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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.

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.

Background Renal cell carcinomas (RCC) are characterized by the deregulation of several hundred hyperosmolality-responsive genes. High expression of a subset of these genes including the Ran binding protein 3 like ( RANBP3L ) is linked to a favorable prognostic outcome in RCC. However, the cellular function of RANBP3L remains largely unknown. Methods We used CRISPR/Cas9-mediated gene editing to generate functional deletions of the Ranbp3l and nuclear factor of activated T cells 5 ( Nfat5 ) gene loci in a murine renal cell line. The NFAT5-KO cells were used to assess the regulation of Ranbp3l by NFAT5 using immunofluorescence, RNA-Seq and promoter assays. RANBP3L-deficient cells were analyzed for changes in cell morphology, proliferation, migration and colony-forming capacity using immunofluorescence and live cell imaging. RANPB3L-dependent changes in gene expression were identified by RNA-Seq. Results We show that NFAT5 directly regulates Ranpb3l under hyperosmotic conditions by binding its promoter. Functional analysis of RANBP3L-deficient cells revealed a loss of epithelial structure, an increased cell migration behavior and colony forming capacity, accompanied by massive alterations in gene expression, all of which are hallmarks for tumor cells. Strikingly, a RANBP3L dependent signature of 60 genes separated samples with clear cell carcinoma (KIRC) from papillary (KIRP), chromophobe renal carcinoma (KICH) and healthy tissue. Conclusions Loss of RANBP3L induces a tumor like phenotype resembles RCC, especially KIRC, on the morphological and gene expression level and might promote tumor development and progression. Therapeutic reconstitution or elevation of osmoregulated RANBP3L expression might represent a novel treatment strategy for RCC or KIRC.

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis. C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.

Despite the constant development of innovative therapeutic options for hematological malignancies, the gold-standard therapy regimen for curative treatment often includes allogeneic hematopoietic stem cell transplantation (HSCT). The graft-vs.-leukemia effect (GVL) is one of the main therapeutic goals that arises from HSCT. On the other hand, graft-vs.-host disease (GVHD) is still one of the main and most serious complications following allogeneic HSCT. In acute myeloid leukemia (AML), HSCT together with high-dose chemotherapy is used as a treatment option. An aggressive progression of the disease, a decreased response to treatment, and a poor prognosis are connected to internal tandem duplication (ITD) mutations in the Fms like tyrosine kinase 3 (FLT3) gene, which affects around 30% of AML patients. In this study, C3H/HeN mice received an allogeneic graft together with 32D-FLT3 AML cells to induce acute GVHD and GVL. It was examined if pre-incubation of the graft with the anti-human cluster of differentiation (CD) 4 antibody MAX.16H5 IgG prevented the development of GVHD and whether the graft function was impaired. Animals receiving grafts pre-incubated with the antibody together with FLT3 AML cells survived significantly longer than mice receiving untreated grafts. The observed prolonged survival due to MAX.16H5 incubation of immune cell grafts prior to transplantation may allow an extended application of additional targeted strategies in the treatment of AML.

Skin cancer is the most common cancer worldwide, with rapidly increasing incidence and consistent mortality. Skin cancer encompasses melanoma and non-melanoma skin cancer, which in turn is mainly divided into cutaneous squamous cell carcinoma and basal cell carcinoma. Small noncoding micro­RNAs (miRNAs) regulate protein expression after transcription and play a role in the development and progression of skin cancer. Deregulated expression of miRNAs in skin cancer is associated with cell proliferation, angiogenesis, metastasis, apoptosis, immune response, and drug resistance. Specific patterns of miRNAs in specific skin cancer types can be used as diagnostic markers. For therapeutic purposes, both miRNA and chemically modified variants thereof as well as miRNA antagonists (antagomiRs) or RNA inhibitors may be applied topically. Due to their specific physicochemical properties, physical or chemical diffusion promoters are used with varying degrees of success. There is no question by now that such preparations have a high potential for the treatment of epithelial skin tumors in particular.

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.

Metastatic primary cutaneous melanoma is a frequently fatal disease despite recent therapeutic advances. Biomarkers to stratify patients’ prognosis are lacking. MicroRNAs (miRNAs) are small, non-coding RNAs. We aimed to determine the expression of miR-211-5p in primary tumors and metastases of malignant melanoma and its potential use as a prognostic biomarker. We performed in situ hybridization for miRNA-211-5p on 109 FFPE melanoma samples from 76 patients, including 31 paired primary tumor/metastasis samples. For validation, we performed in silico analyses of TCGA skin cutaneous melanoma (SKCM) cohort. High miR-211-5p expression was more frequent in primary tumors (70.8%) compared to metastases (39.3%). In metastases, it was associated with a significantly worse overall survival. Data from TCGA SKCM cohort confirmed that high miR-211-5p expression in melanoma metastases, but not primary tumors, is associated with worse overall survival. MiR-211-5p expression in metastases is associated with a shorter survival, emphasizing the potential of miR-211-5p as a risk predictor for a less favorable clinical outcome in metastatic disease. In situ hybridization could be implemented in a routine laboratory workflow and can be performed on diagnostic tissue.

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment, promoting tumor initiation, growth, progression, metastasis, and immune evasion. Recently it was shown that cancer cell-derived exosomes induce a tumor-promoting phenotype in TAMs. Exosome-loaded proteins, DNA, and RNAs may contribute to the macrophage reprogramming. However, the exact mediators and mechanisms, particularly in melanoma, are not known. In this study we examined the effects of cutaneous melanoma-derived exosomes on macrophage function and the underlying mechanisms. First, we showed that exposure to melanoma exosomes induces a tumor-promoting TAM phenotype in macrophages. Sequencing revealed enrichment for several miRNAs including miR-125b-5p in cutaneous melanoma exosomes. We showed that miR-125b-5p is delivered to macrophages by melanoma exosomes and partially induces the observed tumor-promoting TAM phenotype. Finally, we showed that miR-125b-5p targets the lysosomal acid lipase A (LIPA) in macrophages, which in turn contributes to their phenotype switch and promotes macrophage survival. Thus, our data show for the first time that miR-125b-5p transferred by cutaneous melanoma-derived exosomes induces a tumor-promoting TAM phenotype in macrophages.

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2′- O -methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2′- O -methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo . We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis. Zhou et al. show that in the context of AML1-ETO-driven leukaemia, AES and DDX21 induce small nucleolar RNA (snoRNA)–ribonucleoprotein (RNP) formation and this is important for self-renewal of leukaemic cells.