- Click here to visit the home page of the American Urological Association 2011 Annual meeting
- Here are abstracts related to ISIS products that will be presented (note that 3 of the 4 relate to drugs that OGXI is developing- OGX-011 and OGX-427):
125: CLUSTERIN ANTISENSE INHIBITOR OGX-011 SYNERGIZES ACTIVITY OF SECOND GENERATION ANTI-ANDROGEN, MDV3100, IN CASTRATE RESISTANT PROSTATE CANCER MODEL
Hiroaki Matsumoto, Hidetoshi Kuruma, Amina Zoubeidi, Ladan Fazli, Martin Gleave
INTRODUCTION AND OBJECTIVES: Experimental evidence strongly implicate the AR and intra-tumoral androgen synthesis in promoting tumor cell survival and development of castrate resistant prostate cancer (CRPC). The new second generation AR antagonist, MDV3100, has shown activity in preclinical and clinical studies. Our previous studies link androgen ablation therapy with clusterin upregulation and castrate resistance, and we developed the antisense oligonucreotide, OGX-011, that synergistically enhances both castration and chemotherapies in prostate cancer (CaP) models. In this study, we tested whether OGX-011 sensitized MDV3100 and delayed castrate-resistant progression in LNCaP model. METHODS: Effects of single vs combination MDV3100 and OGX-011 regimens on AR-positive LNCaP cell growth rates, protein, and gene expression were analyzed using crystal violet assay, flow cytometry, western blotting and RT-PCR, respectively. AR transcriptional activity was measured by PSA-luciferase reporter assay, while AR degradation was assessed by cycloheximide chase assay. The effects combination treatment on castrate-resistant LNCaP tumor growth was assessed in castrated male athymic mice.
RESULTS: Combination OGX-011 + MDV3100 showed synergistic effect and more potently suppressed LNCaP cell growth rates in a dose and time dependent manner compared to OGX-011 or MDV monotherapy. PARP cleavage, sub G0/G1 apoptotic fraction and repressed AKT phosphorylation was most enhanced with combined therapy. Interestingly, OGX-011 accelerated AR degradation and repressed AR transcriptional activity in combination with MDV3100. In vivo, combined OGX-011 + MDV3100 significantly delayed castration-resistant LNCaP tumor progression compared to scramble antisense oligonucreotide + MDV3100 (p<0.05 and p<0.05 at 12weeks, respectively).
CONCLUSIONS: OGX-011 combined with MDV3100 down-regulated AR levels and activity and suppressed castrate resistant LNCaP cell growth in vitro and in vivo, providing pre-clinical proof-of-principle as a promising approach for AR targeting therapy in CRPC.
2) Sunday, May 15, 2011 3:30 PM-5:30 PM Prostate Cancer: Basic Research
615: HSP27 INHIBITION ACTIVATES THE UNFOLD PROTEIN RESPONSE AND AUTOPHAGY PATHWAY THROUGH INHIBITION OF PROTEASOME ACTIVITY IN PROSTATE CANCER
Masafumi Kumano, Junya Furukawa, Amina Zoubeidi, Shiota Masaki, Eliana Beraldi, Martin Gleave. Vancouver, Canada
INTRODUCTION AND OBJECTIVES: Hsp27 is induced by accumulation of unfolded protein aggregates following various stress conditions, and promotes folding of proteins to their native state or degrading misfolded proteins via the ubiquitin-proteasome pathway (UPP). The specific signaling that occurs between the endoplasmic reticulum (ER) and the nucleus in response to ER stress is known as the unfolded protein response (UPR). During the UPR, accumulated unfolded protein is either correctly refolded or degraded by the UPP. Recently, there has been an increased general interest in understanding the interactions of ER stress, UPP and autophagy pathway in protein clearance. However, it is still unclear whether ER stress-mediated autophagy by proteasome inhibition is involved in cell survival or cell death. Therefore, we hypothesize that inhibition of Hsp27 will enhance ER stress and treatment-induced cancer cell death by increasing unfolded protein burden. We set out to characterize the role of Hsp27 in ER stress and UPP in prostate cancer. METHODS: Effects of Hsp27 silencing using siRNA or antisense inhibitor (OGX-427) or proteasome inhibition (MG132) on PC3 cell apoptosis and putative targets of UPR were compared. Proteasome activity was measured using fluorescent substrate of proteasome in Hsp27 inhibited or overexpressed PC3 cells. We also evaluated autophagy activity after Hsp27 knockdown and cell viability after combination treatment with Hsp27 knockdown +/- inhibition of autophagy by 3-methyladenine (3-MA).
RESULTS: Hsp27 expression and other components of the UPR were activated in PC-3 cells after treatment with MG132 with accumulation of ubiquitinated proteins. Hsp27 knockdown was associated with decreased proteasome activity, increased ubiquitinated protein levels, and activation of UPR, similar to that seen with MG132. In contrast, Hsp27 overexpressing PC3 cells exhibited increased proteasome activity and resistance to MG-132 induced cell death and UPR activation compared to control cells. The inhibition of Hsp27 also led to increased autophagy activity. Furthermore, the combination treatment with Hsp27 silencing and 3MA significantly reduced PC3 cell viability and induced apoptosis.
CONCLUSIONS: These results suggest that Hsp27 enhances the catalytic activity of the proteasome and increases the degradation rate of ubiquitinated proteins. Moreover, the inhibition of both Hsp27 and autophagy enhances prostate cancer cell death and represents a useful therapeutic strategy to target for anti-cancer therapies.
3) Monday, May 16, 2011 8:00 AM-10:00 AM Prostate Cancer: Basic Research
727: ANTISENSE OLIGONUCLEOTIDE TARGETING INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR ENHANCES PACLITAXEL SENSITIVITY IN A PACLITAXEL- RESISTANT PROSTATE CANCER MODEL
Junya Furukawa, Hideaki Miyake, Masato Fujisawa, Christopher Wraight, Brett Monia, Martin Gleave, Michael Cox. Kobe, Japan
INTRODUCTION AND OBJECTIVES: Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in prostate cancer (PCa) and associated with adaptive growth and survival signaling implicated in castrate-resistant disease (CRPC) progression. These activities are mediated by activation of the IGF-I receptor (IGF-IR). While CRPC patients treated with taxanes alone or in combination with estramustine phosphate or predonisone show a survival benefit, development of chemotherapeutic resistance eventually occurs. Therefore novel strategies targeting the molecular basis of androgen deprivation therapy and chemotherapy resistance are required. In this study, we establish paclitaxel-resistant androgen-independent PCa cells (PtxR PC3) and assess the potency and anti-cancer activity of a 2?EMOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, ATL1101, on PtxR PC3 cells in vitro and in vivo. METHODS: PtxR PC3 cells were established by culturing in step-wise increased drug concentrations. IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated PtxR PC3 cells were measured by QT-PCR and immunoblotting. The effect of IGF-1R ASO and paclitaxel combination therapy on PtxR PC3 cell growth and apoptosis in vitro was examined by crystal violet assay and flow cytometry. In vitro combination index (CI) was calculated by Calcusyn® softoware. For in vivo xenograft studies, PtxR PC-3 cells were inoculated s.c. in the flanks of athymic nude mice and tumor volume kinetics were compared for mice treated with paclitaxel injected i.v. and either ATL1101 or control ODN injected i.p.
RESULTS: The IC50 for paclitaxel in PtxR PC3 was 15-fold higher than that in parental cells. PtxR PC3 also shows cross-resistance to docetaxel and mitoxantrone. We observed dose- and sequence-specific suppression of IGF-IR mRNA and protein expression in ATL1101-treated PtxR PC3 cells in vitro which correlated with decreased proliferation and increased apoptosis of PtxR PC3 cells. Combination of ATL1101 with paclitaxel showed CI values below 1 at the IC50, IC75, suggesting the drug interactions resulted in synergy. Compared to control ODN, ATL1101 significantly suppressed PtxR PC3 tumor growth as a combination therapy with paclitaxel in murine xenografts.
CONCLUSIONS: This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO suppresses growth of paclitaxel resistant PC tumors, synergistically enhancing paclitaxel sensitivity in vitro and in vivo.
4) Monday, May 16, 2011 3:30 PM-5:30 PM Prostate Cancer: Basic Research
1279: CLU INHIBITION USING OGX-011 IS A NEW ADJUVANT THERAPEUTIC STRATEGY FOR HSP90 INHIBITION IN PROSTATE CANCER.
Francois Lamoureux, Min-Jean Yin, Amina Zoubeidi, Martin Gleave. Vancouver, Canada
INTRODUCTION AND OBJECTIVES: Prostate cancer (PCa) responds initially to anti-androgen therapies, however, progression to castration resistant disease (CRPC) frequently occurs. Several small molecule inhibitors of HSP90 show promise in CRPC and other cancers. However, most HSP90 inhibitors (17-AAG or PF-04928473 and its prodrug PF-04929113) trigger the elevation of HSPs (HSP90, 70, 27 and clusterin), which lead to tumor cell survival and treatment resistance. We hypothesized that targeting clusterin (CLU) using siRNA or the antisense drug, OGX-011, may enhance HSP90 inhibitors-induced cell death in PCa. METHODS: Inducible and constitutive CLU and other HSP mRNA and protein levels were measured by real-time RT-PCR and immunoblot assays. The combination of OGX-011 with PF-04928473 or 17-AAG was evaluated in vitro on LNCaP and PC3 cells growth and apoptosis. The HSP90 inhibitor PF-04929113 was evaluated in combination with OGX-011 in vivo in athymic mice bearing castration resistant LNCaP xenografts, while the combination of OGX-011 with 17-AAG was evaluated in PC3 xenografts.
RESULTS: In prostate tumor cell lines, PF-04928473 and 17-AAG induced expression of HSPs in a dose and time dependent manner, and especially CLU at RNA and protein level, by increasing HSF-1 nuclear translocation and transcription activity. In vitro, OGX-011 synergistically enhanced the activity of HSP90 inhibitors on cell growth and apoptosis with increased sub-G1 fraction and PARP cleavage. These biologic events were accompanied by decreased expression of HSPs, Akt, AR and PSA, and HSF-1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 15mg/kg), potentiated the effect of orally administered HSP90 inhibitors (PF-04929113: 25mg/kg; 17-AAG: 50mg/kg), significantly inhibiting tumor growth by 80% and prolonging survival in PC3 and castrate resistant LNCaP xenograft model compared to the HSP90 inhibitors alone.
CONCLUSIONS: These results indicate that HSP90 inhibitor-mediated induction of CLU expression can be attenuated by OGX-011, with synergistic effects on delaying progression of CRPC.