Abstract
BackgroundMelanoma, 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.MethodsWestern 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.ResultsA 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.ConclusionOverall 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.
Highlights
Semaphorins (SEMAs), a large family of phylogenetically conserved proteins classified into eight classes, are involved in different physiological and developmental functions, including regulation of the nervous and immune systems and angiogenesis [1]
Semaphorin 5A (Sema5A) affects in vitro melanoma cell invasion, migration and vasculogenic mimicry (VM) By interrogating Protein Atlas database we observed that Sema5A protein and Messenger RNA (mRNA) are expressed in all tested cancer types, including melanoma
We evaluated the expression of Sema5A protein by immunohistochemistry (IHC) in metastatic lesions from 13 patients with melanoma, finding 70% of positive specimens (Fig. 1c-e)
Summary
Semaphorins (SEMAs), a large family of phylogenetically conserved proteins classified into eight classes, are involved in different physiological and developmental functions, including regulation of the nervous and immune systems and angiogenesis [1]. We focused on the functional relation between Sema5A and Bcl-2, an anti-apoptotic protein associated with melanoma progression, resistance to apoptosis and poor prognosis [16]. In addition to its canonical anti-apoptotic role, Bcl-2 is involved in multiple non-canonical functions, including melanoma metastasis, angiogenesis, and autophagy [17]. Bcl-2 overexpression in human melanoma cells increases in vitro and in vivo tumor progression-associated properties and angiogenesis [17,18,19,20,21] and promotes a cancer stem cell phenotype [22]. We investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation
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