Purpose 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 (TAFs). Experimental Design 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, prior to and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. Results Expression of BRAF(V600E) induced transcription of IL-1α and IL-1β in melanocytes and melanoma cell lines. Furthermore, 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-1α/β 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. Conclusions This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and suggests that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.
Immunotherapy approaches for pancreatic ductal adenocarcinoma (PDAC) have met with limited success. It has been postulated that a low mutation load may lead to a paucity of T cells within the tumor microenvironment (TME). However, it is also possible that while neoantigens are present, an effective immune response cannot be generated due to an immune suppressive TME. To discern whether targetable neoantigens exist in PDAC, we performed a comprehensive study using genomic profiles of 221 PDAC cases extracted from public databases. Our findings reveal that: (a) nearly all PDAC samples harbor potentially targetable neoantigens; (b) T cells are present but generally show a reduced activation signature; and (c) markers of efficient antigen presentation are associated with a reduced signature of markers characterizing cytotoxic T cells. These findings suggest that despite the presence of tumor specific neoepitopes, T cell activation is actively suppressed in PDAC. Further, we identify iNOS as a potential mediator of immune suppression that might be actionable using pharmacological avenues.
Curcumin (diferuloylmethane) inhibits tumour cell growth by inducing apoptosis in many tumour types, including melanoma, via complex and ill-defined pathways. Recent studies have shown that curcumin is both a nitric oxide scavenger and an inhibitor of inducible nitric oxide synthase (iNOS) expression, low levels of which correlate with antiapoptotic function and poor survival and which may be regulated by inhibition of nuclear factor-kappaB (NFkappaB) activation. To elucidate the mechanisms by which curcumin inhibits melanoma proliferation, we tested the in vitro effects of curcumin on specific cell cycle pathways and melanoma cell survival, including NFkappaB activation. Curcumin induced melanoma cell apoptosis and cell cycle arrest, which is associated with the downregulation of NFkappaB activation, iNOS and DNA-dependent protein kinase catalytic subunit expression, and upregulation of p53, p21(Cip1), p27(Kip1) and checkpoint kinase 2. Curcumin also downregulated constitutive iNOS activity in melanoma cells. Our results demonstrate that curcumin arrested cell growth at the G(2)/M phase and induced apoptosis in human melanoma cells by inhibiting NFkappaB activation and thus depletion of endogenous nitric oxide. Therefore, curcumin should be considered further as a potential therapy for patients with melanoma.
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