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CAMKK2 Promotes Prostate Cancer Independently of AMPK Via Increased Lipogenesis Lucy Penfold1, Angela Woods1, Phillip Muckett1, Alexander Yu

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CAMKK2 Promotes Prostate Cancer Independently of AMPK Via Increased Lipogenesis Lucy Penfold1, Angela Woods1, Phillip Muckett1, Alexander Yu Published OnlineFirst September 21, 2018; DOI: 10.1158/0008-5472.CAN-18-0585 Cancer Metabolism and Chemical Biology Research CAMKK2 Promotes Prostate Cancer Independently of AMPK via Increased Lipogenesis Lucy Penfold1, Angela Woods1, Phillip Muckett1, Alexander Yu. Nikitin2, Tera R. Kent2, Shuai Zhang1, Rebecca Graham1, Alice Pollard1, and David Carling1,3 Abstract New targets are required for treating prostate cancer, parti- activation of AMPK reduced cell growth in human prostate cularly castrate-resistant disease. Previous studies reported cancer cells by inhibiting de novo lipogenesis. Activation of that calcium/calmodulin-dependent protein kinase kinase 2 AMPK in a panel of human prostate cancer cells inhibited (CAMKK2) expression is increased in human prostate can- cell proliferation, migration, and invasion as well as andro- cer. Here, we show that Camkk2 deletion or pharmacologic gen-receptor signaling. These findings demonstrate that inhibition protects against prostate cancer development in CAMKK2 and AMPK have opposing effects on lipogenesis, a preclinical mouse model that lacks expression of prostate- providing a potential mechanism for their contrasting specific Pten. In contrast, deletion of AMP-activated protein effects on prostate cancer progression in vivo.Theyalso kinase (Ampk) b1 resulted in earlier onset of adenocarci- suggest that inhibition of CAMKK2 combined with activa- noma development. These findings suggest for the first time tion of AMPK would offer an efficacious therapeutic strategy that Camkk2 and Ampk have opposing effects in prostate in treatment of prostate cancer. cancer progression. Loss of CAMKK2 in vivo or in human prostate cancer cells reduced the expression of two key Significance: These findings show that CAMKK2 and its lipogenic enzymes, acetyl-CoA carboxylase and fatty acid downstream target AMPK have opposing effects on prostate synthase. This reduction was mediated via a posttranscrip- cancer development and raise the possibility of a new com- tional mechanism, potentially involving a decrease in pro- bined therapeutic approach that inhibits CAMKK2 and acti- tein translation. Moreover, either deletion of CAMKK2 or vates AMPK. Cancer Res; 78(24); 6747–61. Ó2018 AACR. Introduction Two previous studies (4, 5) reported that calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) gene expression Prostate cancer is the most common cancer in men in the is upregulated at all stages of human prostate cancer, with United Kingdom and United States, and accounts for almost evidence that gene expression positively correlates with grade. 20% of all new cancer cases in men in the United States (1). Both studies provided convincing evidence that the CAMKK2 Prostate cancer development is thought to proceed through a gene is androgen responsive in human prostate cancer cell series of defined stages, including prostatic intraepithelial neo- lines, providing a potential direct molecular mechanism for plasia (PIN), adenocarcinoma, and metastatic cancer (2). Stan- upregulation of CAMKK2 in prostate cancer. Inhibition of dard therapies include androgen ablation therapy, and CAMKK2 was shown to block androgen-stimulated growth, although 80% of patients initially respond favorably, most migration, and invasion in vitro (4, 5), and CAMKK2 inhibition relapse within 1 to 2 years of developing castrate-resistant reduced tumor growth in a prostate cancer xenograft model disease (3). Given the high prevalence and mortality rates (5). CAMKK2 is one of two upstream kinases (the other being associated with prostate cancer, novel drug targets are needed. LKB1) that phosphorylates AMP-activated protein kinase (AMPK) on threonine 172 (T172) within the a subunit, leading to its activation (6, 7). AMPK is a key regulator of energy 1MRC London Institute of Medical Sciences, Imperial College London, Hammer- homeostasis in eukaryotic cells, and activation of AMPK leads smith Hospital, London, United Kingdom. 2Department of Biomedical Sciences to inhibition of protein and lipid synthesis, inhibiting cell 3 and Cornell Stem Cell Program, Cornell University, Ithaca, New York. Institute of growth, and so supporting the hypothesis that AMPK acts as Clinical Sciences, Imperial College London, Hammersmith Hospital, London, a tumor suppressor (8–12). However, there is also evidence United Kingdom. that suggests that under certain circumstances AMPK might Note: Supplementary data for this article are available at Cancer Research help cancer cells survive under adverse nutritional conditions Online (http://cancerres.aacrjournals.org/). and so support tumor growth (13–17). At the present time, the Corresponding Author: David Carling, Medical Research Council London Insti- role of AMPK in prostate cancer remains unclear, and whether tute of Medical Sciences, Hammersmith Hospital, Du Cane Road, London W12 AMPK is involved in mediating the downstream effects of 0NN, United Kingdom. Phone: 020-838-34313; E-mail: [email protected] CAMKK2 signaling in prostate cancer remains enigmatic. doi: 10.1158/0008-5472.CAN-18-0585 Here, we use a mouse model of prostate cancer in which the Ó2018 American Association for Cancer Research. tumor suppressor, Pten, is deleted specifically in prostate www.aacrjournals.org 6747 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 21, 2018; DOI: 10.1158/0008-5472.CAN-18-0585 Penfold et al. epithelial cells (18) in order to investigate the effect of washed with PBS-tween (0.1%) and incubated for 30 min- Camkk2 and Ampk in disease progression in vivo. This model utes with avidin–biotin complex (VECTASTAIN Elite ABC Kit, accurately recapitulates the different stages seen in the human Vector Laboratories) according to manufacturer's instructions. disease albeit on a more rapid time scale making it an excellent Sections were washed with PBS and stained using the DAB model for the human disease. Consistent with the findings Substrate Kit (Vector Laboratories) according to the manu- from human prostate cancer, Camkk2 expression is signi- facturer's instructions before counterstaining with Gill hemat- ficantly increased in mouse prostate following deletion of oxylin (Sigma). Sections were then dehydrated and mounted Pten. We show that CAMKK2 is required for driving protein using DPX mountant (Sigma). expression of lipogenic enzymes, leading to increased de novo lipogenesis in prostate cancer cells. Conversely, AMPK activa- In vivo studies with STO-609 tion inhibits de novo lipogenesis. Genetic deletion of Camkk2 Osmotic minipumps (Models 2004/2006, Alzet Osmotic in vivo slows prostate cancer development, whereas deletion Pumps) were filled with varying concentrations (6, 15, and of Prkab1 (the gene encoding Ampkb1) leads to earlier onset 30 mg/mL) of STO-609 (Enzo Life Sciences) in 200 mmol/L of adenocarcinoma. Our findings suggest that CAMKK2 and NaOH. Addition of STO-609 lowered the pH of the solution to AMPK have opposing effects on prostate cancer progression, approximately pH 12.8, making it compatible with the mini- mediated at least in part by their antagonistic effects on de novo pump specifications. As a vehicle control, 200 mmol/L NaOH lipogenesis. was adjusted to pH 12.8 by the addition of HCl. Minipumps were primed at least 48 hours before surgery and submersed in PBS at 37C for several hours to allow the pump to begin Materials and Methods operating before implantation. C57BL/6J male mice were anes- Animal models thetized with isoflurane (Abbott), the interscapular region All in vivo studies were performed in accordance with the shaved, and an osmotic minipumpinsertedsubcutaneously. United Kingdom Animals (Scientific Procedures) Act (1986) Pump model 2004 (flow rate of 0.25 mL/h; 4-week duration) and approved by the Animal Welfare and Ethical Review Board was used for the initial dosing studies in wild-type mice. Two at Imperial College London. All experimental animals were weeks after surgery, blood samples were collected into hepa- maintained on a C57BL/6J genetic background and fed a chow- rinized microvettes (Sarstedt), and the resulting plasma frac- standard breeding diet number 3 (Special Diets Services). Mice tion stored at À80C before analysis for STO-609 levels. After with prostate-specific deletion of Pten were generated by cross- 4 weeks, liver and prostate tissue was collected. Plasma and fl fl ing female Pten / mice (stock number 006440, Jackson Lab- tissue STO-609 accumulation was analyzed by mass spectrom- oratories) with male mice expressing cre-recombinase under etry as described previously by PK/Bioanalytics Core Facility, the control of a modified rat probasin promoter (Pbsn-Cre4; Cancer Research UK Cambridge Institute, UK. For the inter- stock number 026662, Jackson Laboratories). Mice with a vention studies in Pten-null mice, pump model 2006 (flow rate global deletion of Camkk2 (deletion of exon 5) were as of 0.15 mL/h; 6-week duration) was used. Pumps were filled described previously (19). Prkab1-floxed mice were generated with vehicle (200 mmol/L NaOH, pH adjusted to 12.8) or by crossing Prkab1tm1a(KOMP)Wtsi ["knockout first" mice gener- 30 mg/mL STO-609 in 200 mmol/L NaOH and implanted into ated by the trans-NIH Knock-Out Mouse Project (KOMP)] and male mice, aged 13 weeks. Six weeks after implantation, ani- obtained from the KOMP Repository (www.komp.org) with mals were sacrificed and prostates removed for subsequent mice expressing Flp-recombinase (stock number 003946, analysis. Jackson Laboratories).
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