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Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up- Regulation of Nuclear Factor-␬B and Tumor Progression1

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Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up- Regulation of Nuclear Factor-␬B and Tumor Progression1 [CANCER RESEARCH 62, 7335–7342, December 15, 2002] Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up- Regulation of Nuclear Factor-␬B and Tumor Progression1 Punita Dhawan, Amar B. Singh, Darrel L. Ellis, and Ann Richmond2 Departments of Veterans Affairs [A. R.], Cancer Biology [P. D., A. R.], Nephrology [A. B. S.], and Medicine, Division of Dermatology [D. L. E.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232 ABSTRACT iting a cumulative melanoma risk of 1.9% compared with 0.04% of patients without dysplastic nevi (7). The current lifetime incidence The serine/threonine kinase Akt/protein kinase B and the pleiotropic of melanoma in the United States is estimated at 1:74 (8). The high transcription factor nuclear factor-␬B [NF-␬B (p50/p65)] play important incidence of dysplastic nevi and melanoma presents a large public roles in the control of cell proliferation, apoptosis, and oncogenesis. Pre- vious studies from our laboratory have shown the constitutive activation health problem. of NF-␬B in melanoma cells. However, the mechanism of this activation is Whereas histopathology is the current gold standard for the diag- not clearly understood. The purpose of this study was to explore the role nosis of atypical melanocytic lesions, interobserver correlation in the of Akt in the activation of NF-␬B during melanoma tumor progression. diagnosis of dysplastic nevi is variable (9, 10). The malignant poten- Based on our observation that two of the five melanoma cell lines exam- tial of dysplastic nevi and early melanomas is difficult to predict with ined exhibit constitutive Akt activation, we evaluated Akt activation by standard histological staining. Therefore, better techniques are de- immunohistochemistry in a series of pigmented skin lesions using an sired. Standard immunohistochemical markers such as HMB-45 have antibody specific for phospho-Akt Ser-473. Normal and slightly dysplastic not been useful in differentiating a dysplastic nevus from melanoma in nevi exhibited no significant Akt expression, in marked contrast to the situ (11). Some newer immunohistochemical markers such as Ki 67, dramatic Akt immunoreactivity seen in severely dysplastic nevi and mel- anomas (66.3% positive). When these same lesions were stained for nu- proliferating cell nuclear antigen, cyclin A, matrix metalloprotein- clear p65, a similar expression pattern was observed. In addition, inter- ase-2, osteonectin, p16, and gp100 may be helpful prognostically in ruption of Akt activation resulted in increased apoptosis and decreased some subgroups of melanoma patients (12). However, the molecular NF-␬B promoter activity. These results indicate that activation of Akt events that correlate with the progression of benign melanocytic nevi kinase is linked to enhanced NF-␬B nuclear localization and transactiva- to dysplastic nevi to malignant melanomas currently are not well tion. We propose that activation of Akt may be an early marker for tumor understood and are vital for clarifying ambiguities in histopathology progression in melanoma. and clinical prognosis as well as offering potential new therapeutic interventions. INTRODUCTION Many human solid tumor cell lines display increased nuclear NF- ␬B3 levels and/or increased NF-␬B transcriptional activity (13–15). The incidence of cutaneous melanoma has risen logarithmically in the NF-␬B is activated in Hodgkin’s lymphoma, head and neck squamous last few decades, and melanoma accounts for the most deaths from skin cell carcinoma, non-small cell lung cancer, colorectal cancer, thyroid cancer (1–3). Melanoma affects both relatively young and older popula- cancer, pancreatic carcinoma, leukemia, multiple myeloma, prostrate tions, metastasizes rapidly, and is highly resistant to chemotherapy and cancer, and breast cancer (16–18). Our laboratory has previously other treatments. To develop new diagnostic strategies, it is very impor- shown constitutive NF-␬B expression in melanoma cells (19). In most tant to understand the events involved in the progression from dysplasia nontransformed cell types, NF-␬B complexes (a heterotrimer com- to malignancy. The transition from benign lesions to invasive, metastatic posed of p50 and RelA/p65 subunits bound to an inhibitor, I␬B) are tumors occurs through a stepwise process involving changes in expres- largely cytoplasmic. On activation, the I␬B proteins become phos- sion and/or function of oncogenes or tumor suppressor genes, as well as phorylated, ubiquitinated, and subsequently degraded. This liberates constitutive expression of chemotactic cytokines, growth factors, and NF-␬B, allowing it to accumulate in the nucleus, where it enhances metalloproteinases (reviewed in Ref. 4). the transcription of specific genes. The I␬B kinase, or IKK complex Clinically, melanocytic lesions may be divided into benign mela- ␣ (Mr 700,000–900,000), consists of two catalytic units, IKK- and nocytic nevi (i.e., junctional nevi, compound nevi, and dermal nevi), IKK-␤, bound to a regulatory subunit IKK-␥ or NEMO (20–23). lesions with possible malignant potential (i.e., dysplastic nevi, con- Akt/PKB, a serine/threonine kinase, is a core component of the genital nevi, and lentigo maligna), and malignant lesions [i.e., primary PI3K signaling pathway that is activated through phosphorylation of melanoma such as superficial spreading melanoma, nodular mela- Ser-473/474 and Thr-308/309 (24). Several studies have shown that noma, lentigo maligna melanoma, and acral lentiginous melanoma as Akt/PKB activates the transcription of a wide range of genes, espe- well as metastatic melanoma (5, 6)]. For this study, we selected the cially those involved in immune activation, cell proliferation, apo- following types of clinical lesions to study melanocytic tumor pro- ptosis, and cell survival (25). Mechanisms associated with the ability gression: (a) benign melanocytic nevi; (b) dysplastic nevi; (c) lentigo of Akt to suppress apoptosis include the phosphorylation and inacti- maligna; and (d) metastatic melanomas. vation of many proapoptotic proteins such as Bad (26), caspase-9 The incidence of dysplastic nevi in the general population has (27), and the forkhead family of transcription factors (28) and acti- been estimated at 7%, with patients having dysplastic nevi exhib- vation of NF-␬B (29). Based on its role as a key regulator of cellular survival, Akt is Received 5/16/02; accepted 10/18/02. emerging as a central player in tumorigenesis. In the last decade, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with evidence from different studies suggests that Akt perturbations play 18 U.S.C. Section 1734 solely to indicate this fact. an important role in human malignancy. Akt1 and Akt2 amplification 1 Supported by a Career Scientist Award from the Department of Veterans Affairs (to A. R.), by National Cancer Institute Grants CA56704 (to A. R.) and CA34590 (to A. R.), and NIH Center Grants CA 68485 and P30 AR 41943. 3 The abbreviations used are: NF-␬B, nuclear factor-␬B; PKB, protein kinase B; PI3K, 2 To whom requests for reprints should be addressed, at Department of Cancer Biology, phosphatidylinositol 3Ј-kinase; GST, glutathione S-transferase; WM, wortmannin; RPE, Vanderbilt University School of Medicine, MCN T-2212, Nashville, TN 37232. Phone: retinal pigment epithelial; NHEM, normal human epidermal melanocyte; IKK, I␬B (615) 343-7777; Fax: (615) 343-4539; E-mail: [email protected]. kinase. 7335 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. ROLE OF AKT IN MELANOMA TUMOR PROGRESSION and/or mRNA overexpression was observed in human gastric cancer was used to visually capture the immunolocalization of antigen using Novared and 10–20% of human ovarian, breast, and pancreatic cancers, re- substrate, and cell contents were counterstained with hematoxylin. For immu- spectively (30, 31). Activation of the Akt2 kinase is reported in 40% nofluorescence, primary antibodies were used at the following dilutions: rabbit of ovarian cancers (32, 33), whereas Akt1 kinase activity is often anti p-Akt (1:50; Cell Signaling Technology); and mouse anti RelA/p65 increased in prostate and breast cancers (34). Amplification of AKT3 (1:100; Chemicon). All primary antibody incubations were performed in a moisture chamber overnight at 4°C. Secondary antibodies were donkey anti- has not been described. Furthermore, loss of the negative regulator of rabbit Texas red and goat antimouse Alexa 488. The slides were fixed with this pathway, the tumor suppressor PTEN, correlates with increased Vectashield and sealed with clear nail polish. Stained sections were viewed and Akt activity in many types of cancers (35, 36). photographed using the fluorescence microscope. The excitation wavelength The present challenge is to make sense of the network that controls was 549 nm for Texas red and was 488 nm for Alexa 488. apoptosis during melanoma tumorigenesis, which requires evaluating Measurement of Apoptosis. Subconfluent melanoma Hs294T, SKMel28, the Akt status of typical and atypical pigmented lesions, as well as cell and WM115 cells and normal RPE cells were transfected with either vector lines. Although high expression of Akt and/or Rel/NF-␬B has been control or Akt dominant negative constructs. Alternatively, cells were incu- demonstrated in several different tumors, the relevance of constitutive
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