On the basis of our genetic findings that

On the basis of our genetic findings that hexokinase 2 (HK2)-mediated aerobic glycolysis, known as the Warburg effect, drives prostate tumor growth in xenograft model bearing /p53-deficient murine or human prostate cancer myeloperoxidase (Wang et al., 2014), we designed preclinical studies to determine the therapeutic efficacy on /p53-deficiency-driven prostate tumorigenesis by pharmacologically co-targeting HK2-mediated Warburg effect and ULK1-dependent autophagy in genetic mouse models. We report here that pharmacologic inhibition of HK2 enzymatic activity with 2-deoxyglucose (2-DG) phosphorylates AMPK which inhibits mTORC1-S6K1 translation axis to preferentially reduce anti-apoptotic protein MCL-1 synthesis to prime intrinsic apoptosis while simultaneously induces ULK1-dependent pro-survival autophagy to counteract the apoptotic action of anti-Warburg effect. Accordingly, co-targeting HK2-mediated aerobic glycolysis with 2-DG and ULK1-driven autophagy with chloroquine (CQ) selectively kills cancer cells through apoptosis to cause significant tumor regression in xenograft, leads to near-complete tumor suppression and significantly extends survival in /p53-deficiency-driven CRPC mouse models. Thus, our preclinical studies suggest an innovative and efficacious therapeutic strategy for the subsets of currently incurable CRPC carrying PTEN and TP53 mutations.
Results
Discussion Resistance to the second generation anti-AR axis therapy is a serious therapeutic hurdle for CRPC patients (Claessens et al., 2014). Here we report that loss of Pten and p53 in prostate epithelial cells, occurring in 20–30% of human CRPC, leads to de novo murine CRPC that is completely resistant to second generation ADT. In contrast, mechanism-driven co-targeting HK2-mediated Warburg effect with 2-DG and ULK1-dependent autophagy with CQ markedly kill such CRPC cells to suppress tumor growth by selectively inducing apoptosis in multiple independent yet complementary preclinical models. AMPK is a highly conserved Ser/Thr protein kinase complex that sits at a central node in maintaining cellular energy homeostasis by activating catabolic metabolism and inhibiting anabolic metabolism in response to metabolic stress (Hardie et al., 2012; Oakhill et al., 2012). We have shown here that pharmacological or genetic inhibition of HK2 causes AMPK phosphorylation at Thr 172, an indicator of activated AMPK, which might be due to reduction of ATP ((Patra et al., 2013; Wu et al., 2015). However, the role of activated AMPK in tumorigenesis is less clear, as both oncogenic and tumor suppressor functions have been reported (Liang and Mills, 2013). During cancer treatment, the role of AMPK also remains controversial (Liang and Mills, 2013). The possible reasons might be that catalytically active AMPK phosphorylates a plethora of substrates that transduce multiple signals for various cellular responses associated with tumor development and cancer therapy. Our mechanistic studies revealed that activated AMPK functions as a double-edged sword in prostate cancer cells in response to 2-DG-induced metabolic stress. Activation of AMPK induces ULK1-dependent autophagy to protect cells from apoptosis while concomitantly inhibits mTORC1 signaling to reduce MCL-1 protein synthesis to prime mitochondria for apoptosis. These findings provide a strong theoretical rationale for precision targeting of AMPK downstream ULK1-mediated autophagy, rather than AMPK itself, to achieve desired therapeutic outcome. Our studies provided compelling evidence to support that pharmacologic co-targeting HK2-mediated Warburg effect with 2-DG and ULK1-induced autophagy with CQ could be an efficacious treatment for /p53-deficiency-driven CRPC in vivo. Whereas monotherapy with 2-DG or CQ inhibits prostate tumor growth by reducing cell proliferation, combination therapy with 2-DG and CQ causes significant tumor regression by inducing robust cancer cell apoptosis in xenograft models carrying /p53-deificient mouse and human prostate cancer cells and in a spontaneous prostate cancer mouse model. Notably, the combination treatment selectively kills cancer cells by apoptosis while sparing normal prostate epithelial cells. This selective cytotoxicity towards /p53-deficient CRPC cells is due to their addiction to HK2-mediated Warburg effect for tumor growth (Wang et al., 2014) and their dependence on ULK1-induced autophagy for cell survival when HK2 glycolytic enzyme activity is inhibited.