We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.

Preclinical model in HCC: the SGK1 kinase inhibitor SI113 blocks tumor progression in vitro and in vivo and synergizes with radiotherapy

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.

The treatment and management of patients with metastatic melanoma have evolved considerably in the “era” of personalized medicine. Melanoma was one of the first solid tumors to benefit from immunotherapy; life expectancy for patients in advanced stage of disease has improved. However, many progresses have yet to be made considering the (still) high number of patients who do not respond to therapies or who suffer adverse events. In this scenario, precision medicine appears fundamental to direct the most appropriate treatment to the single patient and to guide towards treatment decisions. The recent multi-omics analyses (genomics, transcriptomics, proteomics, metabolomics, radiomics, etc.) and the technological evolution of data interpretation have allowed to identify and understand several processes underlying the biology of cancer; therefore, improving the tumor clinical management. Specifically, these approaches have identified new pharmacological targets and potential biomarkers used to predict the response or adverse events to treatments. In this review, we will analyze and describe the most important omics approaches, by evaluating the methodological aspects and progress in melanoma precision medicine.

Background: A bidirectional crosstalk between tumor cells and the surrounding microenvironment contributes to tumor progression and response to therapy. Our previous studies have demonstrated that bcl-2 affects melanoma progression and regulates the tumor microenvironment. The aim of this study was to evaluate whether bcl-2 expression in melanoma cells could influence tumor-promoting functions of tumor-associated macrophages, a major constituent of the tumor microenvironment that affects anticancer immunity favoring tumor progression. Methods: THP-1 monocytic cells, monocyte-derived macrophages and melanoma cells expressing different levels of bcl-2 protein were used. ELISA, qRT-PCR and Western blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Chromatin immunoprecipitation assay was performed to evaluate transcription factor recruitment at specific promoters. Boyden chamber was used for migration experiments. Cytofluorimetric and immunohistochemical analyses were carried out to evaluate infiltrating macrophages and T cells in melanoma specimens from patients or mice. Results: Higher production of tumor-promoting and chemotactic factors, and M2-polarized activation was observed when macrophages were exposed to culture media from melanoma cells overexpressing bcl-2, while bcl-2 silencing in melanoma cells inhibited the M2 macrophage polarization. In agreement, the number of melanoma-infiltrating macrophages in vivo was increased, in parallel with a greater expression of bcl-2 in tumor cells. Tumor-derived interleukin-1β has been identified as the effector cytokine of bcl-2-dependent macrophage reprogramming, according to reduced tumor growth, decreased number of M2-polarized tumor-associated macrophages and increased number of infiltrating CD4⁺IFNγ⁺ and CD8⁺IFNγ⁺ effector T lymphocytes, which we observed in response to in vivo treatment with the IL-1 receptor antagonist kineret. Finally, in tumor specimens from patients with melanoma, high bcl-2 expression correlated with increased infiltration of M2-polarized CD163⁺ macrophages, hence supporting the clinical relevance of the crosstalk between tumor cells and microenvironment. Conclusions: Taken together, our results show that melanoma-specific bcl-2 controls an IL-1β-driven axis of macrophage diversion that establishes tumor microenvironmental conditions favoring melanoma development. Interfering with this pathway might provide novel therapeutic strategies.