AIM #1: DETERMINE APPROPRIATE METHOD OF MEASUREMENT OF ANDROGEN RECEPTOR IN VITRO To address the discrepancy in determination of AR content in bladder cancer, AR will be measured by IHC, fluorescence in situ hybridization, real-time quantitative reverse-transcription polymerase chain reaction (real-time RT-PCR) for mRNA expression, and western blotting in bladder cancer cell lines, fresh bladder cancer tissue, and frozen paraffin-embedded bladder cancer specimens. Nuclear and cytosolic fractions will both be examined when appropriate. LNCAP prostate cancer cells can serve as a positive control for AR and UMUC-3 and TCC-SUP are well-published bladder cancer cell lines that we have available to us that have high expression of AR relative to other bladder cancer cell lines. We can potentially knockdown AR in one of these cell lines to serve as a negative control. Related Sub-Aim: It is unclear from published reports whether AR expression in the bladder is limited to tissue at the bladder neck and near the prostate in men or uniformly present throughout the bladder. Therefore, using banked specimens, we will do the above measurements of AR from different portions of the bladder to see if expression is the same at the bladder neck compared to other areas of the bladder. We will also look at gender differences and inter-individual differences.
AIM #2 : ASSESS ALTERATIONS IN EXPRESSION OF AR COREGULATORS IN RESPONSE TO MDV3100 An analysis of genes important in AR signaling was performed in 26 bladder tumors from The Cancer Genome Atlas (TCGA) project for which sequencing and array-comparative genomic hybridization (aCGH) data are available and shown below. We included a 10-gene set important in AR signaling (SOX9, RAN, TNK2, EP300, PXN, NCOA2, AR, NRIP1, NCOR1, and NCOR2) and 3 additional coactivators recently published to be associated with bladder cancer (NCOA1, NCOA3, and CREBBP) (Boorjian et al, 2009). Although AR was only altered in 8% of cases, at least one alteration in one of these androgen-signaling genes was seen in 19 (73%) of these cases. Therefore, AR-positive and AR-negative bladder cancer cell lines and xenografts as determined by the methods in AIM #1 will be assessed for alterations in these AR signaling genes. Treatment with MDV3100 will be undertaken and changes in gene and co-regulator expression will be assessed in response to therapy. MDV3100 is a new class of antiandrogen agent that is FDA approved and well-tolerated. We are almost finalized on an MTA with the company in order to receive this drug for clinical research. This may elucidate other targets beside AR in bladder cancers responsive to single-agent MDV3100.
AIM#3 : STUDY RELATIONSHIP OF ESTROGEN RECEPTORS TO ANDROGEN RECEPTOR We are recruiting Iawen Hsu, Ph.D. as our new post-doctoral fellow because she has made novel discoveries in bladder cancer looking at estrogen receptors during her doctoral training. She hypothesized that given the gender disparity in bladder cancer with more men afflicted with the disease, that perhaps estrogen receptors (ER) have an influence on this phenomenon. She found that among the two isoforms of ER that one predisposes towards bladder cancer and the other one protects. She has yet to evaulate targeting these isoforms concurrently. In addition, androgen receptor has also been shown to have an influence on bladder cancer but the phenomenon has not been well studied. I propose to recruit Iawen Hsu given her tremendous experience in bladder cancer research with the task of exploring ER and androgen receptor in bladder cancer. With her efforts, we may be able to develop targeted therapies for these receptors and make a significant impact in bladder cancer.
AIM #3 : ASSESS THE ABILITY OF MDV3100 TO SYNERGIZE WITH COMMON CHEMOTHERAPEUTICS IN VITRO (MDV3100 + doxorubicin/valrubicin, MDV3100 + mitomycin C, MDV3100 + EGFR mAb/TKI, MDV3100 + Src inhibitor, MDV3100 + tamoxifen) A recent report demonstrates that AR expressionmay confer resistance to doxorubicin in some bladder cancer cell lines. In fact, the authors demonstrated that knockdown of AR made the UMUC3 cell line sensitive to doxorubicin (Shiota et al, 2012). Perhaps, the combination of MDV3100 and doxorubicin may be synergistic in bladder cancer cell lines and xenografts that express AR. However, doxorubicin is still investigational in the treatment of bladder tumors but is closely related to valrubicin, which is a FDA-approved agent for intravesical administration in patients with BCG-refractory carcinoma in situ of the bladder. Therefore, studying valrubicin with MDV3100 may prove to be an easier combination therapy to eventually study further in a future phase I study. The current adjuvant therapy given to patients with low-grade bladder tumors that recur frequently is a single peri-operative intravesical administration of mitomycin C after resection of bladder tumors. This regimen could be studied in conjunction with MDV3100 as well. A recent report demonstrated that epidermal growth factor (EGF) could increase AR transcriptional activity in bladder cancer through the formation of an AR/Estrogen Receptor-beta (ER-beta)/Src complex that is mediated by NCOA2 (Izumi et al, 2012). This suggests that inhibition of the EGF Receptor (EGFR) via a monoclonal antibody (mAb) or tyrosine kinase inhibitor (TKI) specific for EGFR may be synergistic with inhibition of AR via MDV3100. Likewise a src inhibitor such as dasatinib may be synergistic with MDV3100 if the association between AR and Src is important for tumorigenesis. Finally, few studies have demonstrated a synergistic effect with concurrent administration of an estrogen receptor antagonist (e.g. tamoxifen) with an anti-androgen in prostate cancer (Mangerini et al, 2012) Furthermore the formation of the complex of AR, ER-beta, and src cited in the report above (Izumi et al, 2012) may potentially be disrupted by the concurrent administration of MDV3100 and tamoxifen. Therefore, the combinations suggested above have some pre-clinical evidence to warrant further study.
