2 20 mM CaC1 2 or 0 mM CaC1 2 for 10 minutes to complete de-esterification of the dyes. Confocal images were obtained for one minute to determine basal fluorescence intensity, and then the appropriate treatment was added. Confocal images were captured using a Zeiss LSM 700 confocal system, Plan-Four 20X objective (N.A. = 0.8) and scanned at a resolution of 512x512 pixels (780ms/min). To minimize photo 25 bleaching and photo-toxicity of samples, laser power was reduced to 7%. Excitation was at 488 nm, and emission was at 525 nm. Images were acquired and analyzed with AxioVision and Zen software (Zeiss). Calcium traces were generated by normalizing fluorescence to basal fluorescence intensity. Data presented as mean standard error (n = 10 individual cells). 30 [0316] Protease Sensitivity Assays [0317] ERa LBD (N304-S554) containing an N-terminal 6-His tag, was purified and stored in Tris-HCI buffer (50 mM Tris-HCI pH 8.0, 10% glycerol, 2 mM DTT (dithiothreitol), 1 mM EDTA, and 1 mM sodium orthovanadate). Purified ERa LBD 43 WO 2014/113820 PCT/US2014/012405 protein (10 pg) was incubated with 500 nM E2 for 20 minutes at 37 OC. Subsequently, control DMSO vehicle, BHPI (1 pM) or inactive Compound 88 (1 pM) and incubated for 20 minutes at 370C. For partial protease cleavage, the binding mixture was added with/without protease K at a concentration of 7.5 ng protease K per pg protein. After 5 a 10 minute incubation at 22 C, the digestions were terminated by addition of SDS sample buffer. The denatured samples were analyzed on a 15% SDS-PAGE gel and visualized by coomassie blue staining. [0318] Intrinsic Fluorescent Spectroscopy 10 [0319] The stock solution of full-length ERa domain was diluted to 400 nM in a Tris-Buffer (50 mM Tris/HCI pH8.0, 150 mM KCl, 2 mM DTT, 1 mM EDTA, and 10% glycerol). Intrinsic fluorescence measurements used a Varian Cary Eclipse Fluorescence Spectrophotometer and a 10 mm quartz cuvette. The excitation wavelength for tryptophan fluorescence was 295 nM and excitation and emission 15 slits were 5 nm. Emission spectra were collected at 370C from 310-380 nm. E2 (500 nM), BHPI (500 nM), or inactive compound 88 (500 nM) was added and samples were incubated at 37 C for 10 minutes, and ERa emission spectrum was recorded. All the spectra were corrected for baseline. 20 [0320] Colony Formation Assays [0321] Assays to assess anchorage-independent cell proliferation in soft agar were carried out as described in Cherian, M.T, et al., A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes. J Biol Chem. 2012; 287(28):23368-80. Each treatment condition was evaluated on five 25 independent sets of cells. Culture medium was changed every 3 days. Colonies were visible after 2 weeks, and total colonies were counted at day 21 using a dissecting microscope. Photographs of colonies were taken using a Zeiss Axiolmager2 imaging system at 5X magnification. 30 [0322] Mouse Xenoqraft [0323] All experiments were approved by the Institutional Animal Care Committee (IACUC) of the University of Illinois at Urbana-Champaign. The MCF-7 cell mouse xenograft model is described in detail in Ju, Y.H., et al., Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human 44 WO 2014/113820 PCT/US2014/012405 breast cancer (MCF-7) cells implanted in athymic mice. Cancer research. 2002; 62(9):2474-7. At least 12 animals, usually with 4 tumors per animal, were required per experimental group to maintain significant statistical power to detect >25% difference in tumor growth rates. Briefly, estrogen pellets (1 mg: 19 mg estrogen: 5 cholesterol) were implanted into 60 athymic female OVX mice, which were 7 weeks of age. Three days after E2 pellet implantation 1 million MCF-7, human breast cancer cells per site in matrigel were subcutaneously injected at 2 sites in each flank for a total of 4 potential tumors per mouse. When the average tumor size reached 17.6 mm 2 (4.7 by 4.7 mm), E2 pellets were removed and a lower dose of E2 in 10 sealed silastic tubing (1:31 estrogen: cholesterol, 3 mg total weight) was implanted in the same site. When the average tumor size reached 23.5 mm 2 (5.5 by 5.5 mm), mice were divided into 4 groups with tumor size normalized: E2 group, no treatment control (NC) group, B_10 group and B_1/B_15 group. E2 silastic tubes in the NC group were removed, while E2 silastic tubes in the E2, B_10, and B_1/B_15 groups 15 were retained. The E2 and NC group received intraperitoneal injection every other day with 10 ml/kg vehicle (2% DMSO, 10% Tween-20, and 88% PBS). The B_10 group received 10 mg/kg BHPI by intraperitoneal injection every other day. The B_1/B_15 group received 1 mg/kg BHPI by intraperitoneal injection every other day for 14 days. Since this extremely low BHPI dose had no effect, (average tumor 20 cross-sectional area -45 mm 2 ) they then received 15 mg/kg BHPI every day for another 10 days. Food intake and body weight were measured every 4 days and food intake is presented as grams/day. Tumors were measured every 4 days with a caliper. Tumor cross sectional area was calculated as (a/2)*(b/2)*3.14, where a and b were the measured diameters of each tumor. On termination of the experiments 25 mice were euthanized and the tumors were excised and weighed. Two of sixty mice were removed during the course of the study, one that failed to form tumors and the other due to unrelated illness. No tumors were excluded from analysis, and blinding was not performed. 30 [0324] Tumor Microarray Data Analysis [0325] Analysis was performed using several publically available tumors cohorts. ERa and UPR correlation analysis was performed on 278 breast cancer tumors (GSE20194). (See, Shi, L., et al., The MicroArray Quality Control (MAQC)-Il study of common practices for the development and validation of microarray-based 45 WO 2014/113820 PCT/US2014/012405 predictive models. Nat Biotechnol. 2010; 28(8):827-38.) Analysis of UPR gene expression profiles in normal, DCIS, and IDC samples was carried out on 19 samples (GSE21422). (See, Kretschmer, C., et al., Identification of early molecular markers for breast cancer. Mol Cancer. 2011; 10(1):15.) A "UPR Gene Signature" 5 was constructed to carry out risk prediction analysis. The UPR gene signature was evaluated for its ability to predict: (i) tumor relapse in 261 early-stage ERa+ breast cancers (GSE6532) (see, Loi, S., et al., Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol. 2007; 25(10):1239-46); (ii) tumor relapse in 474 ERai+ patients receiving 10 solely tamoxifen therapy for 5 years (GSE6532, GSE1 7705), (see, Loi, S., et al., Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol. 2007; 25(10):1239-46; and Symmans, W.F., et al., Genomic index of sensitivity to endocrine therapy for breast cancer. J Clin Oncol. 2010; 28(27):4111-9); and (iii) overall survival in a mixed-cohort 15 of 236 breast cancer patients (GSE3494) (see, Miller, L. D., et al., An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc Natl Acad Sci U S A. 2005; 102(38):13550-5). Microarray data analysis was performed using BRB ArrayTools (version 4.2.1) and R software version 2.13.2. Gene expression values from CEL 20 files were normalized by use of the standard quantile normalization method. (Bolstad, B.M., et al., A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003; 19(2):185-93.) Pearson correlation tests and Spearman log rank tests were used to determine gene expression correlation coefficients. Wald tests were used to test 25 whether UPR genes were predictive of tumor recurrence and overall survival. Univariate and multivariate hazard ratios were estimated using Cox regression analysis. Covariates statistically significant in univariate analysis were further assessed in multivariate analysis. A patient was excluded from multivariate analysis if data for one or more variables were missing. Risk prediction using the UPR gene 30 signature was carried out using the supervised principle components method (Bair, E., and Tibshirani, R., Semi-supervised methods to predict patient survival from gene expression data. PLoS biology. 2004; 2(4):E1 08) and visualized using Kaplan-Meier plots and compared using log-rank tests. 46 WO 2014/113820 PCT/US2014/012405 [0326] Statistical Analysis [0327] Calcium measurements reported as mean ± SE. All remaining data is reported as mean ± S.E.M. Two-tailed student's t-test was used to compare the means between groups. P < 0.05 was considered significant. 5 [0328] Example 1 - High Throughput Screening to Identify Novel Small Molecule Inhibitors of Estrogen Receptor a (ERa). [0329] Example 1 a - High Throughput Screening 10 [0330] High throughput screening (HTS) and follow-up testing for specificity, toxicity, and potency were performed. The screening was a cell-based high throughput screen of approximately 150,000 small molecules for inhibitors of E2-ERa regulated gene expression. Candidate compounds were then "filtered" through additional tests for specificity, toxicity, potency, and site of action. A schematic 15 representation of the screening process is shown in Figure 1. Several "filtering" assays were carried out. Preliminary "hits" were re-screened to eliminate most inhibitors that might act in the same way as tamoxifen and other selective estrogen receptor modulators (SERMs) by competing with estrogens for binding to ERa. To test for specificity, the ability of the hits to inhibit estrogen-ERa-regulated gene 20 expression and androgen (dihydrotestosterone)-androgen receptor (DHT-AR) regulated gene expression was compared. Androgen receptor (AR) was chosen because of its importance in androgen-stimulated growth of prostate cancer cells. Small molecules that inhibited both ERa and AR were not carried forward because they were considered less likely to have the requisite specificity. 25 [0331] To test for toxicity, we initially screened the "hits" in MDA-MB-231 breast cancer cells. These are triple negative breast cancer cells and do not contain ERa. MDA-MB-231 cells are a stringent system in which to test for non-specific toxicity. MDA-MB-231 cells are highly sensitive to growth inhibition by non-specific small molecules. (Kretzer, N.M., et al., A noncompetitive small molecule inhibitor of 30 estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem. 2010; 285(53):41863-73.) Also, in a test for non-specific toxicity of small molecules that do not inhibit ERa, more small molecules inhibited the growth of the MDA-MB-231 cells than the ERa-containing MCF-7 cells or T47D cells. 47 WO 2014/113820 PCT/US2014/012405 [0332] One goal was to identify small molecules that inhibit estrogen-ERa dependent growth of cancer cells. Since the cell growth assays were not suitable for true high throughput screening, HTS was performed using the ability of small molecules to inhibit an estrogen-ERa inducible gene as a surrogate marker. About 5 2,000 small molecule "hits" were tested for their ability to inhibit estrogen-ERa (1 7p estradiol [E2]-ERa)-dependent growth of MCF-7 human breast cancer cells. Twenty three (23) compounds, comprising eighteen (18) structurally distinct families, specifically inhibited estrogen-dependent growth of MCF-7 cells with IC50s <1 pM. 10 [0333] Example 1b - Identification of BHPI [0334] The most promising compounds identified through HTS were BHPI and its close relatives. The structure of the lead compound, BHPI, 3,3-bis(4 hydroxyphenyl)-7-methyl-1,3,dihydro-2H-indol-2-one, is shown in Figure 2. (The structure and characterization of some other BHPI family members is shown in 15 Figure 15.) [0335] Example 2 - BHPI and structurally related compounds selectively inhibit estrogen-delpendent cell proliferation and E2-ERa mediated gene expression. [0336] BHPI specifically inhibits estrogen-ERa induced expression of an 20 estrogen response element-luciferase reporter gene with no effect on androgen (dihydrotestosterone)-androgen receptor (AR) induction of a prostate specific antigen (PSA) response element-luciferase reporter gene. [0337] A dose-response study of BHPI inhibition of the ERE-luciferase reporter gene was performed. The cell maintenance and luciferase assay were 25 carried out as follows. Five to six days before the experiment, T47D-KBluc cells (ATCC: CRL-2865) were subcultured and plated at high density (-30-40% confluence) in RPM11640 + 10% fetal bovine serum (FBS) and grown at 370C in 5% C02. Two days later, to reduce the level of endogenous estrogens, the medium was changed to RPMI + 10% charcoal dextran-treated (cd)-FBS. After three or four days, 30 with a medium change on day two, the cells were harvested, counted, and 50,000 cells in 100 pl of medium were added to each well of 96-well white-wall-clear bottom plates (BD Biosciences, NJ) in RPMI + 10% CD-calf serum (CS). The medium was replaced the next day with medium containing a test compound (here BHPI), with or without 10 nM E2. After 24 hours, the medium was aspirated off, and 30 pl of Bright 48 WO 2014/113820 PCT/US2014/012405 Glow (Promega, WI) was added. The plate was shaken for 5 minutes to help lyse the cells and then subjected to centrifugation at 2500 RPM for 2 minutes to remove any bubbles in the wells. Samples contained ethanol vehicle, or 10 nM E2 in ethanol plus the indicated concentrations of BHPI in DMSO. 5 [0338] On addition of E2, endogenous ERa in T47D human breast cancer cells drives the induction of the stably transfected (ERE)3-lucif erase reporter gene. The results are shown in Figure 3. The data represents the average + S.E.M. of quadruplicate assays. Figure 3A shows the dose-response study of E2 induction of the ERE-luciferase reported gene. Figure 3B shows the dose-response study of 10 BHPI inhibition of E2-ERa induction of the ERE-luciferase reporter gene. BHPI and structurally related compounds selectively inhibit estrogen-dependent cell proliferation and E2-ERa mediated gene expression. Figure 3B shows results from dose response studies of the effect of BHPI on 1 7p-estradiol (E2) induction of ERE luciferase activity in ERa positive T47D-kBluc breast cancer cells (black bars) and for 15 dihydrotestosterone-androgen receptor (DHT-AR) induction of prostate specific antigen (PSA)-luciferase in ERa negative HeLaA6 cells (open bars). BHPI strongly inhibited E2-ERa induction of an estrogen response element (ERE)-lucif erase reporter and had no effect on androgen induction of an androgen response element (ARE)-luciferase reporter. 20 [0339] Example 3 - BHPI is not a competitive inhibitor for binding to ERa [0340] BHPI is not a competitive inhibitor, and it does not act by competing with estrogens for binding to ERa. Figure 4 shows the effect of BHPI on expression of an estrogen-regulated gene in the presence of low and high concentrations of 25 estrogen. ERa positive MCF-7 human breast cancer cells were maintained in 5% CD-FBS for 4 days to deplete the medium of endogenous estrogens. Cells were harvested in 10% CD-CS, and plated into 6-well plates at a density of 450,000 cells per well. The following day, the medium was replaced with fresh 10% CD-CS. The next day, wells were pre-treated for 30 minutes with either 100 nM ICI 182,780 (ICI), 30 100 nM BHPI, or an 0.1% DMSO/EtOH-vehicle control. Cells were then induced with either 1 nM E2 (Figure 4, open bars), 1,000 nM E2 (Figure 4, black bars), or 0.1% ethanol vehicle control (Figure 4, grey bar) for two hours. Cells were lysed and RNA was extracted. qPCR (qPCR: quantitative polymerase chain reaction) analysis for pS2 (pS2: Trefoil factor 1) mRNA was performed. Data represents the average of 3 49 WO 2014/113820 PCT/US2014/012405 independent sets of cells, each assayed in triplicate. Date reported as mean + S.E.M. [0341] BHPI interacts with ERa and inhibits E2-ERa-regulated gene expression. BHPI is a non-competitive ERa inhibitor. Increasing the concentration of 5 the estrogen, 17p-estradiol (E2), by 1,000 fold abolished the ability of the competitor antiestrogen ICI 182,780 to inhibit gene expression with no effect on BHPI inhibition of the E2 induction of pS2 mRNA. [0342] It has also been shown that BHPI does not compete with estrogens for binding to ERa in vitro. Radioligand competition assays comparing the ability of 10 increasing concentrations of unlabeled E2 and BHPI to compete with 0.2 nM [ 3
H]
estradiol for binding to ERa show that BHPI is at least 10,000 fold weaker competitor than E2. (Carlson, K. E., Choi, I., Gee, A., Katzenellenbogen, B. S., and Katzenellenbogen, J. A., Altered ligand binding properties and enhanced stability of a constitutively active estrogen receptor: evidence that an open pocket conformation 15 is required for ligand interaction. Biochemistry (1997) 36: 14897-14905. Describes the method of doing the binding assay to determine whether a compound competes with estrogen.) [0343] Example 4 - BHPI binds directly to ERa and appears to change its 20 shape [0344] To test for in vitro interaction of BHPI with purified ERa, the ability of BHPI to alter the fluorescence emission spectrum of purified ERa was evaluated. Consistent with BHPI binding E2-ERa, BHPI, but not an inactive relative, significantly altered the fluorescence emission spectrum of ERa. Figure 5 shows the structures of 25 BHPI (Figure 5A) and of an inactive related compound, termed Compound 8 (Figure 5B). Figure 6A shows the effect of BHPI and a control compound on the fluorescence emission spectrum of full-length ERa. Fluorescence emission spectra of full-length ERa in the presence of E2 and (i) DMSO; (ii) 500 nM BHPI; or (iii) 500 nM of the BHPI-related inactive Compound 8 are shown. 30 [0345] Whether BHPI could alter the sensitivity of purified ERa ligand-binding domain (LBD) to protease digestion was also tested. ERaLBD was subjected to protease digestion in the presence of DMSO or BHPI. Figure 6B and Figure 6C show the effect of BHPI on protease sensitivity of the ERa ligand binding domain (LBD) analyzed by SDS polyacrylamide gel electrophoresis. Bands were visualized by 50 WO 2014/113820 PCT/US2014/012405 Coomassie staining. Figure 6B shows the protease digestion pattern after cleavage with proteinase K. Addition of BHPI followed by cleavage with proteinase K revealed a 15 kDA band in BHPI treated ERa LBD that was absent in the control LBD. Figure 6C shows the protease digestion pattern after cleavage with chymotrypsin. 5 [0346] Example 5 - BHPI inhibits ER-requlated qene expression [0347] Example 5a - BHPI inhibits induction of E2-ERa induced genes in breast and ovarian cancer cells 10 [0348] The best-understood result of binding of the estrogen, 17p-estradiol (E2), to ERa is regulation of gene expression in the cell nucleus. BHPI, but not the protein synthesis inhibitor cycloheximide (CHX), inhibits E2-ERa-mediated induction of pS2, GREB1 (GREB1: gene regulated by estrogen in breast cancer 1), and SDF-1 (SDF-1: stromal cell-derived factor 1) mRNAs in three ERa(+) cell lines. 15 [0349] The ability of BHPI to inhibit induction of three E2-ERa induced genes in breast and ovarian cancer cells was tested. The pS2 gene was selected because it is the most heavily studied and best characterized estrogen-regulated gene in human cells. GREB-1 was selected because it has been shown to be an estrogen regulated early response gene. SDF-1 was selected because of its emerging role in 20 cancer metastases. [0350] BHPI is an ERa-dependent inhibitor of protein synthesis. The ability of BHPI to inhibit E2-ERa induction of endogenous gene expression independent of its ability to inhibit protein synthesis in cells that contain ERa was tested. Cycloheximide inhibition of protein synthesis was used as a control in these experiments. If E2-ERa 25 induction of an mRNA was not inhibited by cycloheximide, then inhibition resulting from BHPI is due to its ability to inhibit E2-ERa mediated gene expression, not its ability to inhibit protein synthesis in ERa containing cells. Cycloheximide did not inhibit E2-ERa induction of pS2, SDF-1, or GREB1 mRNAs (Figure 7). It also did not inhibit E2-ERa down-regulation of IL1-R1 mRNA (IL1 R1 or IL1-R1: interleukin 1 30 receptor type 1) (Figure 8). These results show the ability of BHPI to inhibit E2-ERa regulated gene expression independent of its effects on protein synthesis. [0351] More specifically, qRT-PCR (qRT-PCR: quantitative reverse transcriptase polymerase chain reaction) analysis of the effect of BHPI on E2-ERa mediated induction of mRNAs in several cell lines was performed. The three ERa 51 WO 2014/113820 PCT/US2014/012405 positive human breast and ovarian cancer cell lines were maintained in cd stripped serum for 4 days to deplete endogenous estrogens and then plated. Cells were harvested in 10% cd-CS, and plated into 6-well plates at a density of 450,000 cells per well. On day 5, the medium was replaced with fresh 10% cd-CS. On day 6, the 5 cells were treated with either an ethanol vehicle, 10 nM E2, 10 nM E2 + 10 pM cycloheximide (CHX), or 10 nM E2 + 1 pM BHPl. Cells were lysed and RNA was extracted at the appropriate time point (2 hours for MCF-7 and T47D breast cancer cells and 4 hours for BG-1 ovarian cancer cells). Cell lysis, cDNA synthesis, and quantitative RT-PCR (RT-PCR: reverse transcriptase-polymerase chain reaction) 10 were carried out. The level of each mRNA in the presence of ethanol vehicle without E2 was set equal to 1. The data represent the average of 3 independent sets of cells, each assayed in triplicate. Data are reported as mean + S.E.M. [0352] Example 5b - BHPI inhibits E2-ERa-down-requlation of IL1 -R1 mRNA 15 in ERa positive T47D breast cancer cells [0353] BHPI, but not cycloheximide, inhibits E2-ERa-down-regulation of ILl R1 mRNA in ERa positive T47D breast cancer cells. ERa positive T47D human breast cancer cells were maintained in 10% cd-FBS for 4 days to deplete the medium of endogenous estrogens. Cells were harvested in 10% cd-CS, and plated 20 into 6-well plates at a density of 400,000 cells per well. The following day, the medium was replaced with fresh 10% cd-CS. The next day wells were treated with either an ethanol vehicle, 10 nM E2, 10 nM E2 + 10 pM CHX, or 10 nM E2 + 1 pM BHPl. Cells were lysed and RNA was extracted at the indicated time points. Cell lysis, cDNA synthesis, and quantitative RT-PCR were carried out. The level of ILl 25 R1 mRNA in the presence of ethanol vehicle without E2 was set equal to 1. Data represent the average of 3 independent sets of cells, each assayed in triplicate. Data are reported as mean + S.E.M. Figure 8 shows the results of the qRT-PCR analysis of the effect of BHPI on E2-ERa-mediated down-regulation of an mRNA. [0354] BHPI inhibited E2-ERa induction of pS2, GREB1 and CXCL2 mRNAs 30 in ERa+ MCF-7, T47D and BG-1 cells (Figure 7) and blocked E2-ERa down regulation of ILl-R1 mRNA (Figure 8). The ability of BHPI to inhibit E2-ERa induction and repression of gene expression indicates that BHPI acts at the level of ERa and not by a general inhibition or activation of transcription. 52 WO 2014/113820 PCT/US2014/012405 [0355] Example 5c - BHPI does not inhibit E2-ERa-requlated gene expression by reducing ERa protein levels or by excluding E2-ERa from the nucleus of the cell [0356] BHPI does not inhibit E2-ERa-regulated gene expression by reducing ERa protein levels or by excluding E2-ERa from the nucleus of the cell. Figure 9 is a 5 Western blot analysis of the effect of BHPI on ERa levels (Figure 9A) and subcellular localization (Figure 9B). Figure 9A shows the effects of BHPI treatment on ERa protein levels. MCF-7 cells were maintained in 5% cd-FBS serum + MEM for 4 days prior to cell plating in order to deplete cells of estrogen. On day 5, cells were harvested in 10% cd-CALF + MEM and plated in 6-well plates at a density of 10 250,000 cells per well. The medium was replaced on day 6. The following day, cells were treated with 10 nM E2, with or without 1 pM BHPI and cell lysates were collected at the indicated time for Western blot analysis. Immunoblotting was performed with an antibody that detects ERa, and the membrane was stripped and reprobed with antibody that detects P-Actin. Figure 9B shows that BHPI does not 15 alter the nuclear localization of ERa. MCF-7 cells were maintained in 5% cd-FBS + MEM for 4 days prior to cell plating in order to deplete cells of estrogen. On day 5, cells were harvested in 10% cd-calf serum + MEM and plated in 100 mm dishes at a density of 2.5 milllion cells per dish. The medium was replaced on day 6. On day 7, cells were pre-treated for 30 minutes with a 0.1% DMSO-vehicle control (-E2 and 20 +E2 samples) or 1 pM BHPI (+E2, BHPI and -E2, BHPI samples), followed by treatment for 2 hours with either a 0.1% ethanol-vehicle control (-E2 and -E2, BHPI samples) or 10 nM 17p-Estradiol (+E2 and +E2, BHPI samples). Cell lysates were collected, and nuclear and cytoplasmic fractions of each lysate were separated using a NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoScientific). Western blot 25 analysis of the cytopasmic (C) and nuclear (N) fractions pertaining to each treatment condition were carried out using an antibody to detect ERa protein levels. The membrane was stripped and reprobed with antibody to detect a-Tubulin (control marker of cytoplasmic fraction) and Lamin A/C (control marker of nuclear fraction), to verify the purity of nuclear and cytoplasmic fractions. 30 [0357] As shown in Figure 9A, at early times after treatment with 1 pM BHPI, ERa protein levels are nearly unchanged. As shown in Figure 9B, treatment of MCF 7 cells with 1 pM BHPI does not inhibit nuclear localization of ERa. In summary, in studies of BHPI's mechanism of action, BHPI did not alter ERa protein levels or nuclear localization. 53 WO 2014/113820 PCT/US2014/012405 [0358] Example 5d - Chromatin immunolprecilpitation (ChIP) shows that BHPI inhibits binding of E2-ERa to regulatory regions of responsive genes [0359] Chromatin immunoprecipitation showed that BHPI strongly inhibited 5 E2-stimulated recruitment of ERa and RNA polymerase |I to the pS2 and GREB1 promoter regions. [0360] The effects of BHPI on E2-ERa induction of pS2 mRNA and GREB-1 mRNA in MCF-7 cells is shown in Figure 10. Cells were maintained in 5% cd-FBS for 4 days to deplete the medium of endogenous estrogens. Cells were harvested in 10 10% cd-CS, and plated into 6-well plates at a density of 450,000 cells per well. The following day, the medium was replaced with fresh 10% cd-CS. The next day, wells were pre-treated for 30 minutes with either 0.1% DMSO-vehicle control, 1 pM BHPI, 10 pM cycloheximide (CHX), or 10 pg/mL tunicamycin (TUN). Tunicamycin is a well established indirect inhibitor of protein synthesis through activation of the UPR, and 15 cycloheximide is a well-established direct inhibitor of protein synthesis. Cells were then treated with or without 10 nM E2 for 2 hours. Data represent the average of 3 independent sets of cells, each assayed in triplicate. Data is reported as mean + S.E.M. Figure 1 OA shows qRT-PCR analysis of the effect of BHPI on E2-ERa mediated induction of pS2 mRNA. Figure 1 OB shows qRT-PCR analysis of the effect 20 of BHPI on E2-ERa-mediated induction of GREB-1 mRNAs. [0361] The effects of BHPI on ERa and RNA polymerase II (RNAP) recruitment to the promoters of the pS2 and GREB-1 genes are shown in Figure 10. Cells were maintained in 5% CD-FBS for 3 days to deplete the media of endogenous estrogens. Cells were pre-treated with a 0.1% DMSO-vehicle control or 1 pM BHPI 25 for 75 minutes, before treating cells with either 0.1% ethanol vehicle control or 10 nM E2 for 45 minutes. Cells were then treated with formaldehyde to cross-link DNA protein complexes. ChIP were performed. [0362] Figure 1 OC shows the ChIP study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to the estrogen regulated pS2 gene. Figure 1 OD 30 shows the ChIP study of the effect of BHPI on recruitment of E2-ERa and RNA polymerase to estrogen regulated GREB-1 genes. ERa is shown with black bars; RNA polymerase II is shown with hatched bars. Data represents the average of 3 independent sets of cells, each assayed in triplicate. Date reported as mean + S.E.M. 54 WO 2014/113820 PCT/US2014/012405 [0363] Example 5e - BHPI inhibits binding of E2-ERa to qene regulatory regions and ovexpression of ERa abolishes BHPI inhibition of E2-ERa mediated gene expression 5 [0364] BHPI inhibits binding of E2-ERa to gene regulatory regions and ovexpression of ERa abolishes BHPI inhibition of E2-ERa mediated gene expression (Figure 11). This shows that BHPI reduces recruitment of E2-ERa to regulatory elements by reducing the affinity of E2-ERa for these DNA regions. [0365] MCF7ERaHA cells, which are MCF-7 cells stably transfected to 10 express a Doxycycline (Dox)-inducible ERa, were estrogen-deprived in CD-FBS for 4-days prior to harvesting cells in 10% CD-calf serum. To induce ERa, the MCF 7ERaHA cells were treated with 0.25 pg/mL doxycycline (DOX) for 24 hours. Cells were then treated with either 0.1% DMSO (-E2; +E2) or 1 pM BHPI (+E2, BHPI) for 30 minutes, followed by treatment with either 0.1% ethanol (-E2) or 10 nM E2 (+E2; 15 +E2, BHPI) for 4 hours. RNA was extracted and mRNA levels of the GREB1 mRNA were determined by qRT-PCR, as shown in Figure 11. Data are mean ± SEM (S.E.M: standard error of the mean) (n = 3 sets of cells). * P < 0.05, ** P < 0.01, compared with +E2 samples. n.s. indicates not significant. [0366] In studies of BHPI's mechanism of action, BHPI did not alter ERa 20 protein levels or nuclear localization (Figure 9). Chromatin immunoprecipitation (ChIP) showed that BHPI strongly inhibited E2-stimulated recruitment of ERa and RNA polymerase |I to the pS2 and GREB1 promoter regions (Figure 10). If BHPI induces an ERa conformation exhibiting reduced affinity for gene regulatory regions, expressing high concentrations of ERa might provide sufficient ERa to still bind to 25 regulatory regions, preventing inhibition by BHPI. Ten-fold overexpression of ERa in MCF7ERaHA cells abolished BHPI inhibition of induction of GREB1 mRNA (Figure 11). 55 WO 2014/113820 PCT/US2014/012405 [0367] Example 6 - BHPI inhibits proliferation of ERa containing cancer cells [0368] Example 6a - BHPI inhibits proliferation of ERa containing breast, ovarian, endometrial, and prostate cancer cells 5 [0369] E2-ERa stimulates proliferation of most breast cancers and many ovarian, endometrial cervical, uterine, and liver cancers and likely several other types of cancer. BHPI inhibits proliferation of ERa-containing cancer cells. [0370] As shown in Figure 12A, BHPI selectively inhibits growth of ERa positive breast cancer cells. BHPI fully blocks proliferation of ERa positive MCF-7 10 breast cancer cells at 100 nM (Figure 12A-1), but has no effect on ERa negative MDA MB-231 breast cancer cells at 10,000 nM (Figure 12A-2). As shown in Figure 12B, BHPI selectively inhibits growth of ERa positive ovarian cancer cells. BHPI fully blocks proliferation of ERa positive BG-1 ovarian cells at 100 nM (Figure 12B-1), but has no effect on ERa negative ES2 ovarian cancer cells at 10,000 nM (Figure 12B 15 2). As shown in Figure 12C, BHPI selectively inhibits growth of ERa positive endometrial cancer cells. BHPI fully blocks proliferation of ERa positive ECC-1 endometrial cancer cells at 100 nM (Figure 12C-1), but has no effect on ERa negative HeLa cervical cancer cells at 10,000 nM (Figure 12C-2). As shown in Figure 12D, BHPI selectively inhibits growth of ERa positive prostate cancer cells. 20 BHPI blocks proliferation of ERa positive PC-3 prostate cells at 100 nM (Figure 12D 1), but has no effect on ERa negative DU1 45 prostate cancer cells at 10,000 nM (Figure 12D-2). [0371] For the experiments shown in Figure 12, all ERa positive cancer cell lines were maintained for 4 days in charcoal dextran treated serum and plated at a 25 density of 1,000 cell per well. The following day, cells were treated with or without 17p-estradiol (E2) and/or BHPI. ERa-negative cells were maintained in full serum, and plated at a density of 1,000 cells per well. The following day, cells were treated with or without E2 and/or BHPI. In all assays, cells were treated for 4-days with the indicated treatment solution. After 4 days, cells were assayed with MTS (MTS: (3 30 (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium)) reagent. Cell number was determined using a standard curve of cell number versus absorbance based on plating a known number of cells from each cell line and assaying using MTS as described in Kretzer, N.M., et al., A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer 56 WO 2014/113820 PCT/US2014/012405 cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem. 2010; 285(53):41863-73. Each data point is the average of at least 6 independent samples of cells, and is reported as mean ± SEM. 5 [0372] Example 6b - BHPI inhibits proliferation in diverse ERa positive cancer cell lines and has no effect on cell growth in ERa negative cell lines [0373] Dose-response studies of the effect of BHPI on the proliferation of a variety of ERa-positive and ERa-negative cancer cells were performed. The cell types studied included ERa-positive and ERa-negative human breast cancer cells; 10 ERa-positive and ERa-negative human ovarian cancer cells; ERa-positive human endometrial cancer cells; ERa-negative human cervical cancer cells; ERa-positive and ERa-negative human prostate cancer cells; ERa-negative DU145 human prostate cancer cells; ERa-negative human lung cancer cells; and ERa-negative human hepatoma (liver) cancer cells. 15 [0374] Figure 13 shows the results of MTS assays analyzing the effects of different concentrations of BHPI on proliferation of ERa-positive and ERa-negative cancer cells. Cell proliferation was evaluated as described for Figure 12 in Example 6a. The effects of BHPI on cell proliferation in 14 ERa positive (black bars) and 12 ERa negative (grey bars) cell lines is shown in Figure 13. Cells lines are grouped by 20 tissue of origin (breast, ovary, cervix, prostate, lung and liver). The dot "-" on each graph denotes the number of cells at the start of the experiment. Most cell proliferation studies were for 3 or 4 days in 10 nM E2. Data are the mean ± SEM (n = 6). [0375] The cell lines studied included ERa-positive MCF-7 human breast 25 cancer cells (Figure 13A); ERa-positive T47D human breast cancer cells (Figure 13B); ERa-positive kBluc-T47D human breast cancer cells (Figure 13C); ERa positive HCC1500 human breast cancer cells (Figure 13D); ERa-positive ZR-75-1 human breast cancer cells (Figure 13E); ERa-positive BT-474 human breast cancer cells (Figure 13F); ERa-positive MCF1 OAERIN9 human breast cancer cells (Figure 30 13G); ERa-positive MCF7ERaHA human breast cancer cells (Figure 13H); ERa negative MDA-MB-231 human breast cancer cells (Figure 131); ERa-negative MCF 10A human breast cancer cells (Figure 13J); ERa-positive BG-1 human ovarian cancer cells (Figure 13K); ERa-positive OVCAR-3 human ovarian cancer cells (Figure 13L); ERa-positive CAOV-3 human ovarian cancer cells (Figure 13M); ERa 57 WO 2014/113820 PCT/US2014/012405 negative ES2 human ovarian cancer cells (Figure 13N); ERa-negative IGROVE-1 human ovarian cancer cells (Figure 130); ERa-positive ECC-1 human endometrial cancer cells (Figure 13P); ERa-positive Ishikawka human endometrial cancer cells (Figure 13Q); ERa-negative HeLa human cervical cancer cells (Figure 13R); ERa 5 positive PC-3 human prostate cancer cells (Figure 13S); ERa-negative DU145 human prostate cancer cells (Figure 13T); ERa-negative 201T human lung cancer cells (Figure 13U); ERa-negative 273T human lung cancer cells (Figure 13V); ERa negative H1793 human lung cancer cells (Figure 13W); ERa-negative A549 human lung cancer cells (Figure 13X); ERa-negative HepG2 human hepatoma (liver) cancer 10 cells (Figure 13Y); and ERa-negative nonmalignant MEF Mouse embryo fibroblasts (Figure 13Z). [0376] Since BHPI inhibits proliferation of 16 of 16 ERa positive cancer cell lines and even 10,000 nM BHPI has no effect on the proliferation of 12 of 12 ERa negative cell lines, the data support the conclusion that the antiproliferative effects of 15 BHPI require ERa. [0377] Example 6c - Effects of BHPI and structurally related compounds on growth of ERa positive human breast cancer cells [0378] BHPI is a structure-selective inhibitor of ERa. Inhibition of proliferation 20 is a structure-selective effect of BHPI acting through ERa and not a non-specific toxic effect. A number of compounds closely related to BHPI were studied with some being effective inhibitors of cell proliferation and others only very weak inhibitors. Study of these related compounds provides information about which chemical substitutions on this chemical scaffold result in compounds effective for the inhibition 25 of proliferation in ERa-containing cancer cells. Figure 14A shows the chemical scaffold of the BHPI-related compounds. Figure 14B is a table showing preferred substitutions at each site on the scaffold. [0379] Inhibition of cell proliferation in ERa positive T47D breast cancer cells by BHPI and structurally related compounds is shown in Figure 14. Cell proliferation 30 was evaluated using MTS as described in the discussion of Figure 12 in Example 6a. BHPI was the most effective of a family of structurally related small molecules. BHPI inhibits E2-ERa stimulated proliferation of T47D cells with an IC50 of 15 nM. Additional members of this structural family were identified as ERa inhibitors through high throughput screening, and additional structurally related compounds were 58 WO 2014/113820 PCT/US2014/012405 purchased. The ability of each of these structurally related compounds to inhibit E2 ERa stimulated proliferation of ERa containing T47D human breast cancer cells was evaluated and compared to BHPI. The compounds are shown in Figure 14 order of relative potency with BHPI shown in Figure 14C, and the next most potent inhibitor 5 shown in Figure 14D. Compound 8, shown in Figure 14L, is the negative control. [0380] Example 6d - BHPI is effective in EGF-resistance models [0381] Epidermal growth factor (EGF) stimulates the growth of many cancer cells. In ERa positive cancer cells, BHPI inhibits EGF-stimulated cell proliferation that 10 is resistant to current antiestrogens. The effects of BHPI and antiestrogens on EGF dependent and E2-dependent growth of T47D breast cancer cells is shown in Figure 15A. Cells were maintained in 10% cd-FBS for 4-days prior to experiment. Cells were plated at a density of 2,000 cells/well, the medium was changed the following day, and the appropriate treatments were added. Plates were incubated for 15 4 days prior to assaying using MTS with a standard curve for cell number. The effects of fulvestrant (ICI) (100 nM) and BHPI (100 nM) on EGF-stimulated (20 ng/ml) and E2-stimulated (100 pM) cell growth of ERa(+) BG-1 ovarian cancer cells is shown in Figure 15B. Cells were maintained in 5% cd-FBS for 4-days prior to experiment. Cells were plated at a density of 200 cells/well, treated the following day, 20 and allowed to grow for 6-days prior to assaying using MTS with a standard curve for cell number. BHPI was effective in epidermal growth factor (EGF) stimulated T47D breast cancer cells that are resistant to 4-OHT, IC11 82,780, and ralixofene (Ral). [0382] Example 6e - BHPI inhibits proliferation of ERa positive breast and 25 ovarian cancer cells resistant to current therapies [0383] Figure 16 shows the dose-response studies of the effect of BHPI on proliferation of ERa positive cancer cell lines resistant to current therapies. Results from tamoxifen- and ICI-resistant BT-474 human breast cancer cells are shown in Figure 16A. Results from tamoxifen- and ICI-resistant ZR-75-1 human breast cancer 30 cells are shown in Figure 16B. Results from cis-platin resistant Caov-3 human ovarian cancer cells are shown in Figure 16C. Results from multi-drug resistant OVCAR-3 human ovarian cancer cells are shown in Figure 16D. Cells were maintained in 10% CD-FBS for 4 days prior to experiments. Cells were plated at a density of 2,000 cells/well. The medium was changed the following day, and the cells 59 WO 2014/113820 PCT/US2014/012405 were treated with 10 nM E2 and/or BHPI, or with 10 nm E2 + ICI or OHT (hatched bars). Plates were incubated for 4 days, with a medium change on day 2, prior to assaying using MTS. Cell number was determined using MTS from a standard curve of absorbance versus cell number for each cell line. The dot ("-") denotes cell 5 number at day 0. The hatched bars denote traditional antiestrogens (4-OHT and ICI). Data represents the average of 6 independent sets of cells, and is reported as mean + S.E.M. [0384] The results shown in Figure 16 show that BHPI is effective in cancer cells that contain ERa and are resistant to conventional chemotherapy or to 10 antiestrogens such as fulvestrant/Faslodex/ICI 182,780. Targeted therapies for ovarian cancer are largely unavailable. NIH-OVCAR-3 (NIH OVCAR-3: National Institutes of Health ovarian carcinoma-3; OVCAR-3) cells are a widely-used model for resistance to chemotherapy agents. NIH-OVCAR-3 cells are resistant to therapeutically relevant concentrations of the DNA intercalator 15 adriamycin/doxorubicin; the nitrogen mustard, melphalan; the DNA cross-linker, cisplatin; the microtubule blocker, taxol; and other agents. NIH-OVCAR-3 cells contain ERa. BHPI effectively inhibited growth of the NIH-OVCAR-3 cells (Figure 16D). Exposure to BHPI for longer periods of time (about a week) results in cell death. In the 5-day experiment using NIH-OVCAR-3 cells, 100 nM BHPI induced 20 death of some of the cells and 1 pM BHPI induced death of more of the cells (Figure 16D). OVAR-3 cells are also resistant to 5 pM ICI (Figure 16D). 1 pM BHPI blocked proliferation of both CaOV-3 cells and OVCAR-3 cells (Figure 16C and Figure 16D). [0385] BHPI was also tested in breast cancer cells that contain ERa and are resistant to estrogen-based therapies. BT-474 are human breast cancer cells 25 containing amplified HER2 (the target for herceptin) and the ERa coregulator amplified in breast cancer (AIB1). BT-474 cells are fully resistant to tamoxifen and are resistant to fulvesterant/Faslodex/ICI 182,780 (ICI). At 100 nM, BHPI effectively inhibited growth of BT-474 cells (Figure 16A). ZR-75-1 cells are often considered partially resistant to antiestrogens. These cells showed no increase in proliferation in 30 the presence of E2. Consistent with the lack of effect of estrogen on cell proliferation, the ZR-75-1 cells were completely resistant to 4-hydroxytamoxifen (OHT; the active form of tamoxifen) and partially resistant to fulvestrant/ICl 82,780 (ICI). At 100 nM BHPI completely blocked proliferation of the ZR-75-1 cells. (Figure 16B). Thus BHPI 60 WO 2014/113820 PCT/US2014/012405 is effective in diverse ERa positive cancer cells - even in cells in which estrogen does not increase cell proliferation. [0386] Example 6f - BHPI inhibits anchorage-independent growth of ERa 5 positive cancer -7 cells in soft agar [0387] Anchorage independent growth is a hallmark of cancer cells. This is often tested by evaluating growth in soft agar. BHPI blocks anchorage-independent growth of ERa positive cancer cells. The ability of BHPI to inhibit colony formation of MCF-7 human breast cancer cells was tested. 10 [0388] 5000 MCF-7 cells were plated into top agar. Cells were treated with medium containing DMSO (vehicle) and either, 10 nM 17p-estradiol (E2) or ethanol (vehicle), or 1 pM BHPI and 10 nM E2. Medium was changed every 3 days. After 21 days, colonies were counted and photographed at 5x magnification. The bar graph represents the average of the total number colonies per well with a diameter 15 >0.5 mm. Assays were carried out in triplicate and are reported as mean ± S.D. E2 robustly stimulated colony formation in soft agar (Figure 17B), and BHPI completely blocked colony formation (Figure 17C). [0389] Example 6q - BHPI induces regression of breast cancer in a mouse 20 xenograft model [0390] The ability of BHPI to inhibit growth and/or induce tumor regression was evaluated in a mouse xenograft model of ERa positive human breast cancer. BHPI rapidly inhibited tumor growth (Figure 19) and induced regression in 48 of 52 tumors (Figure 18). BHPI was well tolerated by the mice. 25 [0391] All mouse xenograft experiments were approved by the Institutional Animal Care Committee of the University of Illinois at Urbana-Champaign (IACUC). The MCF-7 cell mouse xenograft model has been described in detail in Ju, Y.H., et al., Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen dependent human breast cancer (MCF-7) cells implanted in athymic mice. Cancer 30 research. 2002; 62(9):2474-7. [0392] At least 12 animals, usually with 4 tumors per animal, were required per experimental group to maintain significant statistical power to detect >25% difference in tumor growth rates. Briefly, estrogen pellets (1 mg: 19 mg estrogen: cholesterol) were implanted into 60 athymic female OVX mice which were 7 weeks 61 WO 2014/113820 PCT/US2014/012405 of age. Three days after E2 pellet implantation, 1 million MCF-7, human breast cancer cells per site in matrigel were subcutaneously injected at 2 sites in each flank for a total of 4 potential tumors per mouse. When the average tumor size reached 17.6 mm 2 (4.7 by 4.7 mm), E2 pellets were removed and a lower dose of E2 in 5 sealed silastic tubing (1:31 estrogen: cholesterol, 3 mg total weight) was implanted in the same site. When the average tumor size reached 23.5 mm 2 (5.5 by 5.5 mm), mice were divided into 4 groups with tumor size normalized: E2 group, no treatment control (NC) group, B_10 group and B_1/B_15 group. [0393] E2 silastic tubes in the NC group were removed, while E2 silastic tubes 10 in the E2, B_10, and B_1/B_15 groups were retained. The E2 and NC group received intraperitoneal injection every other day with 10 ml/kg vehicle (2% DMSO, 10% Tween-20, and 88% PBS). The B_10 group received 10 mg/kg BHPI by intraperitoneal injection every other day. The B_1 /B_1 5 group received 1 mg/kg BHPI by intraperitoneal injection every other day for 14 days. Since this extremely 15 low BHPI dose had no effect, (average tumor cross-sectional area -45 mm 2 ) they then received 15 mg/kg BHPI every day for another 10 days. [0394] Food intake and body weight were measured every 4 days, and food intake is presented as grams/day. Tumors were measured every 4 days with a caliper. Tumor cross sectional area was calculated as (a/2)*(b/2)*3.14, where a and 20 b were the measured diameters of each tumor. On termination of the experiments, mice were euthanized and the tumors were excised and weighed. Two of sixty mice were removed during the course of the study, one that failed to form tumors and the other due to unrelated illness. No tumors were excluded from analysis, and blinding was not performed. 25 [0395] Figure 18 shows the percent change in tumor size over the course of the experiment, (days 14-24) for each tumor. The control group, which was treated with E2 released from silastic implants and received injections of vehicle, but not BHPI, is represented by the white bars. The size change of the tumors in the experimental group treated with E2 from silastic implants and injected with 15 mg/kg 30 of BHPI daily is shown in black bars. For example, in the mouse whose tumor size data is shown at the far right (mouse: DS-063), two of its 4 tumors decreased in size by -50% and 2 tumors decreased in size by just over 30%. Tumor size at day 14 is set to 0% change. 62 WO 2014/113820 PCT/US2014/012405 [0396] Example 6h - BHPI inhibits growth of human breast cancers in a mouse xenograft model [0397] Human breast cancer MCF-7 tumor growth in athymic nude mice was monitored by measuring tumor diameter with a caliper every 4 days. As shown in 5 Figure 19, the E2 and NC group received vehicle injection (open circles), while the B_10 group received 10 mg/kg BHPI injection every other day (open triangles). The B_1/B_1 5 group received 1 mg/kg BHPI every other day for 14 days, and then received 15 mg/kg BHPI every day until the end of the study (* denotes change in dose, back line) (black triangles). The control group that received no estrogen is 10 shown by the black circles. Tumor size is represented as tumor cross sectional area (mm 2 ). Each tumor was analyzed individually as an independent tumor, and data were expressed as mean ± SEM (n=60) for all the tumors (Figure 19A). Mice were sacrificed and tumor weights were recorded. In Figure 19B, data is expressed as mean ± SEM (n=60) and analyzed using One way ANOVA (analysis of variance) 15 with post hoc Fisher's LSD (least significant difference) test. Different letters indicate significant different among the 4 groups (p < 0.05). Mouse body weight (Figure 19C) and food intake (Figure 19D) were measured every 4 days after drug injection started. Data are expressed as mean ± SEM (n=15 mice/group). BHPI treatment had no effect on body weight or food intake and was, therefore, not overtly toxic. 20 [0398] Using the mouse xenograft model, the tumors in the vehicle treated mice exhibited continued robust growth (Figure 18, white bars). In contrast, BHPI treatment (15 mg/kg daily) resulted in regression of 48 out of 52 tumors (Figure 18, black bars). Tumor regression was evident within a few days after initiation of BHPI treatment (Figure 19A). BHPI easily exceeded the goal of >60% tumor growth 25 inhibition in xenografts which has been proposed as more likely to lead to clinical response. In a related study, BHPI, at the very low dose of 10 mg/kg every other day for 3 weeks, slowed and ultimately stopped tumor growth and final tumor weight was reduced -60% compared to vehicle-treated controls (Figure 19B). BHPI was well tolerated with BHPI-treated and control mice exhibiting similar food intake (Figure 30 19C) and weight gain (Figure 19D). These data show that BHPI can induce rapid regression of large pre-existing breast tumors. 63 WO 2014/113820 PCT/US2014/012405 [0399] Example 7 - BHPI is an ERa-delpendent inhibitor of protein synthesis [0400] Example 7a - BHPI potently inhibits protein synthesis in ERa positive cancer cells 5 [0401] BHPI is an ERa-dependent inhibitor of protein synthesis. In this example, breast, ovarian, cervical, lung, prostate, and liver cancer cells tested. Cells were estrogen-deprived for 4 days in cd-FBS prior to experiments. The top panels of Figure 20 show Western blots for ERa in each cell line. Cells were plated at a density of 10,000 cells/well. The medium was replaced with the appropriate 10 treatment medium the following day, and cells were treated for 24 hours before adding 5 pCi/well of 35 S-methionine. Cell lysates were collected, centrifuged at 13,200 rpm for 10 minutes at 4 C, and sample supernatants were transferred to Whatman filter paper. Radiolabeled protein was isolated via TCA-precipitation of labeled protein in the filters. Free amino acids are not retained in the filters. 15 Incorporation of radioactive methionine into protein was then determined by liquid scintillation counting of the solubilized filters. In control experiments using the well characterized protein synthesis inhibitor cycloheximide (CHX), this method proved reliable in quantitating inhibition of protein synthesis. [0402] Figure 20 shows a comparison of ERa protein levels and the effects of 20 BHPI treatment on protein synthesis. The number of samples was too large to run on a single gel and the data is from 3 identically processed gels. Protein synthesis was determined by incorporation of 35 S-methionine into protein. Incorporation with no added BHPI was set to 100%. In general, protein synthesis in cells expressing moderate or high levels of ERa was robustly inhibited by 100 nM BHPI (hatched 25 bars), while 10,000 nM BHPI (striped bars), the highest concentration tested, had very little or no effect on protein synthesis in ERa negative cells. Cells expressing low levels of ERa, more typical of non-transformed ERa containing cells, such as PC-3 prostate cancer cells, were much less sensitive to BHPI inhibition of protein synthesis. As a control, cycloheximide (CHX) potently inhibited protein synthesis in 30 all the cell lines(checker board bars). For each cell line, incorporation of 35
S
methionine into protein with no added BHPI was set to 100%. Data is the mean S.E.M. for at least 3 sets of cells. 64 WO 2014/113820 PCT/US2014/012405 [0403] Example 7b - BHPI potently inhibits protein synthesis in ERa positive breast cancer cells but has no effect in ERa negative breast cancer cells [0404] BHPI potently inhibits protein synthesis in MCF1 OAERIN9 breast cells which contain ERa but has no effect in MCF10A cells which lack ERa. Figure 21A 5 shows the effect of BHPI on protein synthesis in ERa-positive MCF1 OAERIN9 breast cells and in the parental ERa-negative MCF-10A cells. Figure 21 B shows the effects of the current generation ER inhibitors TPSF, Fulvestrant/faslodex/ICI 182,780 and 4-hydroxytamoxifen on protein synthesis in MCF1 OAERIN9 cells and MCF1 OA cells. [0405] Cells were maintained in 2% DMEM/F12 including 10 pg/ml insulin, 0.1 10 pg/ml cholera toxin, 0.5 pg/ml hydrocortisone, and 20 ng/ml EGF. Cells were plated at a density of 10,000 cells/well in 1% CD-FBS + DMEM/F1 2 without supplements. Medium was replaced with the appropriate treatment medium and the indicated inhibitors the following day. Cycloheximide was at 10 pg/ml. Cells were treated for 24 hours before adding 3 pCi/well of 35 S-methionine radiolabel. Cell lysates were 15 collected, centrifuged at 13,200 rpm for 10 minutes at 4 OC, and sample supernatants were transferred to Whatman filter paper. The samples were exposed to TCA to denature the protein and trap it in the filter paper, and solubilized filters were subjected to liquid scintillation counting to quantitate incorporation of radioactive methionine into protein determined. Protein synthesis was determined by the amount 20 of 35 S-methionine incorporated into protein. Figure 21 B shows the effects of known ERa inhibitors on protein synthesis in ER(+) MCF1 OAERIN9 and ER(-) MCF1 OA mammary cells. Data is the mean ± S.E.M. for at least 3 sets of cells. [0406] Example 7c - Knockdown of ERa abolished BHPI's ability to inhibit 25 protein synthesis [0407] RNAi (RNA interference) knockdown of ERa abolishes BHPI inhibition of protein synthesis. Protein synthesis in MCF1 OAERIN9 cells treated with non-coding (NC) siRNA or ERa siRNA SmartPool followed by 100 nM BHPI is shown in Figure 22A. Protein synthesis in MCF1 OAERIN9 cells pre-treated with 1 pM ICI 30 182,780 for 24 hours to degrade ERa, followed by 100 nM BHPI is shown in Figure 22B. In each case there were 4 samples. Note that Figure 22C shows that ICI, a competitive inhibitor of estrogen, depleted the ERa protein. This complements the data showing that RNA interference knockdown of the mRNA leading to disappearance of the ERa protein abolishes inhibition of protein synthesis. 65 WO 2014/113820 PCT/US2014/012405 [0408] Example 7d - Overexpression of ERa increases BHPI inhibition of protein synthesis [0409] Figure 23B shows the dose-response study of the effect of increasing 5 levels of ERa on BHPI inhibition of the incorporation of 35 S-methionine into protein. Residual protein synthesis (with untreated cells set to 100%) after treatment with 1 pM BHPI in doxycycline-treated MCF7ERaHA cells expressing increasing levels of ERa is shown in Figure 23B (n = 6). Figure 23A is a Western blot analysis showing levels of ERa in cells overexpressing ERa for each sample. Data is mean ± S.E.M. 10 [0410] In summary, these examples show that BHPI is an ERa-dependent inhibitor of protein synthesis. Expression of ERa is necessary to make a cell that is not responsive to BHPI inhibition of protein synthesis sensitive to BHPI inhibition of protein synthesis (Figure 21). Current generation antiestrogens do not inhibit protein synthesis in these cells (Figure 21). Knockdown of the ERa abolishes sensitivity of 15 the cells to BHPI inhibition of protein synthesis (Figure 22). Overexpression of ERa increases BHPI inhibition of protein synthesis (Figure 23). [0411] BHPI nearly abolished protein synthesis in ERa positive cancer cells. If BHPI inhibits protein synthesis through ERa, it should only work in ERa positive cells, and its potency should be related to ERa levels. BHPI inhibited protein 20 synthesis in all 14 ERa positive cell lines and had no effect on protein synthesis in all 12 ERa negative cell lines. BHPI does not inhibit protein synthesis in ERa negative MCF-1 0A cells, but gains the ability to inhibit protein synthesis when ERa is stably expressed in isogenic MCF1 OAERIN9 cells. BHPI loses the ability inhibit protein synthesis when the ERa in the stably transfected cells is knocked down with siRNA 25 or degraded by ICI 182,780. Increasing the level of ERa in MCF7ERaHA cells stably transfected to express doxycycline-inducible ERa progressively increased BHPI inhibition of protein synthesis. Thus, BHPI is likely to be especially effective against the subset of highly lethal breast cancers that contain very high levels of ERa and are often refractory to tamoxifen therapy. BHPI does not work by activating the 30 estrogen binding protein GPR30. BHPI has no effect on cell proliferation or protein synthesis in HepG2 cells that contain functional GPR30, and activating GPR30 with a selective activator, G1, did not inhibit protein synthesis. Thus, ER is necessary and sufficient for BHPI inhibition of protein synthesis. 66 WO 2014/113820 PCT/US2014/012405 [0412] Example 8 - BHPI activates the endoplasmic reticulum stress sensor, the unfolded protein response (UPR) in ERa positive cancer cells [0413] Example 8a - UPR activators inhibit protein synthesis seen with BHPI 5 [0414] Known UPR activators, thapsigargin and ionomycin that activate the UPR by depleting endoplasmic reticulum calcium and increasing cytosol calcium exhibit the potent inhibition of protein synthesis seen with BHPl. [0415] Figure 24 shows the effect of UPR activators on protein synthesis. MCF-7 cells were treated for 2 hours with the indicated UPR activators and 10 incorporation of 35 S-methionine into protein measured. Vehicle control was set to 100% (n = 6). [0416] Known UPR activators tunicamycin and homocysteine, which cause accumulation of unfolded protein, brefeldin, which alters protein transport and DTT, which alters redox potential, all induce modest depletion of EnR Ca 2 *, while 15 thapsigargin and ionomycin activate the UPR by inducing a massive decline in EnR Ca 2 * Thapsigargin and ionomycin elicited the rapid and profound decline in protein synthesis similar to what is seen with BHPI as seen in Figure 24. [0417] Example 8b - BHPI depletes endolplasmic reticulum calcium in ERa 20 positive breast cancer cells and activates all three arms of the UPR [0418] The calcium sensitive dye Fluo-4 AM was used to monitor intracellular calcium in order to test whether BHPI alters intracellular Ca 2 *. Treating ERa positive MCF-7 and BG-1 cells with 1 pM BHPI produced a large and sustained increase in intracellular calcium in the presence of extracellular Ca 2 ' and a transient increase in 25 intracellular calcium in the absence of extracellular calcium. [0419] As shown in Figure 25, BHPI rapidly activates the UPR by depleting endoplasmic reticulum Ca 2 ' and increasing cytosol Ca 2 *. To more quantitatively evaluate time-dependent changes in intracellular Ca 2 *, MCF-7 cells were treated with BHPI in the absence or presence of extracellular calcium, and averaged data for 30 each of 10 cells at each time point was taken. Figure 25 shows the effect of BHPI and the UPR activator thapsigargin on intracellular calcium measured using the calcium sensing dye Fluo-4. Figure 25A is a photomicrograph of the effect of a low concentration (1 pM) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium. Figure 25B-1 is a photomicrograph of 67 WO 2014/113820 PCT/US2014/012405 the effect of a high concentration (10 pM) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without extracellular calcium. Figure 25B-2 is a graphical representation of the effect of a high concentration (10 pM) of BHPI on intracellular calcium in MCF-7 cells in the presence of BHPI with and without 5 extracellular calcium. Figure 25C-1 is a photomicrograph of the effect of the UPR activator thapsigargin (2 pM) on intracellular calcium in MCF-7 cells. Figure 25C-2 is a graphical representation of the effect of the UPR activator thapsigargin on intracellular calcium in MCF-7 cells. 10 [0420] Example 8c - BHPI depletes endoplasmic reticulum calcium, in ERa positive cells but not in ERa negative cells [0421] BHPI increases intracellular calcium levels in ERa positive BG-1 ovarian cancer cells but not in ERa negative HeLa endometrial cells. Figure 26 shows the effect of BHPI on intracellular calcium levels in ERa positive BG-1 ovarian 15 cells (Figure 26A) and ERa negative HeLa cervical cells (Figure 26B). Figure 26A is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in ERa positive BG-1 cells with and without extracellular calcium. Figure 26B is a photomicrograph of the effect of a high concentration of BHPI or thapsigargin (THG) on intracellular calcium in ERa negative 20 HeLa cells without extracellular calcium. Cells are visualized with the Ca 2 , sensitive dye Fluo-4. The highest levels of [Ca 2 *] are seen as the most intensely white. The trace in Figure 26C represents calcium following treatment with thapsigargin or BHPI in ERa negative HeLa cells. Fluorescence intensity was normalized to the basal Fluo-4 signal, which was set to 1. Data is mean ± S.E. 25 [0422] Example 8d - BHPI acts by opening the endoplasmic reticulum IP3R channel [0423] BHPI acts by opening the endoplasmic reticulum IP3R (IP3R: inositol 3-phosphate receptor) channel. Locking the channel closed with the inhibitor 2-APB 30 prevents release of calcium into the cytosol. Inhibiting opening of the endoplasmic reticulum IP3R Ca 2 * channel abolished BHPI release of intracellular calcium and inhibition of protein synthesis. Figure 27A shows the effects of inhibitors of calcium channel opening on intracellular calcium levels after BHPI treatment. 2-APB, which Inhibits opening of the endoplasmic reticulum IP3R Ca 2 * channel, abolished BHPI 68 WO 2014/113820 PCT/US2014/012405 release of intracellular calcium. The ryanodine and IP3R Ca 2 * channels were pre blocked with 100 pM ryanodine (RyR) and 100 pM 2-aminoethyl diphenylborinate (2 APB), respectively, followed by 70 nM BHPI for 3 hours (n = 4). The highest calcium concentrations are shown in the brightest white. Figure 27B shows the effect of 5 inhibitors of calcium channel opening on protein synthesis measured by incorporation of 35 S-methionine into protein. Inhibiting opening of the endoplasmic reticulum IP3R Ca 2 * channel abolishes BHPI inhibition of protein synthesis. The ryanodine and IP3R Ca 2 * channels were pre-blocked with 100 pM ryanodine (RyR) and 100 pM 2-aminoethyl diphenylborinate (2-APB), respectively, followed by 70 nM 10 BHPI for 3 hours. Data are mean ± S.E.M. (n = 4; ***p < 0.001 vs. BHPI alone; n.s., not significant. [0424] Inhibitor studies show BHPI-ERa acts by opening the IP3R Ca 2 * channels in the EnR. With or without extracellular Ca 2 *, locking the IP3R Ca 2 * channels closed with 2-APB prevented the observed increase in intracellular calcium 15 following BHPI treatment (Figure 27A) and abolished BHPI inhibition of protein synthesis; locking the EnR ryanodine channels with ryanodine (RyR) has a modest effect, (Figure 27B). [0425] In summary, known UPR activators tunicamycin and homocysteine induce modest depletion of EnR Ca 2 *, while thapsigargin and ionomycin activate the 20 UPR by inducing a massive decline in EnR Ca 2 *. Thapsigargin and ionomycin elicited a rapid and profound decline in protein synthesis similar to what is seen with BHPI (Figure 24). Treating ERa positive MCF-7 and BG-1 cells with 1 pM BHPI produced a large and sustained increase in intracellular calcium in the presence of extracellular Ca 2 *, and a transient increase in intracellular calcium in the absence of 25 extracellular calcium (Figure 25). To more quantitatively evaluate time-dependent changes in intracellular Ca 2 *, MCF-7 cells were treated with BHPI in the absence or presence of extracellular calcium, and averaged data for each of 10 cells at each time point (Figure 25) When extracellular Ca 2 * is absent, BHPI and thapsigargin (THG) induced similar large increases in fluorescence intensity (Figure 25). Since 30 BHPI elicits a large increase in cytosol Ca 2 * when there is no extracellular Ca 2 *, BHPI is acting by depleting the Ca 2 * store in the endoplasmic reticulum. BHPI had no effect on intracellular Ca 2 * in ERa negative HeLa cells, which remained sensitive to thapsigargin (Figure 26). Inhibitor studies show BHPI-ERa acts by opening the IP3R Ca 2 * channels in the EnR. With or without extracellular Ca 2 *, locking the IP3R 69 WO 2014/113820 PCT/US2014/012405 Ca 2 channels closed with 2-APB prevented the observed increase in intracellular calcium following BHPI treatment (Figure 27A) and abolished BHPI inhibition of protein synthesis; locking the EnR ryanodine channels with ryanodine (RyR) has a modest effect, (Figure 27B). 5 [0426] Example 9 - Model for the Activation of the UPR [0427] Figure 28 presents a model of the activation of the unfolded protein response (UPR). Endoplasmic reticulum (EnR) stress activates the three arms of the UPR. In ERa positive cancer cells, BHPI activates all 3 arms of the UPR. By 10 activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis. Supporting this view, RNAi knockdown of PERK reduces BHPI-stimulated phosphorylation of elF2a (Figure 29). Also the time course of BHPI inhibition of protein synthesis (Figure 30A) is well correlated with the increase in phosphorylation of elF2a (Figure 30B). BHPI does not induce phosphorylation of 15 elF2a in ERa negative cells (Figure 30C). In ERa positive cancer cells additional proof BHPI activates the UPR is shown by the induction of the downstream factors CHOP (CHOP: C/EBP homology protein) and GADD34 (GADD34: Growth arrest and DNA damage-inducible protein 34) (see model in Figure 28B and Figure 28C, and data in Figure 30D and Figure 30E). 20 [0428] Example 9a - Endoplasmic reticulum (EnR) stress activates the three arms of the UPR [0429] As shown in Figure 28A, EnR stress induces the oligomerization and phospho-activation of the transmembrane kinase PERK. P-PERK phosphorylates 25 eukaryotic initiation factor 2a (elF2a), leading to inhibition of protein synthesis and a reduction in the endoplasmic reticulum protein folding load. Reduced protein synthesis increases levels of the transcription factor, ATF4 (ATF4: activating transcription factor 4), which induces the transcription factor CHOP, which induces GADD34 and several pro-apoptotic genes. As shown in Figure 28B, EnR stress 30 induces the oligomerization and phospho-activation of the transmembrane protein, IRE1a. Activated IRE1a removes an intron from full-length XBP1 (fl-XBP1: full length X-box binding protein 1) mRNA, producing spliced (sp)-XBP1 mRNA, which is subsequently translated into sp-XBP1 protein (sp-XBP1: spliced X-box binding protein 1). sp-XBP1 increases the protein-folding capacity of the EnR and turnover of 70 WO 2014/113820 PCT/US2014/012405 misfolded proteins by inducing EnR resident-chaperone protein genes (BiP, HEDJ, SERP1) (SERP1: stress-associated endoplasmic reticulum protein 1) (HEDJ: heat shock protein 40 co-chaperone domain J), EnR-associated degradation (ERAD) genes and alters mRNA decay and translation. As shown in Figure 28C, EnR stress 5 promotes the translocation of the transmembrane protein, ATF6a, from the EnR to the Golgi Apparatus, where it encounters proteases that liberate the N-terminal fragment of ATF6a (sp-ATF6a: spliced activating transcription factor 6a). sp-ATF6 increases the protein-folding capacity of the EnR by inducing EnR-resident chaperones, including BiP and GRP94 (GRP94: glucose regulated protein 94 kilo 10 Daltons; also known as HSP90B1). [0430] BHPI inhibits protein synthesis by activating the unfolded protein response (UPR). E2-ERa elicits transient anticipatory activation of the endoplasmic reticulum stress sensor, the UPR. The possibility that BHPI elicits sustained near quantitative inhibition of protein synthesis by distorting the normal ability of E2-ERa 15 to induce transient activation of the UPR was tested. Moderate and transient activation of the UPR is usually protective, while extensive and sustained UPR activation induces cell death. In response to cell stress, the UPR is activated by multiple mechanisms, including release of Ca 2 , from the lumen of the EnR into the cytosol. This activates the transmembrane kinase PERK by autophosphorylation. P 20 PERK phosphorylates eukaryotic initiation factor 2a (elF2a), inhibiting translation of most mRNAs (Figure 28A). The other arms of the UPR initiate with activation of the transcription factor ATF6 (Figure 28C), leading to increased protein folding capacity and activation of the splicing factor IRE1 a, which alternatively splices the transcription factor XBP1, resulting in production of active spliced (sp)-XBP1 and 25 increased protein folding capacity (Figure 28B). [0431] Example 9b - BHPI induces RAPID phosphorylation and activation of PERK, and PERK knockdown prevents BHPI from rapidly inhibiting Protein synthesis [0432] In Figure 29, Figure 29A is a Western blot analysis showing the effect 30 of BHPI on protein phosphorylation and levels of PERK and elF2a. Figure 29B-1 is a Western blot analysis showing the effect of RNAi knockdown of PERK on phosphorylation and level of elF2a. Figure 29B-2 shows the of RNAi knockdown of PERK on protein synthesis measured by incorporation of 35 S-methionine into protein. Figure 29C shows the qRT-PCR results of the effect of RNAi knockdown on PERK 71 WO 2014/113820 PCT/US2014/012405 mRNA levels. Figure 29D is a Western blot analysis showing the effect of RNAi knockdown on PERK on PERK protein level. [0433] Shown in Figure 29A, BHPI induces phosphorylation of PERK 30 minutes following BHPI treatment. Western blot analysis using ERa positive MCF-7 5 breast cancer cells was carried out. Blots were probed using an antibody that only detects phosphorylated and activated PERK, and antibodies for total protein levels of PERK, and p-actin. As shown in Figure 29B, siRNA knockdown of PERK reduces the ability of BHPI to inhibit protein synthesis. ERa positive MCF-7 cancer cells were maintained for 4 days in 5% cd-FBS + MEM. Cells were harvested in 10% cd-calf 10 serum + MEM without antibiotics, and plated in 96-well plates at a density of 7,500 cells/well. On day 5, cell medium was replaced with antibiotic-free medium containing 0.2 pg/pl liposome, and either 50 nM non-coding SMARTPOOL control siRNA or 50 nM PERK SMARTPOOL siRNA. Cells were transfected for 16 hours, and the transfection medium was replaced with recovery medium for 6 hours 15 (antibiotic-free 10% cd-calf serum + MEM), before replacing the medium with medium containing antibiotics (1% penicillin/streptomycin). Medium was changed on day 7, and cells were incubated until day 9 to knockdown PERK protein (96 hours from the start of transfection). Cells were treated with 1 pM BHPI for the indicated times, and treated with 3 pCi/well of 35 S-methionine 30 minutes prior to collecting cell 20 lysates. Protein synthesis rates were determined. Data is represented as mean ± S.E.M. Effects of PERK knockdown on PERK mRNA levels are shown in Figure 29C, and effects of PERK knockdown on PERK protein levels is shown in Figure 29D. Following the initiation of the knockdown, mRNA samples and protein lysates were collected every 24-hours. qPCR was performed, and Western blot 25 analysis was carried out using antibodies against PERK and p-actin, which served as a protein loading control. [0434] Example 9c - In ERa positive cell lines, the time course of BHPI inhibition of protein synthesis parallels the time course of increased phosrphorylation 30 of elF2a, and BHPI is active in the presence and absence of estrogen [0435] Figure 30A shows the incorporation of 35 S-methionine into protein as a function of time after addition of BHPI. In ERa positive cell lines, the time course of BHPI inhibition of protein synthesis parallels the time course of increased phosphorylation of elF2a, and BHPI is active in the presence and absence of 72 WO 2014/113820 PCT/US2014/012405 estrogen. Figure 30A shows the time course of BHPI inhibition of protein synthesis. ERa positive MCF-7, T47D, and BG-1 cells were incubated for the indicated times in 1 pM BHPI. Incorporation of 35 S-methionine into protein at time = 0 was set to 100%. Data is mean ± SEM (n = 4 sets of cells). At 30 minutes in BHPI, 3 5 S-methionine 5 incorporation into protein was reduced by approximately 50%. [0436] Figure 30B shows the Western blots of the effect of BHPI on phosphorylation and level of elF2a in different cell types as a function of time after addition of BHPI. In ERa positive cells, BHPI increases P-elF2a at 30 minutes. In the presence [+E2], or absence [-E2], BHPI increases P-elF2a in ERa positive MCF-7 10 cells (Figure 30B-1), BG-1 cells (Figure 30B-2), and T47D cells (Figure 30B-3). In the absence of E2, BHPI increases elF2a phosphorylation in ERa positive MCF-7 cells (Figure 30B-4). [0437] Figure 30C contains Western blots showing the effect of BHPI on phosphorylation and level of elF2a in ERa negative cancer cells. BHPI does not 15 increase P-elF2a in ERa negative MDA MB-231 cells (Figure 30C). Since the UPR activator tunicamycin (TUN) increased P-elF2a in these cells, the absence of BHPI induced phosphorylation of elF2a in the MDA MB-231 cells was not due to the inability of UPR activation to induce elF2a phosphorylation. Phospho-elF2a was visualized by Western blotting using a phosphospecific antibody, which detected 20 phosphorylation at Ser-51. Immunoblotting used antibodies for phospho-elF2a, elF2a and p-actin as an internal standard. [0438] Figure 30D and Figure 30E show the results of qRT-PCR of mRNA levels of UPR-related mRNAs in ERa-positive cancer cells treated with BHPI. The induction of CHOP and GADD34 mRNAs in MCF-7 cells (Figure 30D) and CHOP 25 mRNA in BG-1 cells (Figure 30E) following treatment with 1 pM BHPI are shown. mRNA levels were determined by qRT-PCR with t = 0 hours set to 1. [0439] Example 9d - BHPI induces activation of the IRE1a-branch of the UPR in MCF-7 cells and blocks E2-ERa induction of XBP1 mRNA 30 [0440] UPR activation results in translocation of ATF6a from the endoplasmic reticulum to the Golgi where ATF6a protein is cleaved to yield active sp-ATF6a. The sp-ATF6a then moves to the nucleus where it is a transcription factor that helps increase transcription of genes that encode chaperones that help fold proteins. 73 WO 2014/113820 PCT/US2014/012405 [0441] BHPI induces activation of the IREla-branch of the UPR in MCF-7 cells, and blocks E2-ERa induction of XBP1 mRNA. 10 nM E2 induces XBP1 mRNA which is blocked by treatment with BHPI. ERa positive MCF-7 human breast cancer cell lines were maintained in 5% cd-FBS + MEM for 4 days to deplete cells of 5 endogenous estrogens. Cells were harvested in 10% cd-CS and plated into 6-well plates at a density of 450,000 cells per well. On day 5, the medium was replaced with fresh 10% cd-CS. On day 6, the cells were pre-treated with either 0.1% DMSO vehicle control (-E2, +E2 samples) or 1 pM BHPI for 30 minutes prior to treating cells with either 10 nM E2 (+E2 and +E2, BHPI samples) or a 0.1% ethanol-vehicle 10 control (-E2 and -E2, BHPI samples). qRT-PCR analysis was carried out using primers designed to detect total XBP1 mRNA levels. Figure 31A shows the results of qRT-PCR of unspliced XBP-1 mRNA in MCF-7 ERa-positive cancer cells treated with BHPI and no estrogen. Figure 31 B shows the results of qRT-PCR of spliced XBP-1 mRNA in MCF-7 ERa positive cancer cells treated with BHPI and no 15 estrogen. Figure 31C shows the results of qRT-PCR of unspliced XBP-1 mRNA in MCF-7 ERa-positive cancer cells treated with BHPI with and without estrogen. Figure 31 D shows the results of qRT-PCR of spliced XBP-1 mRNA in MCF-7 ERa positive cancer cells treated with BHPI with and without estrogen. [0442] 10 nM E2 or 1 pM BHPI can activate the IRE1a-branch of the UPR as 20 indicated by increased levels of spliced-XBP1 mRNA (sp-XBP1) (Figure 31 B and Figure 31 D), but co-treatment of cells with both 10 nM E2 and 1 pM BHPI blocks IRE1a activation (Figure 31 D). qRT-PCR analysis was carried out using primers designed to only detect XBP1 mRNA lacking the suppressor intron, which is removed by activated IRE1a. UPR activation results in the activation of the protein 25 sensor, IRE1a. IRE1a is an endoribonuclease, which upon activation, removes a suppressor intron (piece of RNA sequence) from XBP1 mRNA. The spliced form of XBP1 mRNA is translated into XBP1 protein. Removal of the suppressor intron produces a more potent XBP1 protein capable of initiating the gene transcription program of the UPR. Analysis of spliced XBP-1 (sp-XBP1) serves as a downstream 30 readout of IRE1a activation, and thus activation of this branch of the UPR. [0443] In summary, in ERa positive cancer cells, BHPI activates all 3 arms of the UPR. By activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis. RNAi knockdown of PERK reduces BHPI stimulated phosphorylation of elF2a (Figure 29). Also the time course of BHPI 74 WO 2014/113820 PCT/US2014/012405 inhibition of protein synthesis (Figure 30A) is well correlated with the increase in phosphorylation of elF2a (Figure 30B). BHPI does not induce phosphorylation of elF2a in ERa negative cells (Figure 30C). In ERa positive cancer cells, additional proof that BHPI activates the UPR is shown by the induction of the downstream 5 factors CHOP and GADD34 (see model in Figure 28B and Figure 28C and data in Figure 30D and Figure 30E). [0444] Example 9e - BHPI activates the ATF6a branch of the UPR [0445] BHPI activates the ATF6a branch of the UPR. Western blot analysis 10 showing levels of full-length (fl-ATF6a) and spliced-ATF6a (sp-ATF6a) in BHPI treated cells is shown in Figure 32. MCF-7 cells (Figure 32A) and T47D cells (Figure 32B) were incubated for 4-days in 5% cd-FBS to deplete cells of estrogens and plated at a density of 250,000 cells per well in 10% cd-calf serum. Cells were incubated with 10 nM 17p-estradiol or ethanol for 24 hours prior to treatment with 15 either 1 pM BHPI or a DMSO control, and protein samples were collected at the indicated times. ATF6a bands signals were normalized using the signals from the appropriate P-Actin band, and then normalized to the lowest signaling intensity band (24 hours). BHPI induces an increase in spliced ATF6a, 30 minutes post-treatment. Consistent with previous studies using well-establish UPR activators, BHPI also 20 induces an acute decrease in fl-ATF6a, followed by a rebound in fl-ATF6a levels. [0446] In summary, in ERa positive cancer cells, BHPI activates all 3 arms of the UPR. By activating the PERK arm of the UPR, BHPI induces phosphorylation of elF2a and inhibits protein synthesis. RNAi knockdown of PERK reduces BHPI stimulated phosphorylation of elF2a (Figure 29). Also the time course of BHPI 25 inhibition of protein synthesis (Figure 30A) is well correlated with the increase in phosphorylation of elF2a (Figure 30B). BHPI does not induce phosphorylation of elF2a in ERa negative cells (Figure 30C). In ERa positive cancer cells, additional proof that BHPI activates the UPR is shown by the induction of the downstream factors CHOP and GADD34 (see model in Figure 28B and Figure 28C and data in 30 Figure 30D and Figure 30E). 75 WO 2014/113820 PCT/US2014/012405 [0447] Example 10 - BHPI inhibits protein synthesis by inducing phosphorylation of eEF2 [0448] At later times, BHPI inhibits protein synthesis by inducing phosphorylation of eEF2 (Figure 33A and Figure 33B) which is related to activation 5 of AMPK (Figure 33C). This second site of inhibition prevents induction of p58 that reverses UPR activation (Figure 33D) and inhibits induction of the chaperone BiP (Figure 33E). BHPI only induces phosphorylation of eEF2 in cells that contain ERa (Figure 34). Inhibition of protein synthesis by phosphorylation of eEF2 blocks induction of chaperones and proteins that reverse UPR activation leading to toxic 10 persistent activation of the UPR and long-term inhibition of protein synthesis. Traditional UPR activators do not induce phosphorylation of eEF2 and only induce transient activation of the UPR (Figure 35). [0449] Example 1 0a - BHPI inhibits protein synthesis through activation of 15 AMPK, leading to phosphorylation of eEF2 [0450] BHPI inhibits protein synthesis through activation of AMPK, leading to phosphorylation of eEF2 as shown in Figure 33. Figure 33A is a Western blot analysis eEF2 phosphorylation (Thr-56) over time in BHPI-treated ERa+ MCF-7 cells. Figure 33B is a Western blot analysis showing the time course of decreasing 20 eEF2K (Ser-366) phosphorylation in BHPI-treated cells. Ser-366 dephosphorylation activates eEF2K. Figure 33C is a Western blot analysis of the time course of AMPKa (Thr-1 72) and AMPKp (Ser-108) phosphorylation in BHPI-treated cells. Figure 33D-1 shows the results of qRT-PCR analysis showing changes in p 5 8 'PK (p 58 'PK: protein 58 kilo Dalton inhibitor of interferon protein kinase) mRNA with -E2 set to 1 and 25 Figure 33D-2 is a Western blot analysis showing p 5 8 'PK and BiP protein after treatment with 1 pM BHPI. Data is mean ± S.E.M (n = 3). [0451] BHPI phosphorylation of eukaryotic elongation factor 2 (eEF2) is a second site of BHPI inhibition of protein synthesis. After approximately 2 hours, BHPI establishes a secondary pathway for inhibition of protein synthesis in ERa positive 30 cancer cells by phosphorylation and inactivation of eukaryotic elongation factor 2, (eEF2) (Figure 33A, Figure 34A). In ERa negative HeLa cells, BHPI did not elicit formation of P-eEF2, but eEF2 was phosphorylated by the eEF2 kinase activators forskolin (FOR) and rotterlin (Rot) (Figure 34B). While both BHPI and thapsigargin activate the UPR through depletion of EnR calcium, thapsigargin does not increase 76 WO 2014/113820 PCT/US2014/012405 eEF2 phosphorylation (Figure 35A). eEF2 phosphorylation is regulated by a single upstream kinase, eukaryotic elongation factor 2 kinase (eEF2K). In MCF-7 cells, BHPI induced dephosphorylation of eEF2K at Ser-366 and activation of eEF2K (Figure 33B). BHPI-induced phosphorylation and inactivation of eEF2 in ERa positive 5 cancer cells occurs by rapid phosphorylation and activation of the metabolic sensor, AMP kinase (AMPK) (Figure 33C). This activates an established pathway, in which activated AMPK phosphorylates eEF2 through activation of eEF2K. Thus, BHPI activates the pathway P-AMPKT-->eEF2KT-->P-eEF21(inactive), inhibiting elongation and protein synthesis. Also, P-eEF2 is rapidly degraded, reducing eEF2 levels 10 (Figure 33A and Figure 35A). Since protein synthesis is inhibited at both initiation and elongation, eEF2 cannot be replenished. [0452] Example 1 Ob - In ERa positive cells, BHPI treatment induces phosphorylation of eEF2 15 [0453] Figure 34 is a Western blot analysis showing the effect of BHPI on phosphorylated and unphosphorylated eEF2 in ERa positive and ERa negative cancer cells. Western blots of the time course of BHPI effects on phosphorylation of eEF2 (at Thr-56) in ERa positive T47D cell are shown in Figure 34A, and in ERa negative HeLa cells in Figure 34B. Cells were maintained for 4-days in CD-FBS to 20 deplete cells of estrogens, plated at 225,000 cells/well, and induced for 24 hours with E2 before treating cells for the indicated times with a DMSO control (+E2), 1 pM BHPI (+E2, BHPI), or 10 pM forskolin (+E2, FOR). The positive controls, forskolin (FOR) and Rottlerin (ROT) induce robust eEF2 phosphorylation demonstrating eEF2 retains the capacity for phosphorylation in HeLa cells. IB: anti-phospho eEF2; anti 25 eEF2 and a-tubulin as internal control. [0454] BHPI phosphorylation of eukaryotic elongation factor 2 (eEF2) is a second site of BHPI inhibition of protein synthesis. After approximately 2 hours, BHPI establishes a secondary pathway for inhibition of protein synthesis in ERa positive cancer cells by phosphorylation and inactivation of eukaryotic elongation factor 2, 30 (eEF2) (Figure 33A, Figure 34A). In ERa negative HeLa cells, BHPI did not elicit formation of P-eEF2, but eEF2 was phosphorylated by the eEF2 kinase activators forskolin (FOR) and rotterlin (Rot) (Figure 34B). 77 WO 2014/113820 PCT/US2014/012405 [0455] Example 10c - Conventional UPR activators induces transient eIF2a iphosiphorylation and inhibition of protein synthesis and do not induce phosphorylation of eEF2 [0456] Figure 35A is a Western blot analysis showing the time course of 5 Thapsigargin (THG) effects on phosphorylation of eIF2a (Ser-51) and eEF2 (Thr-56). Figure 35B shows incorporation of 35 S-methionine into protein as a function of time in cells treated with THG. Unlike BHPI, thapsigargin does not induce phosphorylation of eEF2, induces transient phosphorylation of elF2a and protein synthesis shows partial recovery after 4 hours. Figure 35C is a Western blot analysis of 10 phosphorylation of elF2a (Ser-51) following treatment with tunicamycin (TUN). The UPR activator, tunicamycin, induces transient phosphorylation of elF2a. Figure 35D shows the induction of CHOP mRNA following treatment of MCF-7 cells with 10 pg/mL of the UPR activator tunicamycin. CHOP mRNA levels were determined by qRT-PCR with the ribosomal protein 36B4 as an internal standard. CHOP, a 15 downstream marker for activation of the PERK arm of the UPR (see Figure 28A) is induced at 4 hours after TUN treatment. Figures 35E and 35F show the analysis of the time course of tunicamycin (TUN) effects on BiP and p58 IPK levels. Figure 35E is a Western blot analysis showing the effect of TUN on levels of BiP protein. To resolve the stress and turn off the UPR after its activated by TUN treatment, the 20 chaperone BiP is induced at 8 and 24 hours after TUN treatment. Figure 35F is a Western blot showing the effect of TUN on levels of p 58 1K. The protein p 58 ', which reverses PERK phosphorylation is also induced at 8 and 24 hours. [0457] BHPI induces persistent activation of the PERK arm of the UPR as shown by elF2a phosphorylation at 24 hours (Figure 30B), inhibition of protein 25 synthesis at 24 hours (Figure 20) and a decline in levels of BiP and p 5 8 'PK after 8 hours (Figure 33E and Figure 33F). This shows that BHPI induces persistent activation of the UPR and is different than classical UPR activators TUN and THG. Persistent activation occurs in part because BHPI also acts through ERa to inhibit protein synthesis at a second site, formation of P-eEF2. 30 78 WO 2014/113820 PCT/US2014/012405 [0458] Example 11 - E2, acting through binding to ERa, opens the IP3R calcium channel in the endoiplasmic reticulum causing an efflux of calcium from the interior of the endoplasmic reticulum into the cytosol [0459] E2, acting through binding to ERa, opens the IP3R calcium channel in 5 the endoplasmic reticulum causing an efflux of calcium from the interior of the endoplasmic reticulum into the cytosol in breast cancer cells (Figure 36) and ovarian cancer cells. Since RNAi knockdown of ERa abolishes the calcium increase in the cytosol (Figure 38), it is mediated through ERa. BHPI also opens the IP3R calcium channel in the endoplasmic reticulum and causes a much more massive calcium 10 efflux than E2. This is consistent with the idea that BHPI is working by distorting a normal action of E2-ERa and converting it from protective to lethal. [0460] When calcium is present in the medium (2 mM CaCl2), there is a formal possibility that the calcium accumulating in the cytosol is coming from outside the cell by opening what are called store operated calcium channels. Therefore, 15 experiments were done with no extracellular calcium (0 mM CaCl2) when the only major source of new calcium that can accumulate in the cell is the calcium in the lumen of the endoplasmic reticulum. [0461] Example 11 a - Estrogen stimulates the release of calcium from the 20 endoplasmic calcium [0462] The effects of 300 nM estrogen (E2) on cytosolic calcium levels in T47D breast cancer cells (Figure 36A) and in PEO4 ovarian cancer cells (PEO4: peritoneal epithelial ovarian 4, ovarian cancer cell line) (Figure 36B) conditioned in the presence (2 mM CaCl 2 ) or absence (0 mM CaCl 2 ) of extracellular calcium are 25 shown. Visualization of intracellular Ca 2 , was performed using Fluo-4, and the highest Ca 2 , concentrations are shown in the brightest white (Figure 36A-1 and Figure 36B-1). Quantitation of cytosolic calcium levels in ERa positive T47D breast cancer cells and ERa positive PEO4 ovarian cancer cells treated with E2 in the presence of absence of extracellular calcium is shown. E2 was added at 60 seconds, 30 and fluorescence intensity prior to 60 seconds was set to 1. Data is mean ± S.E. (n=10). Figure 36A-1 and Figure 36A-2 are photomicrographs and Figure 36A-2 and Figure 36B-2 are graphical representations of the effect of estrogen on intracellular calcium levels visualized using the dye Fluo-4. 79 WO 2014/113820 PCT/US2014/012405 [0463] Example 11 b - Estrogen stimulates calcium release from the endoiplasmic reticulum through IP3R Ca 2 *-channels [0464] Estrogen stimulates calcium release from the endoplasmic reticulum through IP3R Ca 2 *-channels. Figure 37A shows the effects of 300 nM estrogen (E2) 5 on cytosolic calcium levels in T47D breast cancer cells pre-treated with 2-APB, ryanodine, or ethanol-vehicle for 30 minutes in the absence of extracellular calcium (0 mM CaCl 2 ). Visualization of intracellular Ca 2 * was done using Fluo-4. The highest Ca concentrations are shown with the brightest white (Figure 37A-1). Figure 37A-2 shows the quantitation of cytosolic calcium levels in ERa positive T47D breast 10 cancer cells treated with E2 in the absence of extracellular calcium, and in cells pre treated with 2-APB or ryanodine in the absence of extracellular calcium. E2 was added at 60 seconds, and fluorescence intensity prior to 60 seconds was set to 1. Data is mean ± S.E. (n=1 0). As shown in Figure 37B, blocking calcium release from the endoplasmic reticulum through IP3R Ca 2 *-channels blocks E2-activation of the 15 PERK arm of the UPR. Western blot analysis showing p-elF2a and total elF2a levels in T47D breast cancer cells pre-treated for 30 minutes with 2-APB and/or ryanodine (RyR) or a vehicle control, followed by treatment with 10 nM E2 for 30 minutes. Data is mean ± SEM (n = 3 mRNA experiments; n = 6 cell proliferation). "-" denotes cell number at day 0. * P < 0.05; ** P < 0.01. As seen in Figure 37B, locking the IP3R 20 calcium channel with 2-APB prevents BHPI from inducing phosphorylation of elF2a. Locking the ryanodine channel does not block phosphorylation. [0465] Example 11 c - Removing ERa from breast cancer cells prevents estrogen-induced Ca 2 *-release from the endoiplasmic reticulum. 25 [0466] Removing ERa from breast cancer cells prevents estrogen-induced Ca 2 *-release from the endoplasmic reticulum. Figure 38 shows the effect of estrogen on cytosolic calcium levels after ERa knock down in T47D cells. Cells were treated with 50 nM non-coding (NC) siRNA or ERa siRNA SmartPool followed by 300 nM E2. E2-mediated calcium release from the endoplasmic reticulum was dependent on 30 ERa. In the absence of extracellular Ca 2 *, RNAi knockdown of ERa prevented E2 stimulated calcium release from ERa positive T47D cells (Figure 38). [0467] Since E2-ERa rapidly activates all three arms of the UPR, experiments were performed to identify a mechanism by which estrogen activates the UPR. Since E2-ERa elicited such rapid activation of the UPR, it seemed that the cell did not have 80 WO 2014/113820 PCT/US2014/012405 time to accumulate misfolded proteins, and, therefore, activation occurs as part of the E2-ERa proliferation program in anticipation of misfolded proteins. Since the UPR activators thapsigargin and ionomycin elicit very rapid and extremely profound depletion of Ca 2 , in the EnR lumen, it was hypothesized that more mild and transient 5 depletion of endoplasmic reticulum Ca 2 , stores induced by estrogen may be responsible for UPR activation. To test whether E2 alters intracellular Ca 2 *, the calcium sensitive dye Fluo-4 AM was used to monitor intracellular calcium. In the presence extracellular Ca 2 *, estrogen produced a rapid and transient increase in fluorescence in T47D breast cancer cells (Figure 36A). In the absence of 10 extracellular Ca2+, the increase in fluorescence was slightly larger, but more transient (Figure 36A). Since E2 elicits an increase in cytosol Ca 2 * when there is no extracellular Ca 2 *, and the Ca 2 * store in the EnR lumen is the major Ca 2 * store available to increase cytosol Ca 2 *, E2 is acting by depleting the Ca 2 * store in the endoplasmic reticulum. Estrogen also induced an increase in calcium release in the 15 presence or absence of extracellular calcium in PEO4 ovarian cancer cells (Figure 36B) demonstrating a conserved mechanism of calcium release conserved across ERa positive tissues. Since several estrogen binding proteins are proposed to influence tumorigenesis in ERa positive cancer cells, whether E2-mediated calcium release from the endoplasmic reticulum was dependent on ERa was assessed. In 20 the absence of extracellular Ca 2 *, RNAi knockdown of ERa prevented E2-stimulated calcium release from ERa positive T47D cells (Figure 38). [0468] EnR calcium homeostasis is regulated by the IP3R (inositol triphosphate) channels, by ryanodine activated Ca 2 * channels and by the ATP dependent SERCA family that pumps Ca 2 * from the cytosol into the lumen. The role 25 of the Ca 2 * channels is often probed using the inhibitors 2-APB, which locks the IP3R channel in the closed position and high concentration ryanodine, which locks the ryanodine channel closed. To more precisely localize the site of E2-ERa action at the EnR, the effects of the Ca2+ channel inhibitors, 2-APB and ryanodine were tested. 2-APB nearly abolished the rapid estrogen-stimulated increase in cytosol 30 Ca 2 * (Figure 37A 1 and Figure 37A-2). In contrast, ryanodine had only a modest effect and did not block the increase in cytosol Ca 2 * (Figure 37A 1 and Figure 37A 2). These data indicate that estrogen rapidly depletes Ca 2 * stores in the lumen of the endoplasmic reticulum calcium primarily by opening the EnR IP3R Ca 2 * channel. 81 WO 2014/113820 PCT/US2014/012405 [0469] Example 11d - E2-ERa activates the IREJ a and ATF6a branches of the UPR, inducing the production of the major EnR chaperone, BiP, and others [0470] Figure 39A shows the results of qRT-PCR of the effect of E2 on the level of spliced XBP1 mRNA. E2-ERa induces splicing of XBP1 mRNA. This 5 indicates that E2-ERa activates the IRE1 a branch of the UPR. Activation of the IRE1 a branch of the UPR activates the nuclease activity in IRE1ca, enabling it to splice XBP-1 mRNA (model in Figure 28). Thus, formation of spliced XBP-1 mRNA serves as a readout for activation of the IRE1ca branch of the UPR. Figure 39B shows the results of qRT-PCR of the effect of E2 on the levels of SERP1 and ERDJ 10 (ERDJ: endoplasmic reticulum- (ER-) localized DnaJ) mRNAs. E2-ERC stimulates induction of downstream transcriptional targets of spliced-XBP1, SERP1 and ERDJ. The increase in SERP1 and ERDJ mRNA coincides with increased splicing of XBP1 mRNA, which together indicate that E2-ERa stimulates activation of the IRE1 a of the UPR. -E2 treatment set to 1. * P < 0.05, ** P < 0.01, compared with -E2 samples. 15 Figure 39C shows the results of qRT-PCR of the effect of E2 and antiestrogens on the level of spliced XBP1 mRNA. qRT-PCR comparing the effect of ICI 182,780 and 4-hydroxytamoxifen (4-OHT) on E2-ERa of sp-XBP1 in T47D breast cancer cells is shown (-E2 set to 1). Figure 39D shows the qRT-PCR results of the effect of RNAi knockdown of ERa on the level of spliced XBP1 mRNA. RNAi knockdown of ERa 20 abolishes E2-ERa induction of sp-XBP1. [0471] E2-ERa activates the ATF6a-branch of the UPR, as indicated by increased levels of spliced ATF6a (sp-ATF6a). Western blot analysis showing full length ATF6a (fl-ATF6a) and spliced-ATF6a (sp-ATF6a) in E2-treated ERa positive cells is shown in Figure 39E, Figure 39F, and Figure 39G. Figure 39E is a Western 25 blot analysis of the effect of E2 and antiestrogens on the level of full-length and spliced ATF6a protein in T47D breast cancer cells. Figure 39F is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in BG-1 ovarian cancer cells. Figure 39G is a Western blot analysis of the effect of E2 on the level of full-length and spliced ATF6a protein in PEO4 ovarian cancer cells. 30 Western blot analysis was carried out using an antibody that detects the N-terminal fragment of ATF6a, in both the 90-kDA full-length ATF6a (fl-ATF6a) protein and the 50-kDa spliced or activated form of ATF6a (sp-ATF6a), and an antibody that detects p-actin. The increase in the level of spliced (sp)-ATF6a demonstrates activation of the ATF6a-arm of the UPR. 82 WO 2014/113820 PCT/US2014/012405 [0472] Figure 39H is an qRT-PCR analysis of the effect of E2 on the level of BiP mRNA in ERa positive MCF-7 breast cancer cells and PEO4 ovarian cancer cells. Figure 391 is a Western blot analysis of the effect of E2 on the level of BiP protein in MCF-7 cells. Figure 39J is a qRT-PCR analysis of the effect of RNAi 5 knockdown of ERa and E2 on the level of BiP mRNA. RNAi knockdown of ERa abolishes E2-induction of BiP. [0473] Figure 39D shows that RNAi knockdown of ERa prevents E2 induction of spliced XBP-1. Thus, E2 activation of the UPR is mediated through its binding to ERa. As seen in Figure 391, unlike BHPI, estrogen induces BiP chaperone at 24 10 hours. [0474] Example 11e - E2-ERa activates the PERK arm of the UPR Figure 40 shows that E2-ERa activates the PERK arm of the UPR. Figure 40A Western blot analysis showing the effect of E2 on levels of phosphorylated and 15 unphosphorylated PERK. Figure 40B is a Western blot analysis showing the effect of E2 on levels of phosphorylated and unphosphorylated elF2a. Figure 40C shows incorporation of 35 S methionine into protein in cells treated with E2 and the antiestrogen ICI 182,780 (I0). Protein synthesis was measured in ERa positive T47D breast cancer cells treated with ICI or a vehicle control for 2 hours, followed by 20 treatment with 10 nm 17p-Estradiol (n = 3). Data is mean ± SEM (n = 3 mRNA experiments; n = 6 cell proliferation). A dot ("-") denotes cell number at day 0. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. [0475] Activation of the third arm of the UPR, the PERK arm leads to inhibition of protein synthesis, an action detrimental to increased cell proliferation. Yet, E2 25 surprisingly induces a rapid and transient increase in PERK phosphorylation (Figure 40A), resulting in increased phosphorylation of elF2a (Figure 40B) and a modest and transient decline in overall protein synthesis (Figure 40C). The antagonist ICI 182,780/Faslodex/fulvestrant, which competes with E2 for binding to ERa, and induces degradation of ERa, largely blocked E-2-mediated PERK activation (Figure 30 40A) and subsequent declines in protein synthesis (Figure 40C). 83 WO 2014/113820 PCT/US2014/012405 [0476] Example 12 - E2-ERa-mediated efflux of calcium from the interior of endoiplasmic reticulum into the cytosol is required for E2-ERa-stimulated proliferation of cancer cells and for E2-ERa-regulation of gene expression [0477] Locking the IP3R calcium channel in the endoplasmic reticulum closed 5 with the inhibitor 2-APB, which prevents the increase in cytosol calcium abolishes E2-ERa-stimulated cell proliferation and strongly inhibits E2-ERa-regulation of gene expression. [0478] Figure 41 shows that elevation of cytosolic calcium, mediated through Ca 2 *-release from the endoplasmic reticulum, is required for E2-ERa mediated gene 10 expression and E2-ERa stimulated cell proliferation in breast and ovarian cancer cells. Elevation of cytosolic calcium, mediated through Ca 2 *-release from the endoplasmic reticulum, is required for E2-ERa mediated gene expression and E2 ERa stimulated cell proliferation in breast and ovarian cancer cells. Figure 41A shows the effects of the intracellular calcium chelator BAPTA-AM (BAPTA-AM: 1,2 15 Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester, intracellular calcium chelator) on E2-ERa stimulated cell proliferation. MCF-7 cells were treated with 10 pM BAPTA-AM for 3 days. E2-ERa stimulated proliferation of MCF-7 breast cancer cells (Figure 41 B) and BG-1 ovarian cancer cells (Figure 41C) treated with 200 pM ryanodine (RyR), 200 pM 2-aminoethyl diphenylborinate (2 20 APB), or both inhibitors (RyR + 2-APB) for 4 days. Figure 41 D shows ERE-luciferase activity of kBluc-T47D breast cancer cells treated with E2 and 100 pM ryanodine (RyR), 200 pM 2-APB, or both inhibitors (RyR + 2-APB). Figure 41 E is a qRT-PCR analysis of effects of 200 pM ryanodine, 200 pM 2-APB, or both inhibitors (RyR + 2 APB) on E2-ERa induction of pS2 and GREB1 mRNA in MCF-7 cells. Data is mean 25 ± SEM (n = 3 mRNA experiments; n = 5 cell proliferation; n = 4 luciferase experiments). The dot ("-") denotes cell number at day 0. * P < 0.05; ** P < 0.01. [0479] Closing the endoplasmic reticulum Ca 2 * channels blocks E2-ERa action. Consistent with earlier work suggesting a role of intracellular Ca 2 * in E2-ERa action, whether Ca 2 *-release linked to UPR activation also played an important role 30 in E2-ERa mediated gene expression, and subsequent E2-ERa induced cell proliferation was assessed. Chelating intracellular Ca 2 * with BAPTA-AM blocked E2 stimulated cell proliferation (Figure 41A). To evaluate the role of the Ca 2 * released from the EnR by E2-ERa, the effect of ryanodine and 2-APB on E2-stimulated cell proliferation and gene expression was examined. Locking ryanodine channels with 84 WO 2014/113820 PCT/US2014/012405 ryanodine and IP3R channels with 2-APB produced 20% and 40% declines in E2 stimulated proliferation, respectively. Treatment of MCF-7 cells with 2-APB and ryanodine together blocked E2-ERa induced cell proliferation of MCF-7 breast and BG-1 ovarian cancer cells (Figure 41 B and Figure 41 C), and strongly inhibited E2 5 ERa induced expression of a stably transfected ERE-luciferase reporter gene (Figure 41 D). [0480] To test for rapid effects on expression of endogenous cellular genes, the well-studied estrogen-inducible pS2 and GREB-1 genes were used. Two hours after E2 treatment, both ryanodine and 2-APB strongly inhibited induction of pS2 and 10 GREB1 mRNAs. Locking both channels nearly abolished E2-ERa induction of pS2 and GREB-1 mRNAs (Figure 41 E). This data suggests that E2-ERa-induced movement of calcium from the lumen of the endoplasmic reticulum to the cytosol may couple anticipatory activation of the UPR to activation of ERa-mediated gene expression, which together promote cell proliferation in response to estrogens. 15 [0481] Example 13 - UPR is up-regulated in estrogen-treated tumors [0482] The UPR is up-regulated in estrogen-treated tumors in human xenograft tumors in mice and by using bioinformatics in cell culture samples taken at different stages of tumor progression. E2-ERa regulates the UPR in MCF-7 breast 20 cancer cells and mouse xenograft, and elevated E2-ERa activity is correlated with increased UPR activity in patient tumor samples. [0483] Figure 42A shows qRT-PCR showing effects of E2-ERa over time on mRNAs for each UPR of the arms in MCF-7 cells. Data is mean ± SEM (n = 3). ERO1 LB is endoplasmic reticulum oxidoreductin 1-like protein beta. Figure 42B 25 shows MCF-7 tumor growth in the presence or absence of estrogen in athymic mice. All mice were treated with estrogen to induce tumor formation. On "day 0", E2 in silastic tubes was removed from the -E2 group, while silastic tubes were retained in +E2 treatment group. Figure 42C is a qRT-PCR analysis of classical E2-ERa regulated genes showing levels of GREB-1 and pS2 mRNAs in mouse tumors with 30 and without E2. Figure 42D is a qRT-PCR analysis showing levels of UPR-related mRNAs in mouse tumors collected after 24 days of exposure to estrogen (+E2) or vehicle-control (-E2). -E2 samples set to 1. Data is expressed as mean ± SEM (n = 15). 85 WO 2014/113820 PCT/US2014/012405 [0484] Figure 42E is an analysis of publically available patient microarray data showing levels of estrogen-regulated mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast and invasive ductal carcinoma tissue. Figure 42E shows relative mRNA 5 levels of classical E2-ERa regulated genes. Figure 42F is an analysis of publically available patient microarray data showing levels of UPR-related mRNAs in normal breast epithelium from normal patients, normal breast epithelium in patients with invasive ductal carcinoma of the breast and invasive ductal carcinoma tissue. Figure 42F shows relative mRNA levels of the UPR pathway in patient samples of normal 10 breast epithelium taken from patients undergoing reduction mammoplasty (RM) (n = 18), histologically normal breast epithelium taken from patients diagnosed with invasive ductal carcinoma (IDC) (n = 9), and carcinoma epithelium taken from patients diagnosed with invasive ductal carcinoma (n = 20). p-values represent comparisons to histologically normal breast epithelium from patients who underwent 15 reduction mammoplasty. * P < 0.05; ** P < 0.01; ***P < 0.001; ns, not significant. [0485] E2-ERa action Increases expression and activation of the UPR. Since E2-ERa acts at endoplasmic reticulum to activate all 3 arms of the UPR and induces formation of sp-XBP1, spATF6a and P-PERK (Figure 39 and Figure 40), the effect of E2 on levels of the mRNAs encoding UPR sensors and downstream targets was 20 investigated. E2 rapidly induced mRNAs encoding sensors for all 3 UPR arms and the chaperones BiP and GRP94 (Figure 42A). These were early responses, which generally tapered off at later times points (i.e., 24 hours). However, estrogen produced sustained increases in resident chaperones and some component of the UPR, such as elF2a (Figure 42A). 25 [0486] In vivo relevance of E2-ERa induction of UPR components and UPR activation was tested using an MCF-7 xenograft model. Growth of MCF-7 tumors was initiated using an implanted estrogen pellet. When tumors reached an average size of approximately 23.5 mm 2 , the estrogen pellets were removed. One group of mice received estrogen in cholesterol in a silastic implant and the control mice 30 received cholesterol alone. Tumors in the E2-treated group continued to grow and tumors in the -E2 group began to regress (Figure 42B). After 24 days, mice were sacrificed, and qRT-PCR analysis was performed on harvested tumors. Analysis of pS2 and GREB1 mRNA, which are highly specific surrogate markers of E2-ERa activity, were induced 12-fold and 14-fold in +E2 tumors, respectively (Figure 42C). 86 WO 2014/113820 PCT/US2014/012405 Consistent with the view that E2-ERa weakly activates the UPR, mRNA analysis of tumor samples indicated mild activation of all three arms of the UPR in +E2 tumors (Figure 42D). A 3-fold increase in sp-XBP1 was observed in the +E2 tumors, indicating strong E2-activation of the IRE1 a arm of the UPR (Figure 42D). Since 5 transcriptional activation of ATF6a is primarily responsible for induction of BiP and GRP94 chaperones and consistent with E2-activation of the ATF6a arm of the UPR, +E2 tumors displayed a 2-fold and 1.8-fold increase in BiP and GRP94 mRNAs, respectively (Figure 42D). Levels of CHOP and GADD34 mRNA were 2.1-fold and 1.4-fold higher in +E2 tumors, respectively, indicating weak E2-dependent activation 10 of the PERK arm (Figure 42D). While analysis showed increased activity of each UPR arm in +E2 tumors, the mRNA levels of several UPR genes were lower in estrogen-treated tumors. Levels of IREla and total XBP1 mRNA (IREla arm); ATF6a mRNA (ATF6a arm), and PERK and p58 'PK mRNA (PERK arm) were all significantly lower in E2-treated mice (Figure 42D). 15 [0487] The ability of estrogens to induce tumor formation in mice, coupled with the observation that UPR activity was increased in tumors, prompted the assessment of whether UPR activity is elevated early in the development of ERa positive breast cancer. E2-ERa activity and UPR pathway activity were compared in histologically normal breast epithelium, taken from patients either undergoing 20 reduction mammoplasty or at the time of diagnosis of breast cancer, with carcinoma samples from patients diagnosed with invasive ductal carcinoma (IDC). IDC samples displayed higher levels of ERa mRNA; higher levels of pS2 and GREB-1 mRNA, which are classical E2-upregulated genes; and lower levels of IL1-R1 mRNA, which is an E2-downregulated gene (Figure 42E). The same carcinoma samples displayed 25 significantly higher levels of SERP1 mRNA, a marker of IRE1 a pathway activation; CHOP and GADD34, which are markers of PERK activation; and BiP and GRP94 chaperones, which are markers of ATF6a activation (Figure 42F). Importantly, E2 ERa and UPR activity was only elevated in cancer lesions, and not in normal epithelium samples from IDC patients. Thus, elevated expression and activation of 30 ERa correlated with increased expression and activation of the UPR in IDC samples relative to normal breast epithelial tissue. Using an independent cohort of 278 ERa positive breast cancer patients, whether variable expression of ERa mRNA and protein or E2-ERa pathway activity correlates with expression of UPR genes in ERa 87 WO 2014/113820 PCT/US2014/012405 positive cancer was assessed. Expression of several UPR genes displayed highly significant correlation with expression of ERa and ERa-target genes. [0488] Example 14 - Model of the effects of estrogen, acting through ERa, on 5 activation of the UPR [0489] Figure 43 is a model of the effects of estrogen, acting through ERa, on the activation of the UPR. The model illustrates the pathway identified by which E2 ERa activates the UPR and the consequences of that activation. E2-ERa rapidly opens the IP3R calcium channel in the endoplasmic reticulum. This allows calcium to 10 move from the inside of the endoplasmic reticulum, where it is present in high concentration, into the cytosol, where the calcium concentration is low. The increased calcium cytosol is required for estrogen to stimulate gene expression and cell proliferation (Figure 41). Depleting the calcium in the interior of the endoplasmic reticulum and its passage 15 through the membrane results in activation of all 3 arms of the UPR. Because the calcium flux is small, PERK activation is weak and transient, and protein synthesis is only inhibited modestly and for a short time. The other arms of the pathway are more robustly activated, leading to production of chaperone proteins that help fold and transport proteins within the cell. This contributes to cell proliferation, resistance to 20 stress, and resistance to anticancer therapy (see Figure 42, Figure 46, Figure 48, Figure 49, and Figure 50). [0490] Example 15 - Activation of the UPR is often protective [0491] Activation of the UPR is often protective (Figure 43 and Figure 44). To 25 test whether the UPR was activated in ERa positive human tumors, the UPR index and was developed which consists of UPR sensors and downstream targets (Figure 45) and used microarray data in publically available breast cancer databases consisting of more than 1,000 patients. Expression of UPR genes was correlated with expression of ERa and estrogen-ERa-regulated genes (Figure 47). Elevated 30 expression of the UPR was strongly associated with a reduced time to recurrence (relapse-free survival) (Figure 45, Figure 46A-C, Figure 48A, and Figure 48C); resistance to tamoxifen therapy (Figure 48A and Figure 48B); a poor prognosis and early death (survival: Figure 48B and Figure 48C). These data establish the estrogen-ERa activation of the UPR pathway as a potential novel therapeutic target 88 WO 2014/113820 PCT/US2014/012405 in cancer; identify why BHPI is selective for cancer cells-because the UPR is overexpressed in the cancer cells; and establish the novelty of BHPI as hyperactivating this pathway identified for the first time as estrogen-ERa activated. 5 [0492] Example 15a - Anticipatory activation of the UPR by estrogen protects cells from exposure to higher levels of subsequent cell stress [0493] Figure 44 shows the effect of prior activation of the UPR by E2 and by TUN on subsequent cell proliferation in cells later treated with TUN. Anticipatory activation of the UPR by estrogen protects cells from exposure to higher levels of 10 subsequent cell stress. Weak anticipatory activations of the UPR with estrogen or tunicamycin protects cells from subsequent UPR stress. T47D cells were maintained in 10% CD-FBS for 8 days and treated with either 250 ng/mL tunicamycin, 100 pM E2, or ethanol/DMSO-vehicle. Cells were harvested in 10% CD-CALF serum and treated with 1 pM ICI 182,780 for 48 hours prior to treating cells with the indicated 15 concentrations of tunicamycin. Cells were pre-treated with ICI 182,780 for 48 hours to fully block estrogen-receptor signaling and to degrade ERa protein levels. Data is mean ± SEM (n = 6). [0494] Estrogen protects cells from subsequent exposure to higher levels of stress. Previous studies have demonstrated a UPR pre-conditioning phenomenon, 20 whereby transient exposure to mild UPR stress protects cells from subsequent cell stress. (Rutkowski, D.T. and Kaufman, R.J., That which does not kill me makes me stronger: adapting to chronic ER stress. Trends Biochem Sci. 2007; 32(10):469-76.) In one such study, treatment of cells with very low doses of the UPR activator, tunicamycin, resulted in mild UPR activation, which stimulated an adaptive response 25 that protected cells from subsequent exposure to tunicamycin. (Rutkowski, D.T., et al., Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS biology. 2006; 4(11):e374.) [0495] Similarly, since E2-ERa also weakly activates of the UPR, E2 should also induce an adaptive UPR response, which protect cancer cells from subsequent 30 exposure to UPR stress. This represents a potential mechanism by which anticipatory activation of the UPR by estrogen might subsequently protect ERa positive breast tumors against apoptosis due to hypoxia, nutritional insufficiency, and therapy. To test this hypothesis, the effects of E2 and a low concentration of the UPR activator, tunicamycin (TUN), on the concentration of tunicamycin required to 89 WO 2014/113820 PCT/US2014/012405 subsequently induce substantial death of T47D breast cancer cells was investigated. E2 and tunicamycin had nearly identical effects; each elicited an approximate 10 fold increase in the concentration of tunicamycin required to induce apoptosis (Figure 44). These data indicate that the weak activation of the UPR induced by E2 5 protects breast cancer cells against subsequent cell death. [0496] Example 15b - UPR qene signature [0497] Figure 45 is a table showing the genes that comprise the UPR gene index used in bioinformatics studies. The components of the UPR index include the 3 10 primary UPR sensors, direct readouts of each arm of the UPR, genes whose expression responds to activation of the UPR, chaperone protein that help fold proteins in the endoplasmic reticulum, and ERAD proteins that help degrade unfolded proteins at the endoplasmic reticulum. Thus, the UPR index components represent a broad set of genes related to the UPR and the folding and destruction of 15 unfolded proteins. These UPR genes independently predictive either of relapse free or overall survival (p < 0.05) were used to construct the UPR gene signature, which was then used to carry out risk prediction analysis. [0498] Gene abbreviations appearing in Figure 45 include: RAMP4: Ribosome-associated Membrane Protein 4; ERO1 La: endoplasmic reticulum 20 oxidoreductin 1 -like protein a; ERO1 LP: endoplasmic reticulum oxidoreductin 1 -like protein P; ERAD: endoplasmic reticulum associated protein degradation; EDEM1: endoplasmic reticulum degradation enhancer, mannosidase alpha-like 1; HERPUD1: homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1; and HRD1: HMG-CoA reductase degradation protein 1. 25 [0499] Example 15c - The UPR qenomic index is a new biomarker that predicts relapse free and overall survival of breast cancer patients [0500] Figure 46 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts. Figure 46A-1 and Figure 46A-2 show 30 bioinformatic analysis of data from two microarray chips (U1 33A) (Figure 46A-1) and (U133B) (Figure 46A-2) showing Kaplan-Meier survival plots comparing time of relapse-free survival in breast cancer patients expressing high and low levels of UPR index genes. These data show that high expression of the UPR index is associated with a shorter interval of relapse-free survival. Figure 46B is a bioinformatic analysis 90 WO 2014/113820 PCT/US2014/012405 of data from two microarray chips (U133A and U133B) showing time to relapse in 277 breast cancer patients, hazard ratio, and p-Values for individual components of the UPR gene index. Elevated expression of individual components of the UPR gene index is predictive of reduced survival. Gene abbreviations appearing in Figure 46B 5 include: HUGO: human genome organization; EIF2S1: eukaryotic initiation factor 2 subunit 1; EIF2AK3: eukaryotic initiation factor 2 alpha kinase 3; DDIT3: DNA damage inducible transcript 3; DNAJC3: DnaJ homolog subfamily C member 3; HSPA5: heat shock protein A5; HSP90B1: Heat shock protein 90 B1; and SYVN1: Synovial apoptosis inhibitor 1. 10 [0501] Figure 46C is a bioinformatic analysis of data from microarray chips (univariate analysis) comparing time to relapse in breast cancer patients using the UPR gene signature and current prognostic markers; (multivariate analysis). Testing with the UPR gene signature provides additional information about time to relapse after including information from several current prognostic markers. Gene 15 abbreviations appearing in Figure 46C include: PPP1 R1 5A: Protein Phosphatase 1, Regulatory Subunit 15A (another name for GADD34: Growth arrest and DNA damage-inducible protein 34). [0502] Figure 46D is a bioinformatic analysis of microarray data showing time to relapse in 474 breast cancer patients, hazard ratio, and p-Value for individual 20 components of the UPR gene index. Microarray analysis was performed prior to initiation of tamoxifen therapy. Since all these patients were treated with tamoxifen, elevated expression of the UPR gene index predicts the subsequent response of tumors to tamoxifen years later. Thus, the UPR gene index is a powerful predictor of the future prognosis of ERa positive cancer patients. 25 [0503] Figure 46E is a bioinformatic analysis of microarray data from two microarray chips (U133A and U133B) showing time to relapse in 236 breast cancer patients; shown are hazard ratio and p-Values for individual components of the UPR gene index. All Kaplan-Meier plots assessing UPR risk prediction were computed using leave-one-out cross-validation. UPR signature genes shown in the tables are 30 listed with their respective univariate Cox hazard ratio and p-value testing the hypothesis if expression data is predictive of relapse or overall survival. Expression of individual components of the UPR gene index predicts overall survival of ERa positive breast cancer patients. Because data from several independent cohorts of ERa positive cancer patients was used, and each cohort produces a similar 91 WO 2014/113820 PCT/US2014/012405 outcome, the data is especially strong and is not due to an artifact in producing data from a single patient cohort. [0504] Example 15d - Expression of UPR genes is positively correlated with 5 expression of ERa and ERa-regulated target-genes [0505] Figure 47 is a bioinformatic analysis of microarray data from ERa positive breast cancer patients comparing expression of classical estrogen-regulated genes and UPR index components. Correlations between the UPR and ERa protein levels (ERa), ERa mRNA levels (ESR1), or transcriptional activity of E2-ERa were 10 analyzed. E2-ERa transcriptional activity was assessed using downstream target genes of E2-ERa (pS2, GREB1). Analysis was carried out on a cohort of 278 breast cancer patients (GSE20194), which consists of 164 ERa positive tumors and 114 ERa negative tumors. Quantitation of ERa protein was by IHC. Pearson correlation coefficients and parametric p-values are shown in the table. "n.s." indicates that no 15 significant correlation was observed. Gene abbreviations appearing in Figure 46C include: TRIB3: tribbles homolog 3. [0506] While expression of UPR genes is correlated with ERa levels and expression of ERa-regulated genes, the UPR index is not simply a surrogate marker for ERa activity. In multivariate analysis, the UPR index, but not ERa, or classical 20 ERa-regulated genes, exhibits a statistically significant increase in hazard ratio. Also, UPR index exhibits predictive power to stratify patients into high and low risk groups above ERa status. Thus, while active ERa is important for expression of the UPR signature, it's the UPR signature, not ERa level or activity, that is predictive of reduced time to recurrence and reduced survival. 25 [0507] Expression of ERa-regulated genes in the cancers provides a measure of how active ERa is in the tumors. High activity of ERa, as measured by high expression of the ERa-regulated genes is associated with high expression of the UPR gene index. This helps tie the elevated expression of the UPR in the most aggressive tumors to ERa. 30 [0508] Example 15e - Expression of the UPR gene signature predicts relapse-free and overall survival in ERa positive breast tumor cohorts [0509] Figure 48 is a bioinformatic analysis of publically available microarray data from ERa positive breast cancer cohorts. Figure 48A shows Kaplan-Meier plots 92 WO 2014/113820 PCT/US2014/012405 of time of relapse-free survival for patients grouped by level of expression (low, medium and high) of the UPR gene index using bioinformatic analysis of microarray data. Relapse-free survival as a function of the UPR gene signature for patients with ERa positive breast cancer who subsequently received tamoxifen alone for 5 years 5 is shown. Interquartile range used to assign tumors to risk groups, representing UPR activity from high to low. Hazard ratios are between low and medium and low and high UPR groups (n = 474). Figure 48B shows Kaplan-Meier plots of time of overall survival for patients grouped by level of expression of the UPR gene index using bioinformatic analysis of microarray data. Overall survival as a function of the UPR 10 signature and clinical covariates (node status, tumor grade, ERa-status, tumor size) are shown. p-value is for the difference between the combined model (UPR gene signature and clinical covariates) versus the covariates only model (multivariate analysis) (n = 236). Figure 48C is a bioinformatic analysis of data from microarray chips (univariate analysis) comparing time of relapse-free survival and overall 15 survival in breast cancer patients using the UPR gene signature and current prognostic markers; (multivariate analysis) Testing was done to determine whether the UPR gene signature provides additional information about time of relapse-free survival and overall survival over and above information from several current prognostic markers. Figure 48C shows univariate and multivariate Cox regression 20 analysis of the UPR signature, clinical covariates, and classical estrogen-induced genes for time to recurrence and survival (n.s., not significant). Median used to classify tumors into high and low risk groups. [0510] The data in Figure 48 is the strongest and most dramatic data from the microarray analysis. For example, after 13 years, the cancer has recurred in 25 approximately 90% of the patients with high expression of the UPR index and only about 30% of the patients with low expression of the UPR index (Figure 48A). After 10 years, only about 10% of the patients with low expression of the UPR index have died but about 50% of the patients with high expression of the UPR index have died. Since this graph includes death from all causes, not just the cancer, this difference is 30 even more striking. A few percent of the patients in both groups will have died of causes unrelated to their cancer. This shows the UPR index is a powerful new prognostic marker. [0511] The UPR gene signature predicts clinical outcome in ERa positive breast cancer. Activation of the UPR pathway represents a novel prognostic indicator 93 WO 2014/113820 PCT/US2014/012405 predictive of relapse and survival in ERa positive breast cancer. To evaluate the UPR, a UPR gene signature consisting of genes encoding components of the UPR pathway and downstream targets of UPR activation was developed (Figure 45). We next explored whether the UPR signature was a useful prognostic marker. Using 5 data from 261 ERa positive breast cancer patients, each assigned to a high- or low genomic UPR grade, we observed reduced time to relapse for patients overexpressing the UPR signature (hazard ratio (HR) = 5.5, 95% Cl: 3.1-9.8). To evaluate the UPR signature in patients undergoing tamoxifen therapy, samples collected from 474 ERa positive breast cancer patients prior to starting 5-years of 10 tamoxifen therapy, were assigned to low, medium, or high UPR risk groups. Increased prior expression of the UPR gene signature was tightly correlated with subsequent reduced time to recurrence (Figure 48A). Hazard ratios increased from 2.2 to 3.7 for the medium and high risk groups, respectively, suggesting that recurrence risk is sensitive to levels of the UPR gene signature (Figure 48A). To 15 determine if the UPR index provided prognostic information of potential clinical utility beyond current clinical covariates, multivariate analysis was performed. In a cohort of 236 ERa positive breast cancer patients, UPR overexpression was strongly predictive of reduced survival (HR 2.69, 95% Cl: 1.3-5.6), over and above clinical covariates alone (tumor grade, node involvement, tumor size and ERa status) 20 (Figure 48B and Figure 48C). Thus, the UPR index is a powerful prognostic gene signature in ERa positive breast cancer with predictive power to stratify patients into high and low risk groups. [0512] Example 16 - UPR expression is elevated in highly malignant ovarian 25 cancers compared to normal ovarian cells [0513] Example 16a - Bioinformatic analysis of expression of UPR qenes in aggressive ovarian cancers [0514] Figure 49 is a bioinformatic analysis of publically available microarray 30 data from ovarian cancer patients with early stage and highly malignant tumors. Shown is a comparison of the UPR gene expression in early stage, low malignant potential (LMP) tumors (n = 18) and advanced stage, highly metastatic cancers of the ovary, fallopian tube, and peritoneum (n = 267). UPR expression is elevated in highly malignant ovarian cancers. 94 WO 2014/113820 PCT/US2014/012405 [0515] These data show that expression of UPR sensors (the three membrane proteins that start the individual arms of the UPR pathway, PERK, ATFa, and IREa) and several UPR-related genes in early stage ovarian cancers is reduced compared to late stage metastatic ovarian cancers. Consistent with elevated expression of the 5 UPR, GADD34 and p 5 8 'PK which help shut down the UPR, are actually reduced. [0516] Example 16b - Bioinformatic analysis of UPR expression in late stage ovarian cancer patients [0517] Figure 50 is a bioinformatic analysis of UPR expression in late stage 10 ovarian cancer patients shown as a Kaplan-Meier time to relapse plot. Shown is a Kaplan-Meier plot of relapse-free survival, as a function of the UPR gene signature, in a cohort of stage Ill-IV ovarian cancer patients treated with carboplatin and taxol agents (n = 58). Increased expression of the following UPR genes were predictive of increases risk of relapse: PERK (EIF2AK3), GRP94 (HSP90B1), BiP (HSPA5), 15 ERDJ (DNAJB1 1), TRB3 (TRIB3), and SERP1. Note that PERK belongs to the PERK arm of the UPR, and TRB3 is a downstream readout of PERK activity. GRP94 and BiP are chaperones and downstream readouts of the ATF6a pathway. ERDJ and SERP1 are downstream readouts of sp-XBP1 and the IRE1 a pathway. [0518] Elevated expression of the UPR components is associated with a 20 reduced time to relapse in late-stage ovarian cancer patients. This confirms the advantages of using BHPI to target these tumors that overexpress genes in the UPR pathway. This also confirms the idea of using ERa to target the UPR in ovarian cancer. Because these tumors overexpress genes in the UPR pathway, they will be especially susceptible to BHPI. 25 [0519] BHPI is a novel type of ERa inhibitor that may be beneficial in all diseases in which estrogen-ERa is associated with increased cell proliferation and increased expression of the UPR occurs. Such diseases include breast cancer and ovarian cancer in which estrogen-ERa is associated with increased cell proliferation and with increased expression of the UPR, and cervical, uterine/endometrial, vulval, 30 and liver cancers and endometriosis in which an association between estrogen-ERa stimulated cell proliferation and the underlying pathology of the disease has been identified. Because estrogen-ERa stimulated cell proliferation is the central feature in the pathology of endometriosis, and BHPI strongly inhibits estrogen-ERa stimulated cell proliferation, BHPI is a viable therapy for endometriosis. 95 WO 2014/113820 PCT/US2014/012405 [0520] BHPI is a unique small molecule whose non-competitive interaction with ERa elicits three effects. (1) BHPI potently inhibits protein synthesis by activating the UPR and its PERK-elF2a arm; (2) BHPI inhibits elongation by inducing phosphorylation of eEF2; and (3) BHPI independently inhibits induction and 5 repression of gene expression by E2-ERa. The data indicate these diverse inhibitory effects of BHPI are mediated through ERa. BHPI inhibits protein synthesis in all 14 ERa positive cells tested with no effect on protein synthesis in all 12 ERa negative cell lines. Expression of ERa is sufficient to make ERa negative MCF10 A cells sensitive to BHPI inhibition of protein synthesis, and knockdown of the ERa with 10 siRNA, or degrading ERa with ICI 182,780, abolishes BHPI inhibition of protein synthesis. Overexpression of ERa progressively increases BHPI inhibition of protein synthesis. ChIP shows BHPI reduces binding of E2-ERa to gene regulatory regions, suggesting that BHPI reduces affinity for response elements, and overexpression of ERa reverses BHPI inhibition of gene expression. Altered fluorescence emission 15 spectrum and protease sensitivity demonstrate that BHPI interacts directly with ERa. With wishing to be bound by any theory, it is likely that BHPI binding alters ERa conformation, altering interactions with its many binding partners, and leading to the diverse inhibitory effect of BHPI. [0521] BHPI works by opening endoplasmic reticulum calcium channels, 20 rapidly depleting calcium stores in the lumen of the endoplasmic reticulum, strongly activating the UPR, and potently inhibiting protein synthesis. The UPR plays important roles in tumorigenesis, therapy resistance, and cancer progression. Moderate and transient UPR activation is protective, while strong and sustained activation triggers cell death. Moderate UPR activation promotes an adaptive stress 25 response leading to increased expression of the UPR and antiapoptic chaperones, and this protects cancer cells from subsequent exposure to higher levels of cell stress. While UPR targeting efforts focus on inactivating a protective stress response by inhibiting UPR components, UPR overexpression in cancer suggests that sustained pharmacological activation of the UPR represents a novel alternative 30 anticancer strategy. Classical UPR activators are non-specific and highly toxic. In contrast, BHPI selectively hyperactivates the UPR activation pathway identified for estrogen-ERa. By increasing the amplitude and duration of UPR activation, BHPI converts UPR activation from protective to lethal. 96 WO 2014/113820 PCT/US2014/012405 [0522] Unlike classical UPR activators, BHPI induces sustained activation of the UPR by severing UPR signaling through inhibition of protein synthesis at a second site. BHPI inhibits elongation through activation of the major metabolic energy sensor, AMPK, leading to phosphorylation and inactivation of eEF2. AMPK 5 plays an important role in breast, ovarian, and endometrial cancers, and AMPK activating drugs, such as metformin, exhibit potential as anticancer agents. AMPK activators may have potential as a new way to target the UPR and induce sustained UPR activation in endocrine-related cancers. The ability of BHPI to target two pathways results in long-term inhibition of protein synthesis, blocking proliferation 10 and killing cancer cells. [0523] Independent of its effects on the UPR and inhibition of protein synthesis, BHPI also inhibits E2-ERa-mediated gene expression. Conventional UPR activators do not inhibit E2-ERa-mediated gene expression. Also, at early times when BHPI is fully effective, inhibition of protein synthesis does not inhibit E2-ERa 15 regulated gene expression. Since BHPI inhibits both induction and repression of gene expression by E2-ERa, BHPI inhibition of E2-ERa-regulated gene expression is not due to non-specific toxic effects. [0524] BHPI can selectively target cancer cells because its targets, ERa and the UPR, are both overexpressed in breast and ovarian cancers. Despite a role for 20 ERa in gynecological cancers, most ovarian cancer cells show little dependence on estrogens for growth and endocrine therapy is largely ineffective. Other non competitive ERa inhibitors have not demonstrated effectiveness in therapy resistant ERa positive ovarian cancer cells. BHPI extends the reach of ERa inhibitors to gynecologic cancers that do not respond to current endocrine therapies and is highly 25 effective in several drug-resistance models including: (1) tamoxifen resistant MCF7ERaHA, which overexpress ERa; (2) tamoxifen-resistant BT-474 and ZR-75-1 breast cancer cells; (3) cisplatin, tamoxifen and ICI 182,780-resistant CaOV3 ovarian cancer cells; and (4) multi-drug resistant OVCAR-3 ovarian cancer cells. BHPI is effective in a broad range of ERa-containing cancers, including, but not limited to 30 breast, ovarian, and endometrial cancers. [0525] With its sub-micromolar potency, effectiveness in a broad range of therapy resistant cancer cells, ability to induce substantial tumor regression, and novel mode of action, BHPI is an exceptional candidate for therapeutic exploration. 97 WO 2014/113820 PCT/US2014/012405 [0526] The UPR is classically viewed as a pathway activated in response to intrinsic or extrinsic stresses, which include protein misfolding, environmental stress and drug treatment. In this "reactive mode", UPR sensors are activated in response to endoplasmic reticulum stress. An alternative "anticipatory mode" of UPR activation 5 is observed in B-cell differentiation where UPR activation precedes the massive production and secretion of immunoglobulin by plasma cells. Because the signals responsible for anticipatory activation of the UPR were unknown, this process was not well understood. [0527] In the present application, it has been shown that the mitogen, 10 estrogen, acting via ERa, triggers anticipatory activation of the UPR in breast, ovarian, and other ERa positive cancer cells. Anticipatory activation of the chaperone arms of the UPR by E2-ERa enhances the protein folding capacity of the EnR, and thereby primes cells to meet the higher protein production and sorting demands that characterize the later growth phases of the cell cycle. Estrogen also rapidly and 15 transiently activates the PERK arm of the UPR, resulting in phosphorylation of elF2a, and transient reductions in overall protein synthesis. E2-ERa can rapidly and simultaneously activate all three arms of the UPR. Calcium mobilization serves as the stimulus. Blocking the Ca 2 ' release through IP3R Ca 2 , channels blocks the activation of the PERK arm of the UPR. Since 2-APB also blocks E2-ERa mediated 20 gene expression, and consequently blocks E2-ERa stimulated cell proliferation, it ties UPR activation and activation of ERa to a single event. That is, activation of ERa and the UPR are both dependent on the release of calcium from the EnR. [0528] Anticipatory activation of the UPR activation plays an essential role in E2-ERa stimulated cell proliferation. This new mechanism of estrogen action via 25 anticipatory activation of the UPR is a new paradigm by which estrogens may influence tumor development. Since activation of both ERa and the UPR are dependent on Ca 2 ' release from the endoplasmic reticulum, it suggests that ERa and UPR activation are mutually inclusive events and that UPR activation must accompany E2-ERa activation. Importantly, this also suggests that increased UPR 30 expression accompanies tumorigenesis long before tumor detection, cancer diagnosis, and the initiation of treatment. [0529] The present application shows that early activation of the UPR by E2 ERa has long-term consequences on the pathology of ERa positive breast cancer. As demonstrated, weak activation of the UPR by estrogen, similar to the UPR 98 WO 2014/113820 PCT/US2014/012405 activator tunicamycin, elicits an adaptive response that protects cells from subsequent exposure to higher levels of cell stress. While treating cells with an antiestrogen can block E2-ERa activation of the IRE1 a and ATF6a arms of the UPR, which could block further induction of chaperone proteins, it also appears to relieve 5 negative regulation of the PERK arm by E2-ERa. Inhibition of PERK and elF2a signaling, via RNAi knockdown of PERK, leads to cell death in cells treated with the ERa antagonist, ICI 182,780, indicating a role of PERK and elF2a signaling in antiestrogen resistance. Thus, differential regulation of the UPR pathway by estrogens allows the UPR to protect cells from stress in the presence of estrogens or 10 in the absence of estrogens. [0530] Bioinformatics analysis shows that increased UPR expression and activity is predictive of response to tamoxifen-therapy and overall survival. For ERa positive breast cancers resistant to endocrine therapies, an important objective is development of more specific biomarkers that predict therapeutic response and 15 identification of new therapeutic targets. The UPR is a new biomarker and therapeutic target in ERa positive breast cancer and other ERa positive cancers. [0531] All references cited herein are incorporated by reference in their entireties. The disclosure may be further understood by the included non-limiting examples. It should be understood that although the present disclosure has been 20 specifically disclosed by preferred embodiments, modification and variation of the concepts herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the claims. 25 99