TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells.
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 (IFN?1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFN?1 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.
Project description:There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease.
Project description: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.
Project description:Recent data implicate elevated transforming growth factor-? (TGF?) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGF? signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGF?/TGF? receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.
Project description:In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF).Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression.Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1?) and IL-1? in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1?/? significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs.This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.
Project description:Melanoma is a type of malignant tumor derived from melanocytes, most of which occur in the skin, and a few occur in the mucosa and choroid. BRAF mutations occur in approximately 50% of melanoma patients. Vemurafenib is a specific and potent BRAF inhibitor that significantly prolongs progression-free survival in patients with BRAF mutant melanoma. But most patients have tumor recurrence after 7-9 months. Drug resistance severely limits the long-term clinical effects of targeted drugs. To explore the mechanism of melanoma resistance to Vemurafenib, the transcripts of Vemurafenib-resistant melanoma A375R cells and the parental A375 cells were sequenced. For more insight please see Transcripts 202 and 205 of IL-6 confer resistance to Vemurafenib by reactivating the MAPK pathway in BRAF(V600E) mutant melanoma cells . RNA-seq data has been uploaded to Sequence Read Archive (SRA), which allows researchers to obtain RNA sequence data for these cells.
Project description:Neuroblastoma is the most common solid tumor in childhood and represents 15% of all children's cancer deaths. We have previously demonstrated that tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has significant effects on neuroblastoma proliferation and migration in vitro and tumorigenicity in vivo. However, the mechanism by which this putative tumor suppressor influences cell proliferation and tumorigenicity was undetermined. Here we show, for the first time, TRIM16's striking pattern of expression and dynamic localization during cell cycle progression and neuroblastoma tumor development. In a tyrosine hydroxylase MYCN (TH-MYCN) neuroblastoma mouse model, immunohistochemical staining revealed strong nuclear TRIM16 expression in differentiating ganglia cells but not in the tumor-initiating cells. Furthermore in vitro studies clearly demonstrated that during G 1 cell cycle phase, TRIM16 protein expression is upregulated and shifts to the nucleus of cells. TRIM16 also plays a role in cell cycle progression through changes in Cyclin D1 and p27 expression. Importantly, using TRIM16 deletion mutants, an uncharacterized protein domain of TRIM16 was found to be required for both TRIM16's growth inhibitory effects and its nuclear localization. Taken together, our data suggest that TRIM16 acts as a novel regulator of both neuroblastoma G 1/S progression and cell differentiation.
Project description:Retinoid therapy is used for chemo-prevention in immuno-suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid-sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid-sensitive PHK cells, but not in retinoid-resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C-terminal RET finger protein (RFP)-like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down-regulated by TRIM16 and was required for TRIM16-reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity.
Project description:Recent advances in gene sequencing have shown that activated BRAF mutations are present in more than 50% of malignant melanomas and contribute to constitutive signals in the MAPK pathway. Besides the importance of its mutations in cell proliferation, BRAF is associated with lymph node, brain and liver metastasis, along with the loss of PTEN expression and ATG5. Knowledge of this genetic alteration has led to the development of personalized and targeted therapy strategies which block different pathways driving melanoma pathogenesis. Several targeted therapy agents such as vemurafenib, dabrafenib and encorafenib have been approved by the FDA as BRAF inhibitors, as well as other immunotherapies such as anti-CTLA-4 (ipilimumab). However, one of the main challenges is acquired resistance via reactivation of MAPK via CRAF/COT overexpression. Resistance to current BRAF inhibitors is a clinical challenge and one of the strategies to overcome this phenomenon is combination treatment, with the most recently approved combination being BRAF/MEK inhibitors (dabrafenib and trametinib) and BRAF or MEK inhibitors with immunocheckpoint blockers. This review delineates the current role of BRAF in melanoma progression and metastasis. It discusses targeted therapies and resistance mechanisms to BRAF inhibitors, and illustrates strategies to overcome this mechanism with recently approved agents.
Project description:The evolution of melanoma, the most aggressive type of skin cancer, is triggered by driver mutations that are acquired in the coding regions of particularly BRAF (rat fibrosarcoma serine/threonine kinase, isoform B) or NRAS (neuroblastoma-type ras sarcoma virus) in melanocytes. Although driver mutations strongly determine tumor progression, additional factors are likely required and prerequisite for melanoma formation. Melanocytes are formed during vertebrate development in a well-controlled differentiation process of multipotent neural crest stem cells (NCSCs). However, mechanisms determining the properties of melanocytes and melanoma cells are still not well understood. The nerve growth factor receptor CD271 is likewise expressed in melanocytes, melanoma cells and NCSCs and programs the maintenance of a stem-like and migratory phenotype via a comprehensive network of associated genes. Moreover, CD271 regulates phenotype switching, a process that enables the rapid and reversible conversion of proliferative into invasive or non-stem-like states into stem-like states by yet largely unknown mechanisms. Here, we summarize current findings about CD271-associated mechanisms in melanoma cells and illustrate the role of CD271 for melanoma cell migration and metastasis, phenotype-switching, resistance to therapeutic interventions, and the maintenance of an NCSC-like state.
Project description:BACKGROUND:As the selective inhibitor of BRAF kinase, vemurafenib exhibits effective antitumor activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after its initial treatment. METHODS:Immunohistochemical staining was performed to detect the expression of iNOS and hTERT, p-p65, Epcam, CD44, PCNA in mice with melanoma xenografts. The proliferation and migration of melanoma cells were detected by MTT, tumorsphere culture, cell cycle, cell apoptosis, AO/EB assay and colony formation, transwell assay and scratch assay in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the iNOS/hTERT signaling pathways were detected by western blot. Nucleus-cytoplasm separation, and immunofluorescence analyses were conducted to explore the location of p50/p65 in melanoma cell lines. Flow cytometry assay were performed to determine the expression of CD44. Pull down assay and ChIP assay were performed to detect the binding ability of p65 at iNOS and hTERT promoters. Additionally, hTERT promoter-driven luciferase plasmids were transfected in to melanoma cells with indicated treatment to determine luciferase activity of hTERT. RESULTS:Melatonin significantly and synergistically enhanced vemurafenib-mediated inhibitions of proliferation, colony formation, migration and invasion and promoted vemurafenib-induced apoptosis, cell cycle arresting and stemness weakening in melanoma cells. Further mechanism study revealed that melatonin enhanced the antitumor effect of vemurafenib by abrogating nucleus translocation of NF-?B p50/p65 and their binding at iNOS and hTERT promoters, thereby suppressing the expression of iNOS and hTERT. The elevated anti-tumor capacity of vemurafenib upon co-treatment with melatonin was also evaluated and confirmed in mice with melanoma xenografts. CONCLUSIONS:Collectively, our results demonstrate melatonin synergizes the antitumor effect of vemurafenib in human melanoma by inhibiting cell proliferation and cancer-stem cell traits via targeting NF-?B/iNOS/hTERT signaling pathway, and suggest the potential of melatonin in antagonizing the toxicity of vemurafenib and augmenting its sensitivities in melanoma treatment.