www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 20 TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells Selina K. Sutton1, Jessica Koach1, Owen Tan1, Bing Liu1, Daniel R. Carter1, James S. Wilmott2, Benafsha Yosufi2,3, Lauren E. Haydu3, Graham J. Mann2,3, John F. Thompson3, Georgina V. Long2, Tao Liu1, Grant McArthur4,5, Xu Dong Zhang6, Richard A. Scolyer2,3, Belamy B. Cheung1, Glenn M. Marshall1,7 1Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia 2Melanoma Institute Australia and the University of Sydney, Sydney, Australia 3 Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney and Discipline of Pathology, Sydney Medical School, The University of Sydney, Australia 4 Molecular Oncology Laboratory, Oncogenic Signaling and Growth Control Program, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia 5Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3010, Australia 6Priority Research Centre for Cancer Research, Oncology and Immunology Unit, University of Newcastle, NSW, Australia 7Kids Cancer Centre, Sydney Children’s Hospital, Sydney, Australia Correspondence to: Glenn M. Marshall, e-mail: [email protected] Belamy B. Cheung, e-mail: [email protected] Keywords: Melanoma, TRIM16, BRAF inhibitor, cell migration, IFNβ1 Received: July 29, 2014 Accepted: September 07, 2014 Published: November 03, 2014 ABSTRACT High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16- dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. INTRODUCTION median survival of only 6–9 months historically [2, 3]. Metastatic melanoma is highly resistant to traditional Melanoma is an aggressive malignancy which cytotoxic chemotherapies [3, 4]. New targeted melanoma is responsible for 80% of skin cancer deaths [1]. While treatments, such as BRAF inhibitors, vemurafenib surgical excision can cure localized disease, metastatic (PLX4032) and dabrafenib (GSK2118436) [5, 6] offer melanoma treated with conventional therapies has a higher patient response rates due to specific BRAFV600 www.impactjournals.com/oncotarget 10127 Oncotarget targeting [4, 5]. However, disease progression is usually poor prognosis in melanoma patients with lymph node observed after a median of 5–7 months, highlighting the metastasis, collectively indicating that TRIM16 represents need for a better understanding of disease pathogenesis [7]. a novel therapeutic target in melanoma. Combination of dabrafenib and trametinib is now the standard of care for BRAFV600 patients, with 50% of patients experiencing a progression free survival of RESULTS 9–10 months [8]. The tripartite motif (TRIM) family of proteins TRIM16 protein expression is reduced in are characterized by a RING B-box-coiled-coil protein melanoma cell lines domain architecture and have been implicated in the pathogenesis of multiple cancers, functioning as either We first analyzed the TRIM16 protein expression oncogenes or tumor suppressors [9, 10]. TRIM16 has a level by Western blotting in 6 melanoma cell lines, role in innate immune function [11] and is secreted by and showed a significantly lower level, compared to a keratinocytes in a caspase-1 dependent manner, a process normal human epidermal melanocyte cell line (NHEM) enhanced by interleukin-1β (IL-1β). TRIM16 increased (Fig. 1A). The two protein bands at 67kDa and 64kDa differentiation markers in keratinocytes and was highly on the Western blot using the TRIM16-specific antibody expressed in basal keratinocytes, but was down-regulated represent two isoforms of the TRIM16 protein caused by during the proliferative phase of wound healing [12]. differential post-translational modification [12, 28]. The TRIM16 expression was significantly reduced in vivo proteasome inhibitor MG-132 increased TRIM16 protein during the progression from normal skin to squamous cell levels in melanoma cells (Supplementary Fig. 1A). The carcinoma (SCC), and inhibited SCC cell migration in TRIM16 protein half-life in melanoma cell lines, A375 vitro [13]. In addition, TRIM16 down-regulated protein- and Mel-CV, was 12 hours and 6 hours, respectively, binding partners, cytoplasmic vimentin and nuclear compared with more than 24 hours in NHEM and normal E2F1, in neuroblastoma cells [14]. Together these data human fibroblasts (WI38) (Fig. 1B and Supplementary suggested that TRIM16 repressed cancer cell replication Fig. 1B). and migration. However, the role of TRIM16 in melanoma To determine whether loss of TRIM16 expression is unknown. was also due to DNA methylation, we treated NHEM, The regulatory inflammatory cytokine, interferon WI38 and four melanoma cell lines with the demethylating beta 1 (IFNβ1) has been used in the therapy of agent, 5-aza-2’-deoxycytidine (5-Aza), at 30 µM for melanoma [15]. The human interferon gene region 72 hours. TRIM16 protein levels were significantly (containing Interferon-β1 (IFNβ1)) is deleted in numerous increased in the four melanoma cell lines following 5-Aza cancers, including melanoma [16, 17]. In melanoma, treatment (P=0.018 for Mel-JD, P=0.025 for Mel-RM, IFN-β gene transfer induced cell death in vitro [18–20] P=0.011 for A375 and P=0.0001 for Mel-CV), compared and suppressed melanoma cell proliferation [21, 22]. Both with normal cells (Fig. 1C). TRIM16 transcription IFN-α2b and IFN-β1a inhibited tumor growth and lymph also increased following 5-Aza treatment (Fig. 1D) in node metastasis in human melanoma xenografts [23]. Of melanoma cell lines (P=0.05), indicating direct or indirect all interferons, human IFNβ demonstrated the highest de-repression of the TRIM16 gene transcription upon anti-proliferative activity against human melanoma cell treatment with the demethylating agent. lines [20]. In contrast to IFN-2α, IFN-β bound with higher We next performed DNA sequencing of the affinity to the IFN α/β receptor 1 (IFNAR1), and induced endogenous TRIM16 coding and promoter regions for a greater degree of apoptosis in melanoma cells [15]. 9 melanoma cell lines. Melanoma cell lines IPC-298, IFNβ may also have some clinical activity in patients with MM200, A375, G361, SK-Mel-2, Mel-CV, Mel-JD, resected high risk melanoma [24]. Transcription of IFN-β Mel-RM, and CHL-1 were sequenced. We found an requires the formation of the multi-protein enhanceosome E121D missense mutation in exon 1 of IPC-298, in the complex [25, 26]. The c-Jun/ATF-2 heterodimer was B-Box1 domain of the TRIM16 protein in 1/9 melanoma essential for the formation of the enhanceosome complex, cell lines (Fig. 1E). The E121D mutation has also been and hence, IFN-β transcription [27]. reported in a publicly available database, catalogue of We used human melanoma cell lines and excised somatic mutations in cancer (COSMIC), in 1/71 stomach human melanoma samples to show that loss of TRIM16 cancers [29]. In addition, a missense mutation at V123M expression led to enhanced cell migration in vitro, was reported among 1/31 primary melanomas.[29] The and metastatic disease in vivo. The BRAF inhibitor, B-box1 domain of TRIM protein, MID1, structurally vemurafenib, suppressed melanoma cell growth in a resembles the ring domain of E3 ubiquitin ligases.[30] TRIM16-dependent manner. Overexpression of TRIM16 TRIM16 has a 46% sequence similarity with MID1, can in melanoma cells up-regulated IFNβ1 and c-Jun levels, adopt ring domain-like folds and has E3 ligase function in which was required for the anti-proliferative TRIM16 vitro and in vivo [31], indicating that mutation in TRIM16 effect. Lastly, low TRIM16 expression associated with B-box1 may perturb E3 ligase activity. www.impactjournals.com/oncotarget 10128 Oncotarget Figure 1: TRIM16 protein expression and half-life is decreased in melanoma cell lines compared to NHEM and is increased following treatment with the demethylating agent, 5-Aza. (A) Immunoblotting analysis of TRIM16 protein expression in NHEMs and 6 melanoma cell lines. Whole cell lysates were prepared and Western blots were probed with an anti-TRIM16 antibody. The Western blot was also probed with anti-β-actin as a loading control. (B) TRIM16 protein half-life was analysed by Western blotting in melanoma cell lines (Mel-CV, A375), NHEM and WI-38 human