Extracellular vesicles from highly invasive melanoma subpopulations increase the invasive capacity of less invasive melanoma cells through mir-1246-mediated inhibition of CCNG2.

Invasive growth is a critical process in tumor progression, requiring the activation of various molecular processes in tumor cells at the invasive front. Intercellular communication between heterogeneous tumor cells enhances cellular activation and adaptation to specific microenvironments. One mechanism of intercellular communication is the delivery of miRNAs through tumor cell-derived extracellular vesicles (EVs). In this context we have observed that conditioned media from a highly invasive cell subpopulation (BLM-HI) enhances the invasive capacity of the parental cell line (BLM). Therefore, we hypothesized that this complex change of cellular behavior is influenced by EV-transported miRNAs. The treatment of BLM cells with EVs derived from BLM-HI cells resulted in a significantly enhanced invasive capacity, as observed in Matrigel-embedded spheroids and in 2D Boyden chamber assays, with a dose-dependent effect. Conversely, the invasive capacity of BLM cells was reduced when secretion of EVs was inhibited by a sphingomyelinase inhibitor. To investigate the molecular mechanisms behind this effect, we performed next-generation sequencing and identified an enrichment of miR-1246 in these EVs. In functional analyses we demonstrated that both the EV mediated delivery of miR-1246 as well as overexpression contributes to the enhanced invasiveness of BLM cells. We identified a binding site of miR-1246 in the 3'UTR of cyclin G2 (CCNG2) and demonstrated direct binding by a luciferase reporter assay.Increased expression of CCNG2 has been associated with cancer metastasis and poor patient outcomes in other malignancies. Our study demonstrates that intercellular communication contributes to the transfer of properties, such as increased invasive capacity, between heterogeneous melanoma cells via EV-transported miRNAs.

Resistance to BRAF Inhibitors: EZH2 and Its Downstream Targets as Potential Therapeutic Options in Melanoma.

Activating BRAF mutations occurs in 50-60% of malignant melanomas. Although initially treatable, the development of resistance to BRAF-targeted therapies (BRAFi) is a major challenge and limits their efficacy. We have previously shown that the BRAFV600E signaling pathway mediates the expression of EZH2, an epigenetic regulator related to melanoma progression and worse overall survival. Therefore, we wondered whether inhibition of EZH2 would be a way to overcome resistance to vemurafenib. We found that the addition of an EZH2 inhibitor to vemurafenib improved the response of melanoma cells resistant to BRAFi with regard to decreased viability, cell-cycle arrest and increased apoptosis. By next-generation sequencing, we revealed that the combined inhibition of BRAF and EZH2 dramatically suppresses pathways of mitosis and cell cycle. This effect was linked to the downregulation of Polo-kinase 1 (PLK1), a key regulator of cell cycle and proliferation. Subsequently, when we inhibited PLK1, we found decreased cell viability of melanoma cells resistant to BRAFi. When we inhibited both BRAF and PLK1, we achieved an improved response of BRAFi-resistant melanoma cells, which was comparable to the combined inhibition of BRAF and EZH2. These results thus reveal that targeting EZH2 or its downstream targets, such as PLK1, in combination with BRAF inhibitors are potential novel therapeutic options in melanomas with BRAF mutations.

Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN.

In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.

Comprehensive Analyses of miRNAs Revealed miR-92b-3p, miR-182-5p and miR-183-5p as Potential Novel Biomarkers in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are important mediators in the intercellular communication, influencing the function and phenotype of different cell types within the tumor micro-milieu and thus promote tumor progression. Since EVs safely transport packages of proteins, lipids and also nucleic acids such as miRNAs, EVs and their cargo can serve as diagnostic and prognostic markers. Therefore, the aim of this study was to investigate EV embedded miRNAs specific for melanoma, which could serve as potential biomarkers. In contrast to previous studies, we not only analysed miRNAs from EVs, but also included the miRNA profiles from the EV-secreting cells to identify candidates as suitable biomarkers. While the characterization of EVs derived from normal melanocytes and melanoma cells showed largely comparable properties with regard to size distribution and expression of protein markers, the NGS analyses yielded marked differences for several miRNAs. While miRNA load of EVs derived from normal human epidermal melanocytes (NHEMs) and melanoma cells were very similar, they were highly different from their secreting cells. By comprehensive analyses, six miRNAs were identified to be enriched in both melanoma cells and melanoma cell-derived EVs. Of those, the accumulation of miR-92b-3p, miR-182-5p and miR-183-5p in EVs could be validated in vitro. By functional network generation and pathway enrichment analysis we revealed an association with different tumor entities and signaling pathways contributing melanoma progression. Furthermore, we found that miR-92b-3p, miR-182-5p and miR-183-5p were also enriched in EVs derived from serum of melanoma patients. Our results support the hypothesis that miRNAs derived from EVs can serve as prognostic or diagnostic liquid biopsy markers in melanoma. We identified EV-derived miRNAs and showed that those miRNAs, which were enriched in melanoma cells and EVs, are also found elevated in serum-derived EVs of patients with metastatic melanoma, but not in healthy subjects.

BRAF/EZH2 Signaling Represses miR-129-5p Inhibition of SOX4 Thereby Modulating BRAFi Resistance in Melanoma.

Many melanomas are associated with activating BRAF mutation. Targeted therapies by inhibitors of BRAF and MEK (BRAFi, MEKi) show marked antitumor response, but become limited by drug resistance. The mechanisms for this are not fully revealed, but include miRNA. Wishing to improve efficacy of BRAFi and knowing that certain miRNAs are linked to resistance to BRAFi, we wanted to focus on miRNAs exclusively associated with response to BRAFi. We found increased expression of miR-129-5p during BRAFi treatment of BRAF- mutant melanoma cells. Parallel to emergence of resistance we observed mir-129-5p expression to become suppressed by BRAF/EZH2 signaling. In functional analyses we revealed that miR-129-5p acts as a tumor suppressor as its overexpression decreased cell proliferation, improved treatment response and reduced viability of BRAFi resistant melanoma cells. By protein expression analyses and luciferase reporter assays we confirmed SOX4 as a direct target of mir-129-5p. Thus, modulation of the miR-129-5p-SOX4 axis could serve as a promising novel strategy to improve response to BRAFi in melanoma.

Impact of rs12917 MGMT Polymorphism on [18F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL).

PURPOSE: The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34 months. PROCEDURES: We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18 years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. RESULTS: We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. CONCLUSION: MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.