The estrogen receptor (ER) β variant ERβ2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERβ2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERβ2 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 ERβ2 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 ERβ2 is mediated by HIF-1α and that targeting of this ERβ2 – HIF-1α interaction may be a strategy to treat prostate cancer.

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.

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.

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