[CANCER RESEARCH 64, 4912– 4918, July 15, 2004] Augmenting Chemosensitivity of Malignant Melanoma Tumors via Proteasome Inhibition: Implication for Bortezomib (VELCADE, PS-341) as a Therapeutic Agent for Malignant Melanoma Katayoun I. Amiri, 1,3 Linda W. Horton, 1 Bonnie J. LaFleur, 2 Jeffrey A. Sosman, 4 and Ann Richmond 1 Departments of 1 Veterans Affairs; Departments of Cancer Biology and 2 Biostatistics, Vanderbilt University Medical Center; 3 Department of Microbiology, Meharry Medical College; and 4 Division of Hematology/Oncology, Vanderbilt Ingram Cancer Center, Nashville, Tennessee ABSTRACT Melanoma poses a great challenge to patients, oncologists, and biolo- gists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor B is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor B activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor B by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1–10 nM and in combination with the chemotherapeutic agent temozo- lomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strik- ingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma. INTRODUCTION Melanoma is the most aggressive form of skin cancer and has increased 6-fold in incidence over the past 50 years. Metastatic disease is estimated to have caused 7600 deaths in 2003 and is the second cause of lost productive years among cancers (1, 2). Mela- noma is highly resistant to conventional chemotherapy with dacarba- zine or its derivative temozolomide (TMZ) having the best single agent activity with a response rate of only 15–20% and a short 4-month median response duration. At this time, no randomized clinical trial has shown a survival advantage to any other more complex chemotherapy and/or biotherapy regimens over single agent dacarbazine (3, 4). Thus, it is imperative to investigate new therapeu- tic targets for the treatment of melanoma to improve the dismal prognosis for this disease. One such important target identified in melanoma tumor progression is the nuclear factor B (NFB) path- way (5, 6). Constitutive activation of NFB is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma (7–12). In the healthy human, NFB regulates the expres- sion of genes involved in normal immunological responses (e.g., generation of immunoregulatory molecules such as antibody light chains) in response to proinflammatory cytokines and byproducts of microbial and viral infections (13–15). However, increased activation of NFB results in enhanced expression of proinflammatory media- tors, leading to acute inflammatory injury to lungs and other organs and development of multiple organ dysfunctions. NFB also modu- lates the expression of factors responsible for growth as well as inhibitors of apoptosis (13, 15, 16). There are five known mammalian NFB subunits, each character- ized by ankyrin repeat elements: (a) Rel (c-Rel); (b) p65 (RelA); (c) RelB; (d) p50; and (e) p52. The NFB protein is composed of two subunits, which may vary affecting the transcriptional activity of the protein. In the absence of activation, NFB complexes (homo- and heterodimers composed of above the mentioned subunits) are seques- tered in the cytoplasm because of their association with an inhibitor of B protein (IB). The IB protein binds to the nuclear localization signal of NFB Rel proteins, thereby inhibiting translocation of the complexes into the nucleus (13–15). When the cell is exposed to activating signals, such as tumor necrosis factor-, the IB protein is phosphorylated by IB kinase, ubiquitinated, and then broken down in the 26 S proteasome (17). This frees the NFB to translocate into the nucleus, where it binds to B sites in the promoter/enhancer regions of specific genes, including the promoter/enhancer for IB, to trans- activate transcription (13, 15, 17). Persistent activation of NFB inhibits apoptosis and promotes proliferation leading to hyperplasia (13, 16, 18, 19). Previous studies in our laboratory have shown an elevated basal IB kinase activity in Hs294T melanoma cells, which leads to an increased rate of IB phosphorylation and degradation. This increase in IB- phosphoryl- ation and degradation leads to an 19-fold higher nuclear localization of NFB (20). We have shown that this constitutive activation of NFB facilitates the immortalization and proliferation of melanocytes and provides a means to escape apoptosis (20 –23). These findings suggest that NFB may represent an effective molecular target in melanoma tumorigenesis. To date, many different strategies have been used to inhibit NFB activity in tumors with various degrees of success. We propose to use the target 26 S proteasome for inhibition of NFB activity in mela- nomas. Among proteasome inhibitors, bortezomib (VELCADE), for- merly known as PS-341, inhibits more specifically the chymotryptic enzyme activity of the proteasome. Bortezomib is a low molecular weight, water-soluble dipeptide that binds to the proteasome with very high affinity and dissociates slowly, imparting stable but reversible proteasome inhibition (24, 25). Bortezomib has shown great promise in the preclinical studies for cancers such as ovarian, lung, squamous cell carcinoma, prostate, and pancreatic (26 –30), and many clinical trials for the treatment of these cancers have been initiated (31, 32). More recently, bortezomib received accelerated approval from the United States Food and Drug Administration for the treatment of patients with refractory multiple myeloma who failed prior chemo- therapy (33), highlighting the potential effectiveness of the drug in the treatment of cancer. This is the first study to investigate the efficacy of bortezomib in melanoma cells and in a murine xenograft model of melanoma to inhibit NFB and, in turn, melanoma tumor progression. In particular, Received 2/24/04; revised 4/28/04; accepted 5/7/04. Grant support: Department of Veterans Affairs Career Scientist Award and Merit Award (A. Richmond), National Cancer Institute Grants CA34590 and CA56704 (A. Richmond), National Cancer Institute Grant CA68485 (Vanderbilt Ingram Cancer Center), Grant 5P30 AR41943 (Skin Disease Research Center), and Millennium Pharma- ceuticals. 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 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Ann Richmond, Department of Cancer Biology, 771 PRB, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-7777; Fax: (615) 343-4539; E-mail: Ann.Richmond@vanderbilt.edu. 4912