AR (androgen receptor) signaling is crucial for the development and maintenance of the prostate as well as the initiation and progression of prostate cancer. Despite the AR's central role in prostate cancer progression, it is still unclear which AR-mediated processes drive the disease. Here, we identified 4 core autophagy genes: ATG4B, ATG4D, ULK1, and ULK2, in addition to the transcription factor TFEB, a master regulator of lysosomal biogenesis and function, as transcriptional targets of AR in prostate cancer. These findings were significant in light of our recent observation that androgens promoted prostate cancer cell growth in part through the induction of autophagy. Expression of these 5 genes was essential for maximal androgen-mediated autophagy and cell proliferation. In addition, expression of each of these 5 genes alone or in combination was sufficient to increase prostate cancer cell growth independent of AR activity. Further, bioinformatic analysis demonstrated that the expression of these genes correlated with disease progression in 3 separate clinical cohorts. Collectively, these findings demonstrate a functional role for increased autophagy in prostate cancer progression, provide a mechanism for how autophagy is augmented, and highlight the potential of targeting this process for the treatment of advanced prostate cancer.

Previous work has suggested androgen receptor (AR) signaling mediates prostate cancer progression in part through the modulation of autophagy. However, clinical trials testing autophagy inhibition using chloroquine derivatives in men with castration-resistant prostate cancer (CRPC) have yet to yield promising results, potentially due to the side effects of this class of compounds. We hypothesized that identification of the upstream activators of autophagy in prostate cancer could highlight alternative, context-dependent targets for blocking this important cellular process during disease progression. Here, we used molecular, genetic and pharmacological approaches to elucidate an AR-mediated autophagy cascade involving Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CAMKK2; a kinase with a restricted expression profile), 5’-AMP-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1), but independent of canonical mechanistic target of rapamycin (mTOR) activity. Increased CAMKK2-AMPK-ULK1 signaling correlated with disease progression in genetic mouse models and patient tumor samples. Importantly, CAMKK2 disruption impaired tumor growth and prolonged survival in multiple CRPC preclinical mouse models. Similarly, an inhibitor of AMPK-ULK1 blocked autophagy, cell growth and colony formation in prostate cancer cells. Collectively, our findings converge to demonstrate that AR can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote prostate cancer by increasing autophagy. Thus, this pathway may represent an alternative autophagic target in CRPC.

33Previous work has suggested androgen receptor (AR) signaling mediates cancer 34 progression in part through the modulation of autophagy. Accordingly, we demonstrate 35 that chloroquine, an inhibitor of autophagy, can inhibit tumor growth in preclinical mouse 36 models of castration-resistant prostate cancer (CRPC). However, clinical trials testing 37 chloroquine derivatives in men with CRPC have yet to yield promising results, potentially 38 due to side effects. We hypothesized that identification of the upstream activators of 39 autophagy in prostate cancer could highlight alternative, context-dependent targets for 40 blocking this important cellular process during disease progression. Here, we used 41 molecular (inducible overexpression and shRNA-mediated knockdown), genetic 42 (CRISPR/Cas9), and pharmacological approaches to elucidate an AR-mediated 43 autophagy cascade involving Ca 2+ /calmodulin-dependent protein kinase kinase 2 44 (CAMKK2; a kinase with a restricted expression profile), 5'-AMP-activated protein kinase 45 (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1). These findings are 46 consistent with data indicating CAMKK2-AMPK-ULK1 signaling correlates with disease 47 progression in genetic mouse models and patient tumor samples. Importantly, CAMKK2 48 disruption impaired tumor growth and prolonged survival in multiple CRPC preclinical 49 mouse models. Finally, we demonstrate that, similar to CAMKK2 inhibition, a recently 50 described inhibitor of AMPK-ULK1 signaling blocked autophagy, cell growth and colony 51 formation in prostate cancer cells. Taken together, our findings converge to demonstrate 52 that AR signaling can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote 53 prostate cancer by increasing autophagy. Further, we propose that an inhibitor of this 54 signaling cascade could serve as an alternative, more specific therapeutic compared to 55 4 existing inhibitors of autophagy that, to date, have demonstrated limited efficacy in clinical 56 trials due to their toxicity and poor pharmacokinetics. 57 58 Introduction 59Prostate cancer is the second leading cause of cancer mortality among men in the United 60States(1). While most prostate cancers can be treated effectively with surgery and/or 61 radiation, a significant number of men present with de novo metastatic disease or 62 progress following initial treatment. The standard of care for advanced prostate cancer is 63 androgen deprivation therapy (ADT) due to the central role of the androgen receptor (AR) 64 in almost all prostate cancers(2). Although ADT is initially effective in slowing the cancer, 65 it invariably fails within 2-3 years, after which the disease progresses to a stage referred 66 to as castration-resistant prostate cancer (CRPC). There is currently no cure for CRPC. 67Interestingly, despite the failure of ADT in CRPC, the overwhelming majority of prostate 68 cancers are still driven by AR as a result of a variety of AR reactivation mechanisms (ex. 69 increased intratumoral androgen synthesis, AR gene and...

Objective: To correlate ultrasound characteristics of spastic muscles with clinical and functional measurements in chronic stroke survivors.Methods: Ultrasound assessment and clinical and functional assessments were performed in 28 ambulatory stroke survivors (12 females, mean age 57.8 ± 11.8 years, 76 ± 45 months after stroke).Results: Muscle thickness in the affected side was decreased compared with the contralateral side (p < 0.001). The decrease was more evident in the upper limb muscles. On the affected side, the Heckmatt scale score was lowest (closer to normal) in the rectus femoris (RF) muscle compared with other muscles (biceps brachii (BB), flexor carpi ulnaris (FCU) and medial gastrocnemius (MG)). Muscle thickness and echogenicity of spastic muscles did not correlate with spasticity, as measured with the modified Ashworth scale (MAS), Fugl-Meyer motor assessment scores, age, or time since stroke. There was a significant negative correlation between grip strength and percentage decrease in muscle thickness for the spastic FCU muscle (r = –0.49, p = 0.008). RF muscle thickness correlated with ambulatory function (Timed Up and Go test (r = 0.44, p = 0.021) and 6-metre walk test (r = 0.41, p = 0.032)). There was no significant correlation between echogenicity and functional assessments Conclusion: Ambulatory chronic stroke survivors had function-dependent changes in muscle thickness on the affected side. Muscle thickness and echogenicity of spastic muscles did not correlate with spasticity, Fugl-Meyer motor assessment scores, age, or time since stroke.