Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality.,Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation.,Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis.,A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset.,Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity.,We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures.,We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2.,By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein.,Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression.,Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression.,Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.,The online version of this article (10.1186/s13046-018-0933-x) contains supplementary material, which is available to authorized users.
While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood.,The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells.,An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs).,Here we culture and characterize apparent MTFs from blood of melanoma patients.,We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them.,We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.,Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia.,Ultrastructurally, the cultured MTFs appeared to be macrophages.,They were rich in mitochondria and lysosomes, as well as apparent melanosomes.,The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin.,This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei.,Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206).,They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44).,MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations.,Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions.,MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs.,When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.,Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice.,We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.