Targetable molecular drivers for triple-negative breast cancer (TNBC) have been difficult to identify; therefore standard treatment remains limited to conventional chemotherapy. Recently, new-generation small molecule Hsp90 inhibitors (e.g. ganetespib and NVP-AUY922) have demonstrated improved safety and activity profiles over the first generation ansamycin class. In breast cancer, clinical responses have been observed in a subset of TNBC patients following ganetespib monotherapy, however the underlying biology of Hsp90 inhibitor treatment and tumor response is not well understood. Glucocorticoid receptor (GR) activity in TNBC is associated with chemotherapy resistance. Here we find that treatment of TNBC cell lines with ganetespib resulted in GR degradation and decreased GR- mediated gene expression. Ganetespib-associated GR degradation also sensitized TNBC cells to paclitaxel-induced cell death both in vitro and in vivo. The beneficial effect of the Hsp90 inhibitor on paclitaxel-induced cytotoxicity was reduced when GR was depleted in TNBC cells, but could be recovered with GR overexpression. These findings suggest that GR-regulated anti-apoptotic and pro- proliferative signaling networks in TNBC are disrupted by Hsp90 inhibitors, thereby sensitizing TNBC to paclitaxel-induced cell death. Thus, GR+ TNBC patients may be a subgroup of breast cancer patients who are most likely to benefit from adding an Hsp90 inhibitor to taxane therapy.

Background: Few promising molecular targets exist for chemoresistant triple negative breast cancer (TNBC). Our laboratory has previously demonstrated that a subset of TNBCs express high glucocorticoid receptor (GR) levels. In breast epithelial cells, GR transcriptional activity directly upregulates the expression of several anti-apoptotic target genes including serum/glucocorticoid regulated kinase 1 (SGK1). High GR and/or SGK1 expression are both associated with decreased chemotherapy-induced cell death in TNBC models. A meta-analysis of patients with early-stage TNBC found that high primary tumor GR (NR3C1) expression significantly associates with earlier relapse compared to GR-non expressing TNBC. Inhibiting GR/SGK1 activity is therefore a testable strategy for improving TNBC responsiveness to chemotherapy. Both GR and SGK1 require Hsp90 for their activity; therefore, Hsp90 inhibition is predicted to disrupt both GR and SGK1 cell survival signaling in TNBC. Employing a second-generation Hsp90 inhibitor, ganetespib (Synta Pharmaceuticals), we determined the effectiveness of Hsp90 inhibition on GR/SGK1 activity and chemotherapy sensitivity using in vitro and in vivo TNBC models. Hypothesis: Hsp90 inhibition will decrease GR and SGK1 activity in TNBC in association with improved chemotherapy effectiveness. Results: Using TNBC cell lines treated with ganetespib at clinically relevant concentrations, we found that total GR and the relative proportion of phosphorylated Ser211 GR were significantly reduced beginning 2 hours following treatment. Reduced glucocorticoid-induced (SGK1) mRNA expression was also seen when cells were pre-treated with ganetespib. We next used fluorescent microscopy and live cell imaging to measure TNBC cell death over a 96 hour time course following chemotherapy +/- ganetespib therapy. Addition of ganetespib to paclitaxel treatment of MDA-MB-231 cells led to significant in vitro cytotoxic synergy; as hypothesized, this synergy was diminished in GR-depleted MDA-MB-231 cells. Cytotoxic synergy following ganetespib and chemotherapy treatment was also seen in vivo in MDA-MB-231 xenografts. Interestingly, tumor GR expression following Hsp90 inhibitor and paclitaxel treatment was significantly reduced compared to tumors from mice treated with paclitaxel alone. Using GR-depleted or control MDA-MB-231 xenografts, studies are currently underway to further characterize the role of GR/SGK1 activity as a target of Hsp90 inhibition to reverse TNBC chemoresistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-05.

Glucocorticoid receptor (GR) activity in triple-negative breast cancer (TNBC) is associated with cell survival and chemotherapy resistance. Targetable molecular drivers for TNBC have been difficult to identify and therefore standard treatment remains limited to chemotherapy. Recently, new-generation small molecule Hsp90 inhibitors (e.g. ganetespib and NVP-AUY922) have proven to be both potent inhibitors of Hsp90 client proteins and far less toxic than previous Hsp90 inhibitors. Moreover, favorable clinical responses have been observed in some TNBC patients enrolled in a phase II clinical trial of ganetespib monotherapy. A ganetespib and paclitaxel clinical trial is ongoing in breast cancer; however, it is not known which Hsp90 client proteins contribute to increased tumor chemotherapy sensitivity. Recent studies in our lab found that inhibition of glucocorticoid receptor (GR) activity plays an important role in Hsp90 inhibitor-mediated sensitization of TNBC cells to paclitaxel-induced cytotoxicity. However, we also observed that following significant shRNA-mediated GR depletion, ganetespib still provided some sensitization of TNBC cells to paclitaxel. This suggests that inactivation of additional anti-apoptotic Hsp90 client proteins play a role in mediating ganetespib effects. Proteins closely associated with GR function that are also key Hsp90 inhibitor client proteins include the androgen receptor (AR, implicated in TNBC proliferation and apoptosis) and JAK, a component of JAK/STAT signaling pathway implicated in TNBC tumorigenesis and chemoresistance. Here we hypothesize that AR and JAK/STAT signaling have overlapping but distinct roles in Hsp90-mediated chemotherapy sensitization of TNBCs. Western blot analysis of TNBC xenografts treated with ganetespib +/- paclitaxel showed depletion of AR as well as JAK1 and 2. Ganetespib treatment also led to decreased levels of TMPRSS2 gene expression, an AR target gene. Additionally Western blot analysis showed decreased levels of activated phosphorylated STAT3 transcriptional factor, a downstream mediator of JAK, following ganetespib treatment of TNBC cell lines. These results suggest the involvement of AR and JAK in mediating ganetespib mechanism of action. We are currently beginning to determine how AR and JAK/STAT signaling pathways may interact with GR signaling by establishing CRISPR/Cas9 knockouts of GR, AR, and/or JAK in TNBC cells treated with ganetespib +/- paclitaxel. Understanding the relative roles of Hsp90 client proteins in TNBC is predicted to lead to a more rational selection of patients with TNBCs likely to benefit from Hsp90-inhibitor treatment. Citation Format: Abena S. Agyeman, Wesley J. Jun, Suzanne D. Conzen. Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3100.