Tindall, Donald J.

Abstract Mortality from prostate cancer in 2011 is estimated at 33,720 deaths in United States which occurs primarily in advanced disease stages. The cornerstone treatment of advanced prostate cancer is androgen deprivation, first established by Huggins and Hodges in 1941. Since then, recognition for the central role of the androgen axis and the androgen receptor (AR) during prostate tumorigenesis and progression, has been accepted. The arrest of the androgen-dependent growth stimulus by surgical or medical castration (with LHRH analogues or antagonists) is the result of reduced circulating androgens including dihydro-testosterone (DHT), which induces apoptotic regression of androgen-dependent cancer cells. This control however is short lived with the emergence of castration-resistant prostate cancer (CRPC). Despite achieving sub-castrate levels of circulating androgens as a result of castration, the androgen-AR axis continues to play an active role in tumor progression. A functional AR, despite the paucity of circulating androgens, is evidenced by the elevation of AR mRNA and re-expression of some androgen-regulated genes during clinical castration, indicative of a persistently activated AR cell signaling pathway. Furthermore, although testosterone and DHT are depleted in serum following traditional androgen deprivation therapy, significant levels of androgens have been measured in locally CRPC tissue1 as well as in metastatic CRPC tissue2. These androgens continue to regulate AR transcriptional activity and stimulate expression of androgen-regulated genes thereby re-invigorating tumor progression. Indeed, a number of molecular and cellular changes have been shown to facilitate activation of the AR under these conditions: amplification of the AR gene allow the receptor to respond to lower levels of androgens; splice variants of the AR exhibit constitutive activity; increased expression of several co-activator proteins enhance activation of the AR; other signaling pathways, including Akt-PI3 Kinase and MAP Kinase, activate the AR; and gain-of-function mutations in the AR allow the receptor to become “promiscuous” and then bind to ligands other than DHT, including anti-androgens3 and weaker sex steroids such as androstenedione. Abiraterone acetate, a selective steroidal irreversible inhibitor of CYP17 (17 hydroxylase/C17,20-lyase) blocks two important enzymatic activities in the synthesis of testosterone4. In a phase III trial in patients progressing on docetaxel chemotherapy, the combination of abiraterone plus prednisone resulted in an improvement in overall survival compared to patients receiving prednisone plus placebo (14.8 months vs 10.9 months, HR=0.646;P<0.001)5. The PSA response to the active drug combination was 38% vs 10% for the placebo group. A second phase III trial testing abiraterone with prednisone vs placebo and prednisone in the castration resistant stage in patients who are chemotherapy naïve has completed accrual (COU-AA-302; NCT00887198). These results re-enforce the rationale behind previous similar clinical attempts in which non-selective and reversible inhibitors of sex steroid biosynthesis such as ketoconazole in combination with hydrocortisone were used6 to target androgen biosynthesis after failure of androgen deprivation therapy. Ketoconazole inhibits the cholesterol side chain cleavage enzyme 11 beta hydroxylase as well as CYP 17, albeit reversibly7. While no overall survival difference for the study group was demonstrated in this study, an increased survival was observed in patients treated with ketoconazole experiencing PSA responses (35% with a 50% decline in PSA). Interestingly, CYP 17 inhibition with abiraterone in CRPC patients may retain activity even in patients previously treated with ketoconazole8. Adverse events with abiraterone use include hypokalemia, hypertension, peripheral edema and headaches all sequelae of secondary mineralocorticoid excess which can be minimized with the use of combination prednisone. Additional drugs in development include Ortrenol (previously named TAK-700; Millenium Pharmaceuticals, Cambridge, MA/Takeda, Osaka, Japan), which inhibits CYP 17, 20 lyase more potently than 17-hydroxylase. Two on-going large phase III studies with ortrenol in combination with prednisone in chemotherapy naïve (ClinicalTrials.gov Identifier: NCT 01193244) and in the post docetaxel chemotherapy (ClinicalTrials.gov Identifier: NCT01193257) CRPC populations will evaluate response of the study combination vs prednisone plus placebo with overall survival as the primary endpoint. Another novel drug is ToK-001 (Tokai Pharmaceuticals, Cambridge, MA) which is a combined inhibitor of CYP 17 and AR currently in early clinical phase II trials. Resistance to abiraterone has been observed in patients initially responding to the drug. Xenograft models suggest that the resistance may occur through upregulation of CYP17A1 and/or induction of AR and AR splice variants that confer ligand independent AR transactivation9. Targeting the AR with next generation AR inhibitors has also emerged as a potential therapeutic maneuver in CRPC. MDV310010 (Medivation, San Francisco, CA), a second generation anti-androgen, is now in phase III clinical trials. This new diarylthiohydantoin compound targets AR by binding over-expressed AR with an affinity that is several fold greater than previously obtained with anti-androgens (bicalutamide and flutamide). Another drug, ARN-509 (Aragon, San Diego, CA) is currently in phase I/II clinical trials (NCT01171898) and further development of this drug is to be determined based on the completion of the on-going dose establishing trial. Both these drugs disrupt the nuclear translocation of AR and impair DNA binding to androgen response elements and recruitment of co-activators and thus have multi-functional anti-tumor capabilities10. Results of a phase III study with the primary endpoint of overall survival evaluating MDV3100 vs placebo in the post chemotherapy, castration resistant stage which has completed accrual is now awaited (NCT00974311). Another phase III study is currently accruing patients after androgen deprivation failure who are chemotherapy naïve evaluating MDV3100 vs placebo also with the primary endpoint of overall survival (NCT01212991). In conclusion, the AR signaling axis remains active during the progression to CRPC and should be considered to be a viable target for therapy. References: 1. Mohler JL. Castration-recurrent prostate cancer is not androgen-independent. Adv Exp Med Biol. 2008;617:223–234. 2. Montgomery RB, Mostaghel EA, Vessella R, et al. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res. 2008;68:4447–4454. 3. Dehm SM, Tindall DJ. Molecular regulation of androgen action in prostate cancer. J Cell Biochem. 2006;99:333–344. 4. Reid AH, Attard G, Barrie E, de Bono JS. CYP17 inhibition as a hormonal strategy for prostate cancer. Nat Clin Pract Urol. 2008;5:610–620. 5. de Bono JS, Logothetis CJ, Molina A, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005. 6. Small EJ, Halabi S, Dawson NA, et al. Antiandrogen withdrawal alone or in combination with ketoconazole in androgen-independent prostate cancer patients: a phase III trial (CALGB 9583). Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2004;22:1025–1033. 7. Haidar S, Ehmer PB, Barassin S, Batzl-Hartmann C, Hartmann RW. Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo. J Steroid Biochem Mol Biol. 2003;84:555–562. 8. Ryan CJ, Smith MR, Fong L, et al. Phase I clinical trial of the CYP17 inhibitor abiraterone acetate demonstrating clinical activity in patients with castration-resistant prostate cancer who received prior ketoconazole therapy. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010;28:1481–1488. 9. Mostaghel EA, Marck BT, Plymate SR, et al. Resistance to CYP17A1 Inhibition with Abiraterone in Castration-Resistant Prostate Cancer: Induction of Steroidogenesis and Androgen Receptor Splice Variants. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011;17:5913–5925. 10. Tran C, Ouk S, Clegg NJ, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009;324:787–790. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN08-01.

Abstract Purpose: The aim of this study was to investigate the promoter hypermethylation as diagnostic markers to detect malignant prostate cells and as prognostic markers to predict the clinical recurrence of prostate cancer. Experimental Design: DNA was isolated from prostate cancer and normal adjacent tissues. After bisulfite conversion, methylation of 14,495 genes was evaluated using the Methylation27 microarrays in 238 prostate tissues. We analyzed methylation profiles in four different groups: (i) tumor (n = 198) versus matched normal tissues (n = 40), (ii) recurrence (n = 123) versus nonrecurrence (n = 75), (iii) clinical recurrence (n = 80) versus biochemical recurrence (n = 43), and (iv) systemic recurrence (n = 36) versus local recurrence (n = 44). Group 1, 2, 3, and 4 genes signifying biomarkers for diagnosis, prediction of recurrence, clinical recurrence, and systemic progression were determined. Univariate and multivariate analyses were conducted to predict risk of recurrence. We validated the methylation of genes in 20 independent tissues representing each group by pyrosequencing. Results: Microarray analysis revealed significant methylation of genes in four different groups of prostate cancer tissues. The sensitivity and specificity of methylation for 25 genes from 1, 2, and 4 groups and 7 from group 3 were shown. Validation of genes by pyrosequencing from group 1 (GSTP1, HIF3A, HAAO, and RARβ), group 2 (CRIP1, FLNC, RASGRF2, RUNX3, and HS3ST2), group 3 (PHLDA3, RASGRF2, and TNFRSF10D), and group 4 (BCL11B, POU3F3, and RASGRF2) confirmed the microarray results. Conclusions: Our study provides a global assessment of DNA methylation in prostate cancer and identifies the significance of genes as diagnostic and progression biomarkers of prostate cancer. Clin Cancer Res; 18(10); 2882–95. ©2012 AACR.

AbstractRecently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.

Azadirachta indica(common name: neem) leaves have been found to possess immunomodulatory, anti-inflammatory and anti-carcinogenic properties. The present study evaluates anti-angiogenic potential of ethanol extract of neem leaves (EENL) in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with EENL inhibited VEGF induced angiogenic responsein vitroandin vivo. Thein vitroproliferation, invasion and migration of HUVECs were suppressed with EENL. Nuclear fragmentation and abnormally small mitochondria with dilated cristae were observed in EENL treated HUVECs by transmission electron microscopy. Genome-wide mRNA expression profiling after treatment with EENL revealed differentially regulated genes. Expression changes of the genes were validated by quantitative real-time polymerase chain reaction. Additionally, increase in the expression of HMOX1, ATF3 and EGR1 proteins were determined by immunoblotting. Analysis of the compounds in the EENL by mass spectrometry suggests the presence of nimbolide, 2′,3′-dehydrosalannol, 6-desacetyl nimbinene and nimolinone. We further confirmed antiproliferative activity of nimbolide and 2′,3′-dehydrosalannol in HUVECs. Our results suggest that EENL by regulating the genes involved in cellular development and cell death functions could control cell proliferation, attenuate the stimulatory effects of VEGF and exert antiangiogenic effects. EENL treatment could have a potential therapeutic role during cancer progression.

Discoveries over the past decade suggest that castration-resistant prostate cancer (CRPC) is sensitive, but not resistant to, further manipulation of the androgen–androgen receptor (AR) axis. Several new therapies that target this axis have demonstrated clinical activity. In this article, preclinical and clinical findings occurring in the field of AR-targeted therapies are reviewed. Reviews of scientific and clinical development are divided into those occurring prereceptor (androgen production and conversion) and at the level of the receptor (AR aberrations and therapies targeting AR directly). Intracrine androgen production and AR amplification, among others, are among the principal aberrancies driving CRPC growth. Phase III data with abiraterone acetate and phase II data with MDV-3100, along with other similar therapies, confirm for the clinician that the scientific findings related to persistent AR signaling in a castrate milieu can be harnessed to produce significant clinical benefit for patients with the disease. Studies aimed at optimizing the timing of their use and exploring the mechanisms of resistance to these therapies are under way. The clinical success of therapies that directly target androgen synthesis as well as the most common aberrancies of the AR confirm that prostate cancer retains dependence on AR signaling, even in the castrate state.

AbstractAdvanced prostate tumors, which are androgen dependent, are often initially treated in the clinic with hormone ablation therapy, either through surgical castration or administration of small-molecule antiandrogens. Most tumors respond favorably to these treatments, exhibiting regression of the tumor, amelioration of symptoms, and a decrease of prostate-specific antigen in patient sera. However, with time, the majority of tumors recur in a more aggressive, castration-resistant (CR) phenotype. Currently, no effective treatment exists for this stage of the cancer, and patients ultimately succumb to metastatic disease. The androgen receptor (AR), which is a member of the nuclear hormone receptor superfamily of proteins, is the transcription factor that is responsible for mediating the effects of androgens upon target tissues, and it has been demonstrated to play a central role in the development and progression of prostate cancer. Despite CR tumor cells being able to continue to grow after hormonal therapy in which testosterone and dihydrotestosterone are markedly reduced, they still require the expression and activity of the AR. The AR can become transactivated in this low-androgen environment through a number of different mechanisms, including amplification and mutation of the receptor, cross talk with other signaling pathways, and altered regulation by coregulatory proteins. This review will summarize the most current data regarding non-ligand-mediated activation of the AR in prostate cancer cells. Developing work in this field aims to more clearly elucidate the signals that drive AR activity independently of androgens in CR disease so that better therapeutic targets can be developed for patients with this stage of highly aggressive prostate carcinoma.

Abstract DNA methylation abnormalities, either gain or loss of methylation in promoters or other regulatory regions, contribute to tumorigenesis by altering the activity of tumor suppressor or oncogenes. To identify the molecular alterations in prostate cancer, we have profiled gene methylation using Infinium Human Methylation27 bead chips in benign prostate tissue, primary prostate tumor tissue from patients who developed metastasis within 5 years of prostatectomy, and serum from patients who developed metastasis. Tissue DNA isolation and bisulfite conversion was done using ZR Genomic DNA kit and EZ methylation kit. Serum DNA isolation and bisulfite conversion was done using ZR serum DNA kit and EZ methylation kit. Methylation data was processed using the Beadstudio average normalization algorithm and differentially methylated genes identified using a two-group T-test p-value < 0.05. To evaluate the impact of methylation on mRNA expression levels, we isolated RNA from LCM-derived epithelial cells of 124 patients using Picopure RNA isolation kit and measured expression using HG-U133+2 microarrays. The gene expression data was preprocessed using GCRMA background correction, FASTLO normalization, affinities-only PM correction, and median polish summarization, and differential gene expression was defined by a two-group T-test p-value < 0.05, a log2 fold change > 1, and a maximum average expression value > log2 7. Comparison of the methylation and gene expression data identified 321 differentially expressed Affymetrix probesets which mapped to genes differentially methylated genes; 272 with differential methylation in the prostate cancer tissue and 97 with differential methylation in the prostate cancer serum. In general, we expected a complementary effect between gene methylation and expression, with hyper-methylation driving decreased expression, and hypo-methylation driving increased expression. This was seen in the prostate cancer tissue methylation data, where 228/272 (84%) probesets possessed this methylation-expression complementarity; 212/238 hyper-methylated genes and 16/34 hypo-methylated genes. The effect was not as strong in the prostate cancer serum methylation data, where 30/97 (31%) probesets displayed the methylation-expression complementarity; 19/79 hyper-methylated genes and 11/18 hypo-methylated genes. Overall, our results revealed a significant correlation of the DNA methylation with the down regulation in the mRNA expression levels of the genes, which could play potential roles in prostate cancer progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4950.

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death in men in the United States. Definitive treatment consists of either radical prostatectomy or radiotherapy followed by androgen deprivation. Although majority of the prostate cancers usually responds to these therapies and can be cured, some of them will eventually relapse. It is believed that the relapsed cancers obtained new ability that facilitates their resistance to these therapeutic stresses. Increased expression of VEGF-C and its receptor Neuropilin-2 (NRP-2) in hormone-refractory and metastatic prostate cancers indicates that the VEGF-C/NRP-2 axis could play an important role in the progression of prostate cancer. Indeed, we observed VEGF-C/Neuropilin-2 axis promotes survival of prostate cancer cells during stress induced by hydrogen peroxide or radiation. Detail signaling events downstream of neuropilin-2 for this stress-resistant function have also been determined. Our findings therefore demonstrated a novel and distinct function of NRP-2 in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3950.

Abstract The androgen receptor (AR) is the principal target for treatment of non–organ-confined prostate cancer (PCa). Androgen deprivation therapies (ADT) directed against the AR ligand–binding domain do not fully inhibit androgen-dependent signaling critical for PCa progression. Thus, information that could direct the development of more effective ADTs is desired. Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes, pointing to a role for secondary transcription factors. A combination of microarray and in silico analyses led us to identify a 158-gene signature that relies on AR along with the transcription factor SRF (serum response factor), representing less than 6% of androgen-dependent genes. This AR-SRF signature is sufficient to distinguish microdissected benign and malignant prostate samples, and it correlates with the presence of aggressive disease and poor outcome. The AR-SRF signature described here associates more strongly with biochemical failure than other AR target gene signatures of similar size. Furthermore, it is enriched in malignant versus benign prostate tissues, compared with other signatures. To our knowledge, this profile represents the first demonstration of a distinct mechanism of androgen action with clinical relevance in PCa, offering a possible rationale to develop novel and more effective forms of ADT. Cancer Res; 71(5); 1978–88. ©2011 AACR.