This project is testing the hypothesis that an alternative hormonal receptor, the glucocorticoid receptor, can compensate for the lack of androgen receptor activity in prostate cancer to impart resistance to potent hormonal therapies. There are three specific aims for this two year award:

Aim 1:  To determine whether high GR expression in CaP cell lines accelerates resistance to MDV3100 in vivo.

Aim 2: To assess the expression and nuclear localization of both AR and GR within circulating tumor cells (CTC) from prostate cancer patients with clinical resistance to a potent androgen pathway inhibitor.

Aim 3: To determine if AR target genes become instead regulated by GR-mediated transactivation of these genes following potent anti-AR signaling therapy.

Over the first year of the award (2012) we have achieved significant progress towards these aims.  Aim 1 focuses on in vivo interrogation of GR signaling in the setting of castration resistant prostate cancer progression. Our initial experiment was to characterize the expression of the GR in the setting of castration in vivo, to determine if GR expression increases with castration as expected. As shown below in Figure 1, in multiple prostate cancer cell lines, GR expression increases (RNA (b), protein immunofluorescence (c)) or the protein becomes nuclear localized, as in the case with CWR-22RV1 cells, suggesting increased GR activation. To test if GR has an active role in promoting cell survival in the context of AR inhibition, we have successfully generated stably transfected prostate cancer cell lines engineered to have decreased GR expression. As the ultimate goal is to translate these findings to a clinical trial of concomitant AR and GR inhibition, decreased GR expression was pursued (rather then overexpressed GR as proposed). In both cell lines tested (CWR-22RV1, LAPC4), decreasing GR expression prolonged time to castration resistant progression, supporting our hypothesis. Using preclinical models, we have also begun to interrogate AR/GR target gene regulation as outlined in Aim 3. In addition to regulating prostate specific antigen (PSA) expression, activation of the GR leads to sustained expression of two tested AR-regulated pro-survival genes SGK1 and MKP1. Furthermore, ectopic SGK1 expression enhances castration resistance in vivo.

The major work within Aim 3 involves a series of chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and mRNA expression analyses. We have been optimizing ChIP conditions and are poised to complete these experiments in the subsequent 6 months of this award.

Aim 2’s focus was on interrogation of circulating tumor cells (CTC’s) from men with metastastatic CRPC failing potent hormonal therapy. The initial antibodies selected and outlined for use on these studies for staining of the AR and GR have led to inconsistent results using control cells. As such, we have changed antibodies, using two new antibodies that have been released for commercial use in the last 6 months. We have optimized the conditions for use and have begun staining patient samples. We are actively collecting patient samples for CTC interrogation and will continue work inthis Aim over the next year.

In addition to the progress made, briefly outlined above, work on this project has led to several reportable outcomes. The initial results of the laboratory studies have been compiled into a manuscript that is written and has been submitted for review, with an acknowledgement to the Cancer Research Foundation. Most exciting, the PI has submitted two separate grants for funding through the Department of Defense that have recently been favorably reviewed and recommended for funding. The first is a one year, $75K award to further explore the role of MKP1 as a pro-survival gene, regulated by the AR and GR in castration resistant prostate cancer. The second is a three-year clinical trial award (total budgeted direct costs $450K). It is a phase I/II trial of the AR antagonist enzalutamide in combination with the GR antagonist mifepristone. This large award will allow us to translate the work funded by the Cancer Research Foundation to patient care and would not have been awarded without the Cancer Research Foundation support through this award.