Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Tumor-associated neutrophils (TANs) in human carcinoma-draining lymph nodes: a novel TAN compartment.

OBJECTIVES: The role of tumor-associated neutrophils (TANs) in the nodal spread of cancer cells remains unexplored. The present study evaluates the occurrence and clinical significance of human nodal TANs. METHODS: The relevance, derivation, phenotype and interactions of nodal TANs were explored via a large immunohistochemical analysis of carcinoma-draining lymph nodes, and their clinical significance was evaluated on a retrospective cohort of oral squamous cell carcinomas (OSCC). The tumor-promoting function of nodal TAN was probed in the OSCC TCGA dataset combining TAN and epithelial-to-mesenchymal transition (EMT) signatures. RESULTS: The pan-carcinoma screening identified a consistent infiltration (59%) of CD66b+  TANs in tumor-draining lymph nodes (TDLNs). Microscopic findings, including the occurrence of intra-lymphatic conjugates of TANs and cancer cells, indicate that TANs migrate through lymphatic vessels. In vitro experiments revealed that OSCC cell lines sustain neutrophil viability and activation via release of GM-CSF. Moreover, by retrospective analysis, a high CD66b+ TAN density in M-TDLNs of OSCC (n = 182 patients) predicted a worse prognosis. The analysis of the OSCC-TCGA dataset unveiled that the expression of a set of neutrophil-specific genes in the primary tumor (PT) is highly associated with an EMT signature, which predicts nodal spread. Accordingly, in the PT of OSCC cases, CD66b+TANs co-localised with PDPN+S100A9- EMT-switched tumor cells in areas of lymphangiogenesis. The pro-EMT signature is lacking in peripheral blood neutrophils from OSCC patients, suggesting tissue skewing of TANs. CONCLUSION: Our findings are consistent with a novel pro-tumoral TAN compartment that may promote nodal spread via EMT, through the lymphatics.

Analysis of the genes coding for subunit 10 and 15 of cytochrome c oxidase in Alzheimer's disease.

Decay of mitochondria and oxidative stress are associated with normal aging, but many neurodegenerative diseases, and particularly Alzheimer's disease (AD), are characterized by a significant increase in the intensity of these traits. Recent data suggest the possible contribution of heme deficiency to the progressive derangement of mitochondria in AD brain; shortage of heme, and particularly of heme-a, actually leads to loss of mitochondrial cytochrome c oxidase (COX), abnormal production of reactive oxygen species and altered amyloid precursor protein metabolism. We reasoned that differences in the amount and/or functioning of COX assembly subunit 10 (COX10) and 15 (COX15), the key enzymes involved in heme-a biosynthesis, could be linked to variations of the individual risk to develop AD. We analyzed their mRNA expression in the hippocampus from AD patients and controls, investigated the existence of nucleotide variations in their DNA sequences and analyzed their distribution in large groups of AD and control individuals. COX 15 mRNA was significantly more abundant in the cerebral tissue of AD patients (3.18 +/- 1.70 vs. 1.22 +/- 0.66 microg, normalized dose, P = 0.01). The IVS-178G>A SNP in COX10 and the c+1120C>T SNP in COX15 were significantly less represented in the patient group (P < 0.001 and P = 0.017, respectively) with respective odd ratios of 0.22 and 0.59, suggesting a possible protective role toward the risk for AD.