Alejandro Gomez-Pinillos

mTOR Signaling Pathway and mTOR Inhibitors in Cancer Therapy

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase. It is ubiquitously expressed in cells and is a therapeutic target for the cancer treatment arsenal. Despite the great responses obtained in tumors addicted to specific mutations or overactivation of key members of the mTOR pathway (HiF1α in RCC, cyclin D1 in MCL, or TSC in SEGA), mTOR inhibitors as single agents have modest activity. Dual PI3K/mTOR kinase inhibitors have been developed with the idea of overcoming resistance to the mTOR inhibition through preventing the activation of PI3K/Akt as a result of release negative feedback loops.

KLF6 Loss of Function in Human Prostate Cancer Progression Is Implicated in Resistance to Androgen Deprivation

Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy.

Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Purα‐mediated decrease in androgen receptor levels

Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR in androgen‐independent LNCaP cells (AI‐cells) and tissues from castration‐resistant patients to decreased nuclear levels of Pur‐alpha (Purα) and loss from a protein complex bound to repressor sequences (ARS) in the 5′‐UTR of AR. Strategies to regain control of increased AR transcription may overcome resistance of AI‐cells and improve treatment outcomes.

Effect of inhibition of the PI3K/Akt/mTOR pathway on AR splicing and downstream targets.

101 Background: Increased androgen receptor (AR) and PI3K/Akt/mTOR signaling drive androgen-independent (AI) prostate cancer (PC) progression. AR splice variants (SV) lacking the ligand-binding domain activate ligand-independent transcriptional programs that support castration resistant proliferation and survival. Treatment of the androgen-dependent PTEN null LNCaP (AD-cells), its AI derivative (AI-cells) and wt PTEN RV1 cells with the dual PI3K/mTOR inhibitor BEZ235 (E) combined with the histone deacetylase inhibitor Panobinostat (PAN) induced synergistic growth inhibition and cell death in vitro and in vivo. The effect of these treatments on AR splicing is unknown. METHODS LNCaP AD, AI and RV1 cells were treated with PAN 20nM, E 350nM, rapamycin (Rapa) 20nM and PI3Ki BKM120 (K) 1.5µM for 24h. Total (t) AR, SVs and AR targets mRNA were measured by qtPCR and western. RV1 xenografts received 3 weeks E 35mg/kg/d or PAN 10mg/kg/QOD or PAN+E. RESULTS Baseline levels of tAR mRNA in AI were higher than AD and RV1 cells while SV1, SV7 and SV5-7 were higher in RV1 than AD and AI cells. Treatment with E, Rapa and K increased both tAR and SVs mRNAs and AR and PSA proteins. In contrast, treatment with PAN reduced tAR and SVs levels in AD and AI with no effect on RV1. PAN reduced the induction of tAR by Rapa or K in AD, AI and RV1 but did not abrogate induction of SVs. PAN inhibition was sufficient to abrogate the effect of E and decrease tAR and SVs levels as well as AR protein and AR canonical and non-canonical targets (PSA, TMPRSS2 and UBE2C) and PSA protein below baseline levels in all cells treated with PAN+E. Similarly, RV1 tumors treated with E had a 50% increase in tAR and 20% in SV5-7 but no induction of CDK1 or UBE2C and a 35% reduction in PSA while RV1 tumors treated with PAN+E showed 40% reduction in both SVs 1 and 7 and downstream targets CDK1 and UBE2C. CONCLUSIONS Inhibition of PI3K/mTOR with E increases overall levels of AR transcription that is more pronounced on SVs and uncouples ARs transcriptional activity on canonical downstream targets in vivo. PAN can abrogate the stimulation of E by decreasing both SVs expression and AR transcriptional activity on non-canonical targets. These effects may underlie their synergistic growth inhibition.