Androgen Receptor signaling axis in castration-resistant prostate cancer Lucarelli Giuseppe, MD, PhD Urology and Kidney Transplantation Unit Dept of Emergency and Organ Transplantation University of Bari This program is supported by an educational donation provided by 1

Prostate cancer disease continuum Castration Resistant Prostate Cancer Prostate cancer disease continuum Death Docetaxel/chemotherapy 2nd-line hormonal therapies Castration Bicalutamide Flutamide Nilutamide Tumour volume Local therapy* Abiraterone Cabazitaxel Continued AR signalling Symptoms Asymptomatic Non-metastatic Metastatic Hormone-sensitive Castration-resistant Time AR, androgen receptor Kohli & Tindall. Mayo Clin Proc 2010;85:77–86.

CASTRATE RESISTANT PROSTATE CANCER PATHOPHYSIOLOGY Testosterone serum levels (ng/dl) Disease progression tumor burden (PSA levels) 150 100 50 Serum testosterone Castration threshold 20 Stimulation of Tumor Growth time De Bono ESMO Educational Program, Genitourinary Cancers 2010 modified

The road to resistance Pathway AR dependence Mechanism Hypersensitive AR dependent AR amplification AR splice variants Promiscuous AR dependent AR mutation Outlaw AR dependent Cross-talk with GF signaling pathways Bypass AR indipendent Parallel survival pathways Lurker cells AR indipendent Epithelial stem cells

Five possible pathways to androgen independence Five possible pathways to androgen independence.   a | In the hypersensitive pathway, more androgen receptor (AR) is produced (usually by gene amplification), or AR has enhanced sensitivity (not shown) to compensate for low levels of androgen, or more testosterone is converted to the more potent androgen, dihydrotestosterone (DHT), by 5 -reductase. b | In the promiscuous pathway, the specificity of the AR is broadened so that it can be activated by non-androgenic molecules normally present in the circulation. c | In the outlaw pathway, receptor tyrosine kinases (RTKs) are activated, and the AR is phosphorylated by either the AKT (protein kinase B) or the mitogen-activated protein kinase (MAPK) pathway, producing a ligand-independent AR. d | In the bypass pathway, parallel survival pathways, such as that involving the anti-apoptotic protein BCL2 (B-cell lymphoma 2), obviate the need for AR or its ligand. Finally, e | in the lurker cell pathway, androgen-independent cancer cells that are present all the time in the prostate — possibly epithelial stem cells — might be selected for by therapy.

AR amplification AR gene amplifications are observed in 20-25% of CRPC but they are very rare in primary tumors Chen CD et al, Nature Medicine  10, 33 - 39 (2004)

Higher AR levels convert antagonists to agonists Chen CD et al, Nature Medicine  10, 33 - 39 (2004)

Cell-cycle genes (UBE2C) steroidogenic enzymes AR splice variants Expression of Cell-cycle genes (UBE2C) Upregulation of steroidogenic enzymes

AR mutations

T877A mutant (Thr→Ala) L701H mutant (Leu→His) Double T877A E2/Prog Double T877A L701H mutant (Zhao XY, Nat Med: 6, 2000) E2/Prog H874Y mutant (His→Tyr) Inappropriate interaction with AR coactivators The promiscuous androgen receptor.   a | Mutations that broaden the specificity of the androgen receptor (AR): in the wild-type receptor, testosterone (T) and dihydrotestosterone (DHT) are agonists, whereas flutamide is an antagonist. The T877A mutant is activated by various non-androgenic steroid hormones, and flutamide also behaves as an agonist. The L701H mutant has reduced affinity for DHT and binds corticosteroids, but when the T877A and L701H mutations are combined, the resulting receptor (ARccr) has high affinity for corticosteroids. b | Models of the ligand-binding sites from the wild-type AR (left) and ARccr (right) with DHT bound, showing the extra space generated by the mutation of residues 701 and 877. Residues 701 and 877 are shown as space-filling models (carbon, white; nitrogen, blue; oxygen, red), and DHT is shown as a ball-and-stick model. A hydrogen bond can form between DHT and T877, but not between DHT and A877.

Q640Stop mutation generates a constitutively activated mutant AR DHT 100 nM DHT 100 nM Q640Stop mutation generates a constitutively activated mutant AR

AR Coactivators Coactivators PCa Expression Function ARA70 Controversial data Enhances AR activities or Tumor suppressor? ARA54 Up-regulated Enhances AR activation (Estradiol+Flutamide) ARA55 ″ p300 Ligand indipendent activation by IL-6 SRC1 Up-regulated Enhances AR activation (Adrenal androgens) TIF2 Up-regulated ″ PIAS Up-regulated RAC3 Up-regulated Correlation with grade & stage AR coactivators augment functional activity of the receptor by interacting with histones and are implicated in ligand indipendent activation of the AR

Outlaw receptor RAS ADT results in de-repression of alternative survival pathways and in recalibration of prostate cancer tissue androgen levels leading to partial restoration of AR transcriptional activity. How growth factor signal transduction creates outlaw receptors.   In the tumour cells of a patient receiving androgen ablation therapy, HER-2/neu (and possibly other receptor tyrosine kinases) can become overexpressed. HER-2/neu indirectly activates mitogen-activated protein kinase (MAPK). MAPK might phosphorylate the androgen receptor (AR), creating an androgen-independent 'outlaw' receptor. An alternative means by which HER-2/neu (or other pathways) might activate the AR is by activating the AKT (protein kinase B) pathway. In this pathway, activation of receptor tyrosine kinases, such as HER-2/neu, increase the level of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by activating phosphatidylinositol 3-kinase (PI3K). Another pathway might involve inactivation of the lipid phosphatase PTEN, so that PtdIns(3,4,5)P3 can no longer be converted back to its substrate, PtdIns(4,5)P2. AKT is activated by PtdIns(3,4,5)P3, and might be able to produce an outlaw AR by phosphorylating it. AKT can also activate parallel survival pathways by phosphorylating and inactivating pro-apoptotic molecules such as BAD and procaspase-9. ARE, androgen response element; PSA, prostate-specific antigen. Karantos et al. Oncogene 2013 (in press)

2. Impairs nuclear translocation MDV-3100-Enzalutamide Enzalutamide is an AR signalling inhibitor that inhibits AR signalling in three distinct ways: 3. Blocks DNA binding and activation 1. Blocks AR binding Cytoplasm Nucleus 2. Impairs nuclear translocation Enzalutamide DHT Enzalutamide Enzalutamide AR 2–3 fold lower affinity than DHT Tran et al. Science 2009;324:787–90; Watson et al. Proc Natl Acad Sci USA 2010;107:16759–65.

Androgen metabolism in prostate cancer Intratumoral androgens in CRPC tumors are maintained at levels sufficient to activate the AR signaling pathway Activated steroidogenesis pathways provide an adaptive response to ADT, facilitating CRPC tumor survival in a low exogenous androgen environment. The maintenance of intratumoral androgens can be accounted for in part by intratumoral or intracrine biosynthesis of steroid hormones, either via the uptake and conversion of adrenal androgens or potentially via de novo steroidogenesis.

Classic pathway Backdoor pathway

Expression of steroidogenic enzyme transcripts in primary and metastatic prostate tumors Soft tissue metastases from CRPC shows elevated testosterone compared to primary tumors Transcripts encoding all enzymes involved in the sequential biosynthesis of testosterone and DHT from cholesterol precursors were expressed in the majority of castration-resistant metastatic tumors examined Montgomery RB, Cancer Res 2008;11, 68

Meccanismo d’azione di abiraterone Steroidogenesi ipokaliemia Ipertensione sovraccarico di fluidi Cholesterol Desmolase Renin-angiotensin Deoxy- corticosterone Pregnenolone Pregnenolo Progesterone Corticosterone Aldosterone X CYP17 17α-hydroxylase 17α-OH- pregnenolone 17α –OH- progesterone 11-Deoxy- cortisol Cortisol ACTH CYP17 C17,20-lyase 5α-reductase DHEA Androstenedione Testosterone DHT CYP19: aromatasi Abiraterone Estradiolo Yang, Drugs. 2011; Attard, JCO 2008

Meccanismo d’azione di abiraterone Steroidogenesi Cholesterol Basse dosi di steroidi minimizzano la tossicità mineralcoticoide - correlata Desmolase Renin-angiotensin Deoxy- corticosterone Pregnenolone Progesterone Corticosterone Aldosterone X CYP17 17α-hydroxylase 17α-OH- pregnenolone 17α –OH- progesterone 11-Deoxy- cortisol Cortisol ACTH CYP17 C17,20-lyase 5α-reductase DHEA Androstenedione Testosterone DHT CYP19: aromatasi Abiraterone + prednisone Estradiolo Yang, Drugs. 2011; Attard, JCO 2008

Stromal-epithelial interactions and intratumoral androgen biosynthesis Alterations in androgen transport normal BPH PCa SLCO1B3 Wright JL, Cancer Epidemiol Biomarkers Prev. 2011

Conclusions Persistent activation of AR signaling in CRPC suggests that a majority of prostate cancers progress through pathways that continue to activate the cellular AR program. While the introduction of potent steroidogenic inhibitors such as abiraterone, in combination with novel AR inhibitors such as MDV3100, holds significant promise for the concept of multi-targeted AR pathway blockade, the optimal timing, sequence, and potential combinatorial strategies using new AR pathway inhibitors are critical unanswered questions in the treatment of men with CRPC.

Prostate cancer is not the only cause of death in men