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

Abstract

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 SNMM 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. 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. Copy number variations involving components of the PI3K/Akt/mTOR pathway were found in SN-MM cell lines. Moreover, 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 immunobiology of these neoplasms and, as an extension, to MM from other sites.

Specific oncogene activation of the cell of origin in mucosal melanoma

“…We have generated the first genetically engineered model of MM. While there are current efforts to establish MM human cell lines 31, 32 and PDX models and it was previously known that dogs naturally are at risk for MM 33, 34, 35 ; genetically engineered models can offer important information about causality and are more experimentally tractable. For instance, we observed decreased tumor-cell-intrinsic MHC class I gene expression in patient MMs, which could be caused by the cell state of the tumor cells vs. secondary effects from the tumor microenvironment.…”