Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor protein that functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. The androgen receptor (AR) is expressed in more than 70% of breast cancers and has been implicated in breast cancer pathogenesis. However, little is known about the role of BRCA1 in AR-mediated cell proliferation in human breast cancer. Here, we report that a high expression of AR in breast cancer patients was associated with shorter overall survival (OS) using a tissue microarray with 149 nonmetastatic breast cancer patient samples. We reveal that overexpression of BRCA1 significantly inhibited expression of AR through activation of SIRT1 in breast cancer cells. Meanwhile, SIRT1 induction or treatment with a SIRT1 agonist, resveratrol, inhibits AR-stimulated proliferation. Importantly, this mechanism is manifested in breast cancer patient samples and TCGA database, which showed that low SIRT1 gene expression in tumor tissues compared with normal adjacent tissues predicts poor prognosis in patients with breast cancer. Taken together, our findings suggest that BRCA1 attenuates AR-stimulated proliferation of breast cancer cells via SIRT1 mediated pathway.

Supplementary Table 1 from Tumor Repressive Functions of Estrogen Receptor β in SW480 Colon Cancer Cells

Estrogen receptor beta 1 (ERβ1) is a ligand-activated nuclear receptor, which has been shown to maintain tissue differentiation in the normal prostate, and regulate androgen response and increase expression of tumor suppressors in prostate cancer cell lines. There are three shorter isoforms of ERβ expressed in the human prostate, ERβ2, ERβ4, and ERβ5, which have already been implicated in chemotherapy resistance and disease progression, suggesting a possible oncogenic role. Their ligand-binding domain (LBD) is truncated, so they are unable to activate canonical ERβ1 signaling pathways; however, they were shown to participate in hypoxic signaling and to induce a gene expression signature associated with stemness and hypoxia. To elucidate the role of the truncated ERβ isoforms in prostate cancer, we created a knockout of all isoforms, as well as a truncation of the LBD, to remove the function of ERβ1. We showed that the removal of all isoforms leads to a decrease in the expression of cancer stem cell (CSC)-associated genes, decreased chemotherapy resistance, and a decrease in the CSC population, based on sphere formation ability and SORE6 (CSC reporter) activity, while removing the LBD function only had the opposite effect. Our results suggest a more aggressive phenotype in prostate cancer cell lines expressing ERβ variants.

Supplementary Table 2 from Tumor Repressive Functions of Estrogen Receptor β in SW480 Colon Cancer Cells

Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancers, and survival in patients with recurrence is rarely beyond 12 months due to acquired resistance to chemotherapy, which is the standard of care for these patients. Our hypothesis is that Estrogen Receptor β1 (ERβ1) increases response to chemotherapy but is opposed by ERβ4, which it preferentially dimerizes with. The role of ERβ1 and ERβ4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERβ1 Ligand Binding Domain (LBD) and knock down the exon unique to ERβ4. We show that the truncated ERβ1 LBD in a variety of mutant p53 TNBC cell lines, where ERβ1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERβ4 knockdown cell line was sensitized to Paclitaxel. We further show that ERβ1 LBD truncation, as well as treatment with ERβ1 antagonist 2-phenyl-3-(4-hydroxyphenyl)-5,7-bis(trifluoromethyl)-pyrazolo[1,5-a] pyrimidine (PHTPP), leads to increase in the drug efflux transporters. Hypoxia Inducible Factors (HIFs) activate factors involved in pluripotency and regulate the stem cell phenotype, both in normal and cancer cells. Here we show that the ERβ1 and ERβ4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIFs. We show the increase of cancer cell stemness due to ERβ1 LBD truncation is attenuated when HIF1/2α is knocked down by siRNA. Finally, we show an increase in the breast cancer stem cell population due to ERβ1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERβ4 positive, while only a small proportion of TNBC patients are ERβ1 positive, we believe that simultaneous activation of ERβ1 with agonists and inactivation of ERβ4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.

Summary:After the discovery of ERβ, a novel role for dihydrotestosterone (DHT) in estrogen signaling was revealed. Instead of just being a better androgen, DHT was found to be a precursor of the ERβ agonist 5α-androstane-3β, 17β-diol (3βAdiol), an estrogen which does not require aromatase for its synthesis. ERβ was found to oppose androgen signaling and thus is a potential target for treatment of prostate cancer. ERβ was also found to have effects that were independent of androgen signaling, particularly in the CNS. Although in rodent models of neurodegenerative diseases (Parkinson's disease, multiple sclerosis, and Alzheimer's disease), ERβ agonists are very effective in relieving symptoms and improving pathologies, this has not proven to be the case in humans. In this review we will focus on the main differences in ERβ signaling between rodents and humans and will make the point that a very important difference between the two species is in the splice variants which are expressed in humans and not rodents. The main conclusion at this point is that before we think of using ERβ agonists clinically, much more work on ERβ signaling in the human or in primates needs to be done.

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ–IL4rα and IL2rγ–IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ–IL4rα and IL2rγ–IL13rα1 receptors signal primarily via JAK1–STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. Significance: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell–intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

Estrogen receptor β (ERβ) was first identified in the rodent prostate and is abundantly expressed in human and rodent prostate epithelium, stroma, immune cells and endothelium of the blood vessels. In the prostates of mice with inactivated ERβ, mutant phenotypes include epithelial hyperplasia and increased expression of androgen receptor (AR)-regulated genes, most of which are also upregulated in prostate cancer (PCa). ERβ is expressed in both basal and luminal cells in the prostate while AR is expressed in luminal but not in the basal cell layer which harbors the prostate stem cells. To investigate the mechanisms of action of ERβ and its potential cross-talk with AR, we used RNA-seq to study the effects of estradiol or the synthetic ligand, LY3201, in AR-positive LNCaP PCa cells which had been engineered to express ERβ. Transcriptomic analysis indicated relatively few changes in gene expression with ERβ overexpression, but robust responses following ligand treatments. There is significant overlap of responsive genes between the two ligands, as well as ligand-specific alterations. Gene set analysis of down-regulated genes identified an enrichment of androgen-responsive genes, such as FKBP5, CAMKK2, and TBC1D4. Consistently, AR transcript, protein levels, and transcriptional activity were down-regulated following ERβ activation. In agreement with this, we find that the phosphorylation of the CAMKK2 target, AMPK, was repressed by ligand-activated ERβ. Down-regulation of TBC1D4, a major regulator of glucose uptake in prostate, indicates that ERβ is changing glucose metabolism in the prostate. These findings suggest that ERβ-mediated signaling pathways are involved in the negative regulation of AR expression and activity, thus supporting a tumor suppressive role for ERβ in PCa.

Loss of the tumor suppressor, PTEN, is one of the most common findings in prostate cancer (PCa). This loss leads to overactive Akt signaling, which is correlated with increased metastasis and androgen independence. However, another tumor suppressor, inositol-polyphosphate 4-phosphatase type II (INPP4B), can partially compensate for the loss of PTEN. INPP4B is up-regulated by androgens, and this suggests that androgen-deprivation therapy (ADT) would lead to hyperactivity of AKT. However, in the present study, we found that in PCa, samples from men treated with ADT, ERβ, and INPP4B expression were maintained in some samples. To investigate the role of ERβ1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERβ1. In these cells, INPP4B was induced by ERβ ligands, and this induction was accompanied by inhibition of Akt activity and reduction in cell migration. These findings reveal that, in the absence of androgens, ERβ1 induces INPP4B to dampen AKT signaling. Since the endogenous ERβ ligand, 3β-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERβ1 on AKT signaling, an ERβ agonist should be added along with ADT.

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. This Kras*-driven heterotypic signaling circuit in the early and advance tumor microenvironment enables cooperative protumorigenic interactions in early steps of the neoplastic transformation process as well as in promoting growth of established tumors, providing novel therapeutic targets in the Kras* pathway for this intractable disease. Here, we explored the role of Kras* in protumorigenic signaling interactions between cancer cells and host cells in tumor microenvironment. Early-stage malignant lesions and advanced tumors show close proximity of Kras* expressing cancer cells and abundant invading TH2 (CD4+ Gata-3+) cells, which can produce IL-4 and IL-13. Single-cell analysis of low- and high-grade IPMN and PDAC patient samples revealed CD4+ T-cell infiltration and higher percentage of the CD4+ T cells are Gata3+ TH2 subtypes compared to T-bet+ TH1 cells. Similarly, imaging mass spectrometry analysis showed presence of CD4+ T cells in close proximity to cancer cells. Mechanistic studies identified Jak1-Stat6 as the primary signaling pathway downstream of IL-4/IL-13 activated IL-4 receptor complexes in cancer cells. Integrated transcriptomic and chromatin immunoprecipitation sequencing (ChIP-seq) profiling identified cMyc, specifically its upstream enhancer element, as a direct target of activated Stat6. Metabolomic analysis demonstrated that IL-4/13STAT6cMyc upregulation drives increased glycolysis along with upregulated expression of key glycolysis enzymes, forging a link between Kras*-driven glycolysis via paracrine activation of cMyc. Citation Format: Prasenjit Dey, Jun Li, Jianhua Zhang, Huamin Wang, Wen-Ting Liao, Vincent P Bernard, Giannicola Genovese, Anirban Maitra, Anders Strom, Ronald A. DePinho. CD4 T cells mediated protumorigenic pathway in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A09.

Abstract Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that the variants of ERβ, namely ERβ1, ERβ2, ERβ4, and ERβ5, contributes to aggressiveness of the TNBC cell line HCC1806 by affecting E-Cadherin expression and chemotherapy sensitivity. We have previously shown that the HCC1806 cell line expresses all the ERβ variants. To investigate their function we used the recently developed dCas9-KRAB system with Guide RNA for both ERβ promoters 0N and 0K, using 3 guide RNA's for each promoter and we found that expression of panERβ decreased from a ct. of 28 to 33 indicating loss of total ERβ. Treatment of HCC1806 cells with chemotherapy in combination with an ERβ agonist, LY500307, is more effective in reducing cancer cell viability. Knockdown of total ERβ in HCC1806 cells using CRISPR-Cas9 technology, decreased the proliferation and increased chemo-sensitivity to docetaxel agent in cells with the 0K promoter knock down, while cells with the 0N promoter knock down were less affected. Furthermore, after ERβ knockdown, the cell morphology of HCC-1806 changed to a more epithelial phenotype and there was increase in the expression of the classical epithelial marker, E-cadherin. N-Cadherin was unchanged in the cells with the 0K promoter knock down, while E-Cadherin was slightly decreased in cells with the 0N promoter knock down. We also found that cells with the 0K promoter knock down migrated less than control cells in a migration assay, while cells with the 0N promoter knock down were less affected. These findings are in agreement with reports showing that the 0K promoter is responsible for expression of the variants ERβ2 and ERβ5, while the 0N promoter expresses more ERβ1. Furthermore, we have earlier shown that ERβ2 and ERβ5 increase aggressiveness of TNBC via several oncogenic signaling pathways. In conclusion, knockdown of ERβ variants in HCC-1806 indicated that the variants may be responsible for conferring oncogenic properties to TNBC such as EMT, cancer cell proliferation and chemo-resistance. Therefore, treating TNBC patients with combination therapy including ERβ agonists, may prove to be extremely beneficial for securing better treatment responses and future pre-clinical studies should design appropriate experiments to confirm these findings in vivo. Citation Format: Faria M, Gustafsson J-A, Strom A. Endogenous expression of ERβ variants contributes towards chemotherapy-resistance in the triple negative breast cancer cell line HCC-1806 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-21.

ERbeta (ERβ) celebrated its 20th birthday in 2016 and although the overwhelming data in the literature indicate a role for this receptor in the control of epithelial proliferation, neurodegeneration and immune function, no ERβ agonists have yet made it to the clinics. This is the situation, despite the fact that very good safe ERβ agonists have been synthesized and at least one has been donated to the NIH for distribution to researchers, who want to study its possible clinical use. Clinical trials are ongoing for the use of ERβ agonists in prostate cancer and schizophrenia but even today reviewers of our grants still make comments like "The grant is excellent except that the focus of the grant is ERβ". There are multiple reasons for the non-acceptance of the value of ERβ and in this paper we will discuss issues raised by labs which do not support a role for ERβ in physiology or pathology.

Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that two of the variants of ERβ, namely ERβ2 and ERβ5, control aggressiveness of TNBC by regulating hypoxic signaling through stabilization of HIF-1α. RNA-seq of patient derived xenografts (PDX) from TNBC shows expression of ERβ2, ERβ4 and ERβ5 variants in more than half of the samples. Furthermore, expression of ERβ4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to tumorsphere formation caused transformation and development of tumorspheres. By contrast, ERβ1, ERβ2 or ERβ5 were unable to support tumorsphere formation. We have previously shown that all variants except ERβ1 stabilize HIF-1α but only ERβ4 appears to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERβ4. We propose that ERβ variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer.

Chemotherapy resistant prostate cancer is a major clinical problem. When the prostate cancer has become androgen deprivation resistant, one of the few treatment regimens left is chemotherapy. There is a strong connection between a cancer's stem cell like characteristics and drug resistance. By performing RNA-seq we observed several factors associated with stem cells being strongly up-regulated by the estrogen receptor β variants, β2 and β5. In addition, most of these factors were also up-regulated by hypoxia. One mechanism of chemotherapy resistance was expression of the hypoxia-regulated, drug transporter genes, where especially ABCG2 and MDR1 were shown to be expressed in recurrent prostate cancer and to cause chemotherapy resistance by efficiently transporting drugs like docetaxel out of the cells. Another mechanism was expression of the hypoxia-regulated Notch3 gene, which causes chemotherapy resistance in urothelial carcinoma, although the mechanism is unknown. It is well known that hypoxic signaling is involved in increasing chemotherapy resistance. Regulation of the hypoxic factors, HIF-1α and HIF-2α is very complex and extends far beyond hypoxia itself. We have recently shown that two of the estrogen receptor β variants, estrogen receptor β2 and β5, bind to and stabilize both HIF-1α and HIF-2α proteins leading to expression of HIF target genes. This study suggests that increased expression of the estrogen receptor β variants, β2 and β5, could be involved in development of a cancer's stem cell characteristics and chemotherapy resistance, indicating that targeting these factors could prevent or reverse chemotherapy resistance and cancer stem cell expansion.

Abstract Recent clinical studies indicate that estrogen receptor β2 (ERβ2) and β5 (ERβ5) expression in TNBC correlates to worse prognosis. We have expressed ERβ2 and ERβ5 in the triple negative cell line SUM159. Estrogen receptors β2 and β5 are highly similar to estrogen receptor β1, except for a truncated C-terminus making the remaining ligand -binding domain incapable of binding to estrogen. In addition, at the C-terminus, ER β2 and β5 have a unique peptide each of 27 and 4 amino acids in length respectively. Stably expressing ERβ2 and ERβ5 using a transposon integrated, tetracycline- regulated expression system increases expression of the drug resistance gene, ABCG2 in the triple negative SUM159 cells. Furthermore, we also find that the cells become more resistant to treatments with paclitaxel. We find that ERβ2 and ERβ5 interact with HIF-1α and HIF-2α and are recruited to HIF response elements (HRE's) in chromatin. In agreement with this, we find that ERβ2 and ERβ5 are recruited to the ABCG2 promoter, which contains HRE's and is known to be a target of HIF signaling. We propose that ERβ2 and ERβ5 play a role in chemo - resistance of TNBC and that targeting these factors may reduce chemo resistance thus preventing tumor-relapses and their associated mortalities. Citation Format: Faria M, Gustafsson J-A, Strom A. Estrogen receptor β2 and β5 increase expression of ABCG2 and drug resistance of the triple- negative breast cancer cell line SUM159 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-10.

Recent clinical studies indicate that estrogen receptor β2 (ERβ2) and β5 (ERβ5) expression in TNBC correlates to worse prognosis. We have expressed ERβ2 and ERβ5 in the triple negative cell line SUM159. Estrogen receptors β2 and β5 are highly similar to estrogen receptor β1, except for a truncated C-terminus making the remaining ligand -binding domain incapable of binding to estrogen. In addition, at the C-terminus, ER β2 and β5 have a unique peptide each of 27 and 4 amino acids in length respectively. Stably expressing ERβ2 and ERβ5 using a transposon integrated, tetracycline- regulated expression system increases expression of the drug resistance gene, ABCG2 in the triple negative SUM159 cells. Furthermore, we also find that the cells become more resistant to treatments with paclitaxel. We find that ERβ2 and ERβ5 interact with HIF-1α and HIF-2α and are recruited to HIF response elements (HRE9s) in chromatin. In agreement with this, we find that ERβ2 and ERβ5 are recruited to the ABCG2 promoter, which contains HRE9s and is known to be a target of HIF signaling. We propose that ERβ2 and ERβ5 play a role in chemo - resistance of TNBC and that targeting these factors may reduce chemo resistance thus preventing tumor-relapses and their associated mortalities. Citation Format: Faria M, Gustafsson J-A, Strom A. Estrogen receptor β2 and β5 increase expression of ABCG2 and drug resistance of the triple- negative breast cancer cell line SUM159 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-10.

Glioblastomas (GBM), deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a tumor suppressive role of estrogen; however, estrogen as a potential therapy for GBM is limited due to safety concerns. Since GBM express ERβ, a second receptor for estrogen, targeting ERβ with a selective agonist may be a potential novel GBM therapy. In the present study, we examined the therapeutic effect of the selective synthetic ERβ agonist LY500307 using in vitro and in vivo GBM models. Treatment with LY500307 significantly reduced the proliferation of GBM cells with no activity on normal astrocytes in vitro. ERβ agonists promoted apoptosis of GBM cells, and mechanistic studies using RNA sequencing revealed that LY500307 modulated several pathways related to apoptosis, cell cycle, and DNA damage response. Further, LY500307 sensitized GBM cells to several FDA-approved chemotherapeutic drugs including cisplatin, lomustine and temozolomide. LY500307 treatment significantly reduced the in vivo tumor growth and promoted apoptosis of GBM tumors in an orthotopic model and improved the overall survival of tumor-bearing mice in the GL26 syngeneic glioma model. Our results demonstrate that LY500307 has potential as a therapeutic agent for GBM.