microRNA expression patterns in tumor infiltrating lymphocytes are strongly associated with response to adoptive cell transfer therapy.

Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TILs) was previously shown to yield clinical response in metastatic melanoma patients as an advanced line. Unfortunately, there is no reliable marker for predicting who will benefit from the treatment. We analyzed TIL samples from the infusion bags used for treatment of 57 metastatic melanoma patients and compared their microRNA profiles. The discovery cohort included six responding patients and seven patients with progressive disease, as defined by RECIST1.1. High throughput analysis with NanoString nCounter demonstrated significantly higher levels of miR-34a-5p and miR-22-3p among TIL from non-responders. These results were validated in TIL infusion bag samples from an independent cohort of 44 patients, using qRT-PCR of the individual microRNAs. Using classification trees, a data-driven predictive model for response was built, based on the level of expression of these microRNAs. Patients that achieved stable disease were classified with responders, setting apart the patients with progressive disease. Moreover, the expression levels of miR-34a-5p in the infused TIL created distinct survival groups, which strongly supports its role as a potential biomarker for TIL-ACT therapy. Indeed, when tested against autologous melanoma cells, miRLow TIL cultures exhibited significantly higher cytotoxic activity than miRHigh TIL cultures, and expressed features of terminally exhausted effectors. Finally, overexpression of miR-34a-5p or miR-22-3p in TIL inhibited their cytotoxic ability in vitro. Overall, we show that a two-microRNA signature correlates with failure of TIL-ACT therapy and survival in melanoma patients.

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients.

Adoptive cell therapy (ACT) of tumor infiltration lymphocytes (TIL) yields promising clinical results in metastatic melanoma patients, who failed standard treatments. Due to the fact that metastatic lung cancer has proven to be susceptible to immunotherapy and possesses a high mutation burden, which makes it responsive to T cell attack, we explored the feasibility of TIL ACT in non-small cell lung cancer (NSCLC) patients. Multiple TIL cultures were isolated from tumor specimens of five NSCLC patients undergoing thoracic surgery. We were able to successfully establish TIL cultures by various methods from all patients within an average of 14 days. Fifteen lung TIL cultures were further expanded to treatment levels under good manufacturing practice conditions and functionally and phenotypically characterized. Lung TIL expanded equally well as 103 melanoma TIL obtained from melanoma patients previously treated at our center, and had a similar phenotype regarding PD1, CD28, and 4-1BB expressions, but contained a higher percent of CD4 T cells. Lung carcinoma cell lines were established from three patients of which two possessed TIL cultures with specific in vitro anti-tumor reactivity. Here, we report the successful pre-clinical production of TIL for immunotherapy in the lung cancer setting, which may provide a new treatment modality for patients with metastatic NSCLC. The initiation of a clinical trial is planned for the near future.

A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme.

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance.Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab.These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment.

miR-17 regulates melanoma cell motility by inhibiting the translation of ETV1.

Melanoma is an aggressive malignancy with a high metastatic potential. microRNA-17 (miR-17) is a member of the oncogenic miR-17/92 cluster. Here we study the effect of miR-17 on melanoma cell motility. Over expression of the mature or pri-microRNA form of miR-17 in WM-266-4 and 624mel melanoma lines enhances cell motility, evident in both wound healing and transwell migration assays. TargetScan algorithm predicts the PEA3-subfamily member ETV1 as a direct target of miR-17. Indeed, a 3-4-fold decrease of ETV1 protein levels are observed following miR-17 transfection into the various melanoma lines, with no significant change in ETV1 mRNA expression. Dual luciferase experiments demonstrate direct binding of miR-17 to the 3'-untranslated region of ETV1, confirmed by abolishing point mutations in the putative binding site. These combined results suggest regulation of ETV1 by miR-17 by a direct translational repression. Further, in both melanoma cell lines ETV1 knockdown by selective siRNA successfully pheno-copies the facilitated cell migration, while overexpression of ETV1 inhibits cell motility and migration. Altered ETV1 expression does not affect melanoma net-proliferation. In conclusion, we show a new role for miR-17 in melanoma, facilitating cell motility, by targeting the translation of ETV1 protein, which may support the development of metastasis.

Differential regulation of aggressive features in melanoma cells by members of the miR-17-92 complex.

The various roles of microRNAs (miRNAs) in controlling the phenotype of cancer cells are the focus of contemporary research efforts. We have recently shown that miR-17 directly targets the ADAR1 gene and thereby enhances melanoma cell aggressiveness. miR-17 and miR-20a belong to the miR-17/92 complex, and their mature forms are identical except for two non-seed nucleotides. Nevertheless, here we show that these two miRNAs carry markedly different effects on melanoma cells. A strong positive correlation was observed between the expression of miR-17 and miR-20a among various melanoma cultures. Luciferase assays showed that miR-17 but not miR-20a directly targets the 3' untranslated region of the ADAR1 gene. Ectopic expression of these miRNAs in melanoma cells differentially alters the expression of five exemplar TargetScan-predicted target genes: ADAR1, ITGB8, TGFBR2, MMP2 and VEGF-A. Whole-genome expression microarrays confirm a markedly differential effect on the transcriptome. Functionally, over-expression of miR-20a but not of miR-17 in melanoma cells inhibits net proliferation in vitro. The differential functional effect was observed following ectopic expression of the mature miRNA or of the pre-miRNA sequences. This suggests that the two non-seed nucleotides dictate target sequence recognition and overall functional relevance. These miRNAs are clearly not redundant in melanoma cell biology.

CEACAM1 promotes melanoma cell growth through Sox-2.

The prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies.

MicroRNAs in cancer: lessons from melanoma.

Melanoma is a high-grade, poorly differentiated malignant tumor of pigment-producing cells (melanocytes), accounting for more than 70% of the skin cancer related deaths. Although new lines of targeted therapy and immunotherapy were introduced lately, durable responses are not common as it is hard to target the elusive metastatic phenotype. microRNAs (miRNAs) are short non-coding RNA molecules that function as specific epigenetic regulators of the transcriptome. miRNAs are involved in a broad spectrum of physiological and pathological processes, including cancer-related functions such as proliferation, cell cycle, migration, invasion, immune evasion and drug resistance. These functions are mostly regulated in melanoma through four molecular deregulated pathways, including the RAS/MAPK pathway, the MITF pathway, the p16INK4A-CDK4-RB pathway and the PI3K-AKT pathway. miRNAs provide a strong platform for delineation of cancer mechanisms. Here we review the diverse roles of miRNAs in melanoma cell biology. Studying miRNA-mediated regulation of aggressive and tumor related features is expected to provide novel mechanistic insights that may pave the way for new diagnostic, prognostic and predictive tools as well as new molecular targets for future therapy.

A comparative analysis of total serum miRNA profiles identifies novel signature that is highly indicative of metastatic melanoma: a pilot study.

CONTEXT: Quantification of circulating microRNAs (miRNAs) has recently become feasible and reliable, with most efforts focusing on miRNAs overexpressed by cancer cells. OBJECTIVE: Identification of a characteristic circulating miRNAs profile in melanoma patients. METHODS: We conducted a pilot study comprised of unbiased qPCR comparison of serum miRNA profiles between metastatic melanoma patients and healthy donors. RESULTS: Loss of two normal serum-miRNAs, miR-29c and miR-324-3p, is highly indicative of metastatic melanoma. Hierarchical clustering analysis supported the results and clearly distinguished melanoma patients from healthy donors, metastatic colon and renal cancer patients. DISCUSSION AND CONCLUSIONS: This approach is independent of tumor heterogeneity and is expected to have superior biomarker performances.

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding­dependent, yet RNA editing­independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

Nicotinamide inhibits vasculogenic mimicry, an alternative vascularization pathway observed in highly aggressive melanoma.

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.

Mutagen-specific mutation signature determines global microRNA binding.

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene products at the post-transcriptional level. It is thought that loss of cell regulation by miRNAs supports cancer development. Based on whole genome sequencing of a melanoma tumor, we predict, using three different computational algorithms, that the melanoma somatic mutations globally reduce binding of miRNAs to the mutated 3'UTRs. This phenomenon reflects the nature of the characteristic UV-induced mutation, C-to-T. Furthermore, we show that seed regions are enriched with Guanine, thus rendering miRNAs prone to reduced binding to UV-mutated 3'UTRs. Accordingly, mutation patterns in non UV-induced malignancies e.g. lung cancer and leukemia do not yield similar predictions. It is suggested that UV-induced disruption of miRNA-mediated gene regulation plays a carcinogenic role. Remarkably, dark-skinned populations have significantly higher GC content in 3'UTR SNPs than light-skinned populations, which implies on evolutionary pressure to preserve regulation by trans-acting oligonucleotides under conditions with excess UV radiation.

Regulation of cancer aggressive features in melanoma cells by microRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.