Published OnlineFirst April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Cancer Therapeutics, Targets, and Chemical Biology Research NUAK2 Amplification Coupled with PTEN Deficiency Promotes Melanoma Development via CDK Activation Takeshi Namiki1,2,3, Tomonori Yaguchi2, Kenta Nakamura2,4, Julio C. Valencia1, Sergio G. Coelho1, Lanlan Yin1, Masakazu Kawaguchi1, Wilfred D. Vieira1, Yasuhiko Kaneko5, Atsushi Tanemura6, Ichiro Katayama6, Hiroo Yokozeki3, Yutaka Kawakami2, and Vincent J. Hearing1 Abstract The AMPK-related kinase NUAK2 has been implicated in number of cells in S phase. NUAK2 silencing and inactivation of melanoma growth and survival outcomes, but its therapeutic the PI3K pathway efficiently controlled CDK2 expression, where- utility has yet to be confirmed. In this study, we show how as CDK2 inactivation specifically abrogated the growth of its genetic amplification in PTEN-deficient melanomas may NUAK2-amplified and PTEN-deficient melanoma cells. Immu- rationalize the use of CDK2 inhibitors as a therapeutic strategy. nohistochemical analyses confirmed an association of CDK2 Analysis of array-CGH data revealed that PTEN deficiency is expression with NUAK2 amplification and p-Akt expression in coupled tightly with genomic amplification encompassing the melanomas. Finally, pharmacologic inhibition of CDK2 was NUAK2 locus, a finding strengthened by immunohistochemical sufficient to suppress the growth of NUAK2-amplified and evidence that phospho-Akt overexpression was correlated with PTEN-deficient melanoma cells in vitro and in vivo. Overall, our NUAK2 expression in clinical specimens of acral melanoma. results show how CDK2 blockade may offer a promising therapy Functional studies in melanoma cells showed that inactivation for genetically defined melanomas, where NUAK2 is amplified of the PI3K pathway upregulated p21 expression and reduced the and PTEN is deleted. Cancer Res; 75(13); 1–8. Ó2015 AACR. Introduction migrationinmelanomacells(2,5–14). The significance of NUAK2 in melanomagenesis is highlighted by the fact that high Recent advances in cancer genomics facilitate the elucidation expression of NUAK2 has an impact on the survival of patients of aberrant downstream pathways in tumor cells with genomic with acral melanomas, in addition to the fact that NUAK2 aberrations and pave the way to develop specifictherapiesfor participates in the regulation of cell proliferation of melanomas novel oncogenes and tumor-suppressor genes in many types of in general (12). On the other hand, synergistic effects of several cancers (1–4). In melanomas, several genomic aberrations, genomic aberrations are also quite important to facilitate such as mutations, amplifications, and deletions in BRAF, tumorigenesis of cancer cells such as that the PI3K pathway NRAS, INK4A, MITF, PREX2, GNAQ,andKIT,havebeen participates in melanomagenesis (15, 16). reported, and recent analyses using array-CGH data also sug- The elucidation of genomic aberrations, including mutations, gested that NUAK2, which resides at chromosome 1q32, is an has progressed using systematic approaches (17). However, important gene that regulates cell-cycle progression and cell detailed mechanisms controlling cell-cycle progression by NUAK2 and additional genes remain to be elucidated. Analyses of cell-cycle progression in NRAS-mutated and MITF-amplified 1Laboratory of Cell Biology, National Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland. 2Division of Cellular Signaling, melanomas showed that control of the cell cycle is differently Institute for Advanced Medical Research, Keio University School of regulated by CDKs in melanoma cells, where CDK4 is a key driver Medicine, Tokyo, Japan. 3Department of Dermatology, Tokyo Medical in NRAS-mutant melanomas, whereas CDK2 has a pivotal role in and Dental University Graduate School and Faculty of Medicine, melanomas with high expression of MITF (18–20) Those results Bunkyo-ku, Tokyo, Japan. 4Department of Dermatology, Shinshu Uni- versity School of Medicine, Matsumoto-shi, Nagano, Japan. 5Research imply that elucidation of mechanisms regulating the cell cycle by Institute for Clinical Oncology, Saitama Cancer Center, Kitaadachi, different genomic aberrations should reveal the different impact 6 Saitama, Japan. Department of Dermatology, Osaka University Grad- of CDKs on the cell cycle. uate School of Medicine, Suita-shi, Osaka, Japan. In melanomas, BRAF mutations have been identified as Note: Supplementary data for this article are available at Cancer Research activating mutations that facilitate melanomagenesis, and this Online (http://cancerres.aacrjournals.org/). discovery accelerated molecular targeted therapies against mela- Corresponding Author: Yutaka Kawakami, Keio University School of Medicine, nomas using drugs such as vemurafenib and dabrafenib (1, 2). 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3777; However, BRAF mutations have diverse discrepancies among Fax: 81-3-5362-9259; E-mail: [email protected] subtypes of melanomas (21). Some subtypes of melanomas, such doi: 10.1158/0008-5472.CAN-13-3209 as acral and mucosal melanomas, have low frequencies of BRAF Ó2015 American Association for Cancer Research. mutations and are speculated to respond poorly to those therapies www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Namiki et al. targeting BRAF mutations. Molecular targeted therapies aimed counted at days 0, 2, and4 after transfection of siRNA. For cell at genomic aberrations other than BRAF mutations should be number analyses treated with roscovitine, cells were seeded at 2.0 developed for the better management of patients with those  105 cells/well in 6-well plates. Cell numbers were counted at subtypes of melanomas. days 3, 5, and 7 after treatment with roscovitine. In this study, we explore additional genomic aberrations and For proliferation assays of C32 and SM-KT1 cells, cells were downstream pathways of NUAK2 and demonstrate that NUAK2 seeded at 1.0  105 cells/well in 24-well plates in quadruplicate. and the PI3K pathway coordinately control CDK2. In addition, we At 48 hours, cell proliferation was measured using the MTS assay showed that CDK2 is an efficient therapeutic target by abrogating according to the manufacturer's protocol (Takara Bio). the growth of cutaneous melanomas. For colony growth assays with roscovitine, cells were seeded at 1.0  105 cells/well (C32, mel2, and mel18) or 5.0  104 cells/ Materials and Methods well (A375, SKMel28, and SKMel23) in 6-well plates in triplicate. After treatment with roscovitine for 14 days, cells were fixed and Tumor specimens stained with crystal violet; measurements were performed at an We obtained 91 paraffin-embedded specimens of primary optical density of 610 nm. melanomas from three institutions. This study was approved by Cell-cycle profile analyses were performed as previously the Tokyo Medical and Dental University Research Committee, described (12). Cells were treated with LY294002 at 20 mmol/L the Osaka University Clinical Research Committee, and the Sai- for 24 hours. tama Cancer Center Research Ethics Committee. Fifty-six tumors were classified as acral melanomas and 35 as non-chronic sun- induced damage (CSD) melanomas, but none was a CSD mel- Animal model anoma according to the definition by Curtin and colleagues (22). All animal experiments were approved by the Animal Care and Use Committee of the Keio University. Of note, 3  106 C32 melanoma cells, 3.0  106 SM2-1 melanoma cells, and 3.0  106 Cell lines mel18 melanoma cells were injected subcutaneously into nude Normal human melanocyte and melanoma cell lines were mice (4 or 5 per group, as noted). Seven days after injection of cultured and maintained as previously described (23). C32, tumor cells, mice were orally treated with 2 mg/dose roscovitine A375, and Malme-3M melanoma cells were purchased from the (every day for 10 days). Tumor sizes were then measured at day 10 ATCC. SKMel28 and SKMel23 melanoma cells were kindly pro- of treatment. vided by the Surgery Branch, NCI/NIH (Bethesda, MD). SM2-1 melanoma cells were kindly provided by Dr. H. Murata (Shinshu University, Matsumoto, Japan). The Mel2 melanoma cell line was Immunoblotting established from a lymph node metastasis of a 68-year-old Immunoblotting was performed as previously described (20). Japanese male acral melanoma patient in 1998, and the mel18 Antibodies used included a rabbit monoclonal anti-phospho melanoma cell line was established from a lymph node metastasis (Ser473) Akt antibody (1:1,000; Cell Signaling Technology), a of a 51-year-old Japanese male acral melanoma patient in 1998 in rabbit polyclonal anti-NUAK2 antibody (1:1,000; Proteintech our laboratory, as described previously (24). C32, mel2, mel18, Group), a mouse monoclonal anti-actin antibody (1:1000; and SM2-1 melanoma cell lines were cultured in RPMI1640 Abcam), a rabbit polyclonal anti-CDK2 antibody (1:2,000; Santa supplemented with 10% heat-inactivated FBS, 100 IU/mL pen- Cruz Biotechnology), a rabbit monoclonal anti-CDK4 antibody icillin, and 100 mg/mL streptomycin at 37 Cina5%CO2 (1:1,000, Cell Signaling Technology), a mouse monoclonal anti- incubator. All other melanoma cells were cultured in DMEM with CDK6 antibody (1:500; Abcam), a rabbit monoclonal anti-p21 5% FBS. The original C32, A375, and Malme-3M melanoma cells antibody (1:1,000; Cell Signaling Technology), a rabbit