Withaferin A activates TRIM16 for its anti-cancer activity in melanoma

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease.

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease.
Malignant melanoma is the deadliest cutaneous neoplasm. Acquired drug resistance frequently develops after a period of objective tumor response, justifying the need for novel therapies 1,2 . Withaferin A (WFA), a steroidal lactone extracted from Withania somnifera, has been described as a potential anti-cancer drug both in vivo and in vitro 3,4 through its diverse anti-tumour properties and low cytotoxicity to non-malignant cells. WFA is a promising therapeutic agent for a broad range of cancers, however its mechanism of action is understudied. Recent research has demonstrated a number of possible mechanisms of action for WFA, such as direct inhibition of the intermediate filament vimentin 5,6 . Inhibition of vimentin reduces formation of metastasis in pre-clinical models of breast cancer, osteosarcoma and lung cancer, melanoma and hepatocellular carcinoma [7][8][9][10][11] . We have reported 12 that the expression of TRIM16 (tripartite motif 16) is significantly reduced during normal skin transition to squamous cell carcinoma. We have also shown that TRIM16 acts as a tumour suppressor and reduces cell motility via down-regulation of vimentin expression 12 . We have also shown that high TRIM16 protein expression is associated with favourable outcome in melanoma patients with stage III disease 13 . Moreover, suppression of TRIM16 expression increased migration of normal human epidermal melanocytes, while overexpression of TRIM16 reduced melanoma cell migration and proliferation 13 . The above detailed evidence suggested that increased TRIM16 expression is a potential molecular target for the treatment of melanoma.

WFA has increased cytotoxicity for melanoma cells compared with fibroblast cell lines.
To determine whether WFA had selective cytotoxicity to melanoma cells over non-malignant cells, WFA was screened at a range (0-5 μM) of concentrations for its effects on cell viability (Fig. 1A) against five melanoma cell lines (MelCV, MelJD, G3601, A375 and MM200) compared with normal human lung fibroblasts (MRC-5 and WI-38). WFA demonstrated marked single agent activity against three of the five melanoma cell lines (MelJD, MelCV, G361) and reduced toxicity toward normal fibroblasts (Fig. 1A). Comparison of overall IC50s of the melanoma cell lines and fibroblast cells (Fig. 1B) showed a significant increase in cytotoxicity for melanoma cells. WFA also exerted a concentration-dependent anti-proliferative effect (Fig. 1C) measured by the BrdU cell proliferation assay in the case of MelJD and MelCV melanoma cells when compared to fibroblasts. Anti-proliferative actions developed at much lower WFA concentrations (Fig. 1C). To determine the effect of WFA treatment on WFA is partially dependent on TRIM16 for its cytotoxic activity. To evaluate whether induction of TRIM16 expression was essential for WFA to exert its effect on melanoma cell survival, we silenced TRIM16 gene expression using two TRIM16-specific siRNAs ( Fig. 4A and Supplementary Fig. 2) in MelJD and MelCV melanoma cells, followed by treatment with WFA. As expected, WFA treatment significantly reduced cell viability of both MelJD and MelCV cells (Fig. 4B), when compared to DMSO treated siControl (Vehicle) cells. Conversely, we found that knockdown of TRIM16 using TRIM16-specific siRNAs blocked the reduction of cell viability induced by WFA treatment in both MelJD and MelCV cells ( Fig. 4B) when compared to DMSO treated siControl (Vehicle) cells. BrdU cell proliferation ( Fig. 4C) and colony formation assays (Fig. 4D, E) also showed that while WFA significantly reduced both short and long-term proliferation of both MelJD and MelCV cells, knockdown of TRIM16 rescued the cells from the effects of WFA. Furthermore, WFA treatment significantly decreased the survival of MelJD and MelCV cells as measured by their mitochodrial membrane potential ( Fig. 4F), however, cells transfected with TRIM16 specific siRNAs were less sensitive to WFA treatment. We further assessed the effects of TRIM16 expression on necrotic events (SYTOX Green accumulation assay) induced by WFA. We found that in line with our cell viability data, WFA induced necrosis in WFA treated melanoma cells (Fig. 4G), however, knockdown of TRIM16 rescued the cells from the cytotoxic effects induced by WFA.

Discussion
Although, kinase inhibitors and immunotherapies using checkpoint inhibitors have greatly improved survival of melanoma patients with advanced disease, treatment failure still represents a major clinical issue 1,2 . Hence, there is an urgent need to introduce novel therapeutics for the treatment of melanoma. The therapeutic effects of WFA in regulating cell survival, migration, angiogenesis, proliferation and metastasis in many types of cancers 14 has drawn more attention toward WFA as an anti-cancer compound. WFA induces mitochondrial dysfunction as well as apoptosis in leukaemia cells 15 , melanoma cells 16 and breast cancer cells 17 . WFA was also found to prevent angiogenesis by binding to vimentin filaments 5 and nestin 18 . Although these studies have shown that WFA is an effective anti-cancer compound for a variety of cancers, the molecular mechanism of WFA's drug action in melanoma is not fully understood. The objective of the present study was to further investigate the anti-cancer potential of WFA against human melanoma and to decipher the molecular mechanisms involved.
Although the effect of WFA on melanoma cell viability 16 and proliferation 19,20 has been investigated before, in this study, we conducted a thorough investigation using a wide range of melanoma cell lines, as well as normal fibroblasts to investigate the therapeutic window and concentration range for WFA to be administered without the risk of adverse side-effects. Previous studies have shown that WFA administration inhibits in vivo growth of a variety of tumor xenografts 17,19,21 including uveal melanoma 19 , as well the sensitizing effect of WFA of B16F1 www.nature.com/scientificreports/ melanoma cells to radiotherapy 22 . Further investigation using preclinical animal models to test WFA efficacy for melanoma is a natural extension to our current study. G2/M cell cycle arrest and apoptosis induction following treatment with WFA has been reported for melanoma 16 and uveal melanoma 19 . In melanoma cell lines, the apoptotic process triggered by WFA involved the mitochondrial pathway and was associated with Bax mitochondrial translocation, cytochrome-c release, transmembrane potential changes, and caspase 9 and caspase 3 activation. WFA cytotoxicity may also require www.nature.com/scientificreports/ early reactive oxygen species (ROS) production 16 . Consistent with this data, both melanoma cell lines in our study had a concentration-dependent increase in apoptosis. Interestingly, MelJD cells (BRAFWT) were more sensitive to the cytotoxic effect of WFA compared to MelCV cells, suggesting that BRAF mutation status might regulate sensitivity to WFA. A more intensive investigation is required to understand the link between WFA sensitivity and BRAF mutational status both in vitro and in vivo.
Here, we have shown that WFA treatment induced TRIM16 mRNA expression in melanoma cell lines and that TRIM16 was required to induce maximal cytotoxic effect. We found that MelCV melanoma cells were less sensitive to WFA treatment in comparison to MelJD cells. Interestingly, MelCV cells showed a lower basal TRIM16 expression compared to the MelJD cells 13 . We hypothesize that MelCV cells may be intrinsically less sensitive to WFA treatment due to pre-existing lower basal of TRIM16 and that the apoptotic action of TRIM16 may be suppressed by other means in these cells. The mechanism by which TRIM16 expression is lost in melanoma cells is currently unknown. Previous data indicated multiple mechanisms in neuroblastoma, including promoter methylation and reduced protein stability 23 , similar dysregulation may occur in melanoma. We showed that with increasing WFA concentration, the induction of TRIM16 mRNA expression was only mild, suggesting it is also possible that WFA can act by other regulatory mechanisms such as post-translational modifications that increase the stability or inhibit proteosomal degradation of TRIM16. The latter hypothesis was supported by research that shows WFA can inhibit the proteasome, the site of TRIM16 degradation 12,24 .
WFA treatment effectively reduced the growth of 4T1 mouse mammary tumor xenografts 11 . Growth inhibition was also accompanied by degradation of vimentin 11 . WFA was also found to prevent angiogenesis by binding to the intermediate F-actin and vimetin filaments 5 and nestin 18 . It has been previously described that TRIM16 bound directly to cytoplasmic vimentin 23 . We hypothesize that the inhibition of this interaction is necessary for the action of WFA on melanoma cells. Stage III melanoma patients with lymph node metastases have high TRIM16 expression with a longer median survival (59 months) compared to patients with low TRIM16 expression (16 months) 23 , therefore we investigated the effect of WFA on melanoma cell migration. Consistent with studies conducted in breast cancer cell lines 11 , our migration assay results demonstrated that WFA inhibited melanoma cell migration. Therefore, we suggest that WFA treatment and upregulation of TRIM16 expression could be a potential method to prevent disease progression and serve as maintenance therapy for Stage II melanoma patients.
Despite promising in vitro data summarized in this article, several steps are still necessary to introduce WFA for prevention and/or therapy of melanoma. Studies on WFA efficacy in vivo would further validate this compound as a candidate for therapeutic development. Second, more intensive investigation is required to understand the link between WFA sensitivity and BRAF mutational status. Last, it is unknown whether TRIM16 reactivation by WFA potentiate another targeted anti-melanoma therapy.

Materials and methods
Cell culture. Melanoma cell lines, MelJD, MelCV and MM200 were kindly gifted from Professor Xu Dong Zhang at the University of Newcastle (Newcastle, NSW, Australia). Melanoma cell line G361 and A375 were purchased from ATCC. All melanoma cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (LIFE TECHNOLOGIES Australia, VIC, Australia) with 5% fetal calf serum (FCS) (LIFE TECHNOLOGIES). MRC-5 and WI-38 normal human fibroblasts were purchased from ATCC and grown in alpha-minimum essential media (MEM) (LIFE TECHNOLOGIES) supplemented with 10% heat inactivated FCS. All cells were freshly thawed from initial seeds, cultured at 37 °C/5% CO 2 in a humidifier incubator for not more than 2 months.  The cytotoxic effects of WFA treatment were determined by SYTOX Green staining (LIFE TECHNOLO-GIES). Melanoma cells (2 × 10 4 cells per well) were cultured in 96-well plates and treated with WFA for 48 h. Supernatants were then discarded, and the cells were incubated with SYTOX Green working solution (30 nM/ well) for 30 min. The fluorescence of SYTOX Green was measured at 490-nm excitation and 520-nm emission wavelengths using VICTOR Multilabel counter (PERKINELMER).
Cell migration assay. MelJD and MelCV cells were serum starved prior to seeding into a trans-well insert (BD BIOSCIENCES) along with WFA. Trans-well inserts were placed in a companion plate with 5% FCS media as chemo-attractant, plates were then incubated for an additional 18 h. After incubation, cells were fixed with methanol, and stained with May-Grunwald (SIGMA-ALDIRCH, Australia) and Giemsa Stain (SIGMA-ALDRICH). Data was generated by counting cells under microscope (OLYMPUS, × 20 objective). Migration index was calculated by dividing migrated cell with total number of cells in wells.

RNA isolation and quantitative real-time PCR (RT-qPCR).
Total RNA was extracted using the PURELINK RNA kit (LIFE TECHNOLOGIES) according to the manufacturer's protocol. 1 μg total RNA was reverse-transcribed using the TETRO cDNA synthesis kit (BIOLINE, Australia). 1 μL of purified cDNA (0.1-1 μg) was added to a reaction mix containing, 2.5 μL 10× PCR buffer, 1.5 μL 25 mM MgCl, 2.5 μL of 10 mM dNTPs, 0.5 μL GOLD TAQ (INVITROGEN) and 1 μL of each of forward and reverse TRIM 16 primers (10 nM): Forward CAG GCT CCA GGC TAA CCA AAAG and Reverse TCC TCT AAG AAG GGC ATC ACA TTG . Gene expression was verified using Power SYBR GREEN MIX (APPLIED BIOSYSTEMS, LIFE TECHNOLO-GIES) PERFORMED ON ABI7500 THERMO-CYCLER (APPLIED BIOSYSTEMS, LIFE TECHNOLOGIES) with a standard protocol. Differential gene expression was measured using the log2∆∆Ct analysis. All mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Statistical analysis. Data were analysed with Prism 7 software (GRAPHPAD) and results are presented as the mean ± SEM. All statistics were based on continuous variables. For single comparisons, differences were determined by using a two-tailed, unpaired Student's t-test with a confidence interval (CI) of 95%. For multiple comparison one-way ANOVA was used. P ≤ 0.05 was denoted as statistically significant. Drug dose-response curves were analysed with a nonlinear regression curve fit model. The p values are as indicated on images. Analyses were not performed in a blinded manner.