Estrogen Receptor ß4 Regulates Chemotherapy Resistance and Induces Cancer Stem Cells in Triple Negative Breast Cancer.

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 ß1 (ERß1) increases response to chemotherapy but is opposed by ERß4, which it preferentially dimerizes with. The role of ERß1 and ERß4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERß1 Ligand Binding Domain (LBD) and knock down the exon unique to ERß4. We show that the truncated ERß1 LBD in a variety of mutant p53 TNBC cell lines, where ERß1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERß4 knockdown cell line was sensitized to Paclitaxel. We further show that ERß1 LBD truncation, as well as treatment with ERß1 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 ERß1 and ERß4 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 ERß1 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 ERß1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERß4 positive, while only a small proportion of TNBC patients are ERß1 positive, we believe that simultaneous activation of ERß1 with agonists and inactivation of ERß4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.

Estrogen receptor ß regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3.

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, ERß, and INPP4B expression were maintained in some samples. To investigate the role of ERß1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERß1. In these cells, INPP4B was induced by ERß 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, ERß1 induces INPP4B to dampen AKT signaling. Since the endogenous ERß ligand, 3ß-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERß1 on AKT signaling, an ERß agonist should be added along with ADT.

Estrogen receptor ß exerts tumor suppressive effects in prostate cancer through repression of androgen receptor activity.

Estrogen receptor ß (ERß) 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 ERß, mutant phenotypes include epithelial hyperplasia and increased expression of androgen receptor (AR)-regulated genes, most of which are also upregulated in prostate cancer (PCa). ERß 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 ERß 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 ERß. Transcriptomic analysis indicated relatively few changes in gene expression with ERß overexpression, but robust responses following ligand treatments. There is significant overlap of responsive genes between the two ligands, estradiol and LY3201 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 ERß activation. In agreement with this, we find that the phosphorylation of the CAMKK2 target, AMPK, was repressed by ligand-activated ERß. These findings suggest that ERß-mediated signaling pathways are involved in the negative regulation of AR expression and activity, thus supporting a tumor suppressive role for ERß in PCa.

The ERß4 variant induces transformation of the normal breast mammary epithelial cell line MCF-10A; the ERß variants ERß2 and ERß5 increase aggressiveness of TNBC by regulation of hypoxic signaling.

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 ERß 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 ERß, namely ERß2 and ERß5, 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 ERß2, ERß4 and ERß5 variants in more than half of the samples. Furthermore, expression of ERß4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to tumorsphere formation caused transformation and development of tumorspheres. By contrast, ERß1, ERß2 or ERß5 were unable to support tumorsphere formation. We have previously shown that all variants except ERß1 stabilize HIF-1α but only ERß4 appears to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERß4. We propose that ERß variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer.

Selective Estrogen Receptor ß Agonist LY500307 as a Novel Therapeutic Agent for Glioblastoma.

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 ERß, a second receptor for estrogen, targeting ERß with a selective agonist may be a potential novel GBM therapy. In the present study, we examined the therapeutic effect of the selective synthetic ERß 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. ERß 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.

The FKBP52 Cochaperone Acts in Synergy with ß-Catenin to Potentiate Androgen Receptor Signaling.

FKBP52 and ß-catenin have emerged in recent years as attractive targets for prostate cancer treatment. ß-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that putatively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that ß-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and ß-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone-independent and -dependent AR signaling. Our data demonstrate that FKBP52 promotes ß-catenin interaction with AR and is required for ß-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks ß-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and ß-catenin. Our data suggest that co-regulation of AR by FKBP52 and ß-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

Estrogen Receptor ß2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1α in Prostate Cancer.

The estrogen receptor (ER) ß variant ERß2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERß2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERß2 interacts with and stabilizes HIF-1α protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1α is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ERß2 interacts with HIF-1α and piggybacks to the HIF-1α response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ERß2 is mediated by HIF-1α and that targeting of this ERß2 - HIF-1α interaction may be a strategy to treat prostate cancer.