Deep-proteome mapping of WM-266-4 human metastatic melanoma cells: From oncogenic addiction to druggable targets

<div><p>Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis. Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy. Metastatic melanoma’s heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease. To this direction, we have herein employed a nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) proteomics technology to profile the deep-proteome landscape of WM-266-4 human metastatic melanoma cells. Our advanced melanoma-specific catalogue proved to contain 6,681 unique proteins, which likely constitute the hitherto largest single cell-line-derived proteomic collection of the disease. Through engagement of UNIPROT, DAVID, KEGG, PANTHER, INTACT, CYTOSCAPE, dbEMT and GAD bioinformatics resources, WM-266-4 melanoma proteins were categorized according to their sub-cellular compartmentalization, function and tumorigenicity, and successfully reassembled in molecular networks and interactomes. The obtained data dictate the presence of plastically inter-converted sub-populations of non-cancer and cancer stem cells, and also indicate the oncoproteomic resemblance of melanoma to glioma and lung cancer. Intriguingly, WM-266-4 cells seem to be subjected to both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) programs, with 1433G and ADT3 proteins being identified in the EMT/MET molecular interface. Oncogenic addiction of WM-266-4 cells to autocrine/paracrine signaling of IL17-, DLL3-, FGF(2/13)- and OSTP-dependent sub-routines suggests their critical contribution to the metastatic melanoma chemotherapeutic refractoriness. Interestingly, the 1433G family member that is shared between the BRAF- and EMT/MET-specific interactomes likely emerges as a novel and promising druggable target for the malignancy. Derailed proliferation and metastatic capacity of WM-266-4 cells could also derive from their metabolic addiction to pathways associated with glutamate/ammonia, propanoate and sulfur homeostasis, whose successful targeting may prove beneficial for advanced melanoma-affected patients.</p></div

Molecular reconstruction of a WM-266-4-specific network that is tightly associated with the EMT program.

<p>The green box indicates the most decisive EMT biomarker VIM/VIME in the WM-266-4 metastatic melanoma setting. Thirty-seven EMT components could not be incorporated into the cluster. dbEMT, INTACT and CYTOSCAPE were the bioinformatics resources applied.</p

Molecular reassembly of networks that regulate: (A) “glioma”, (B) “non-small cell lung cancer” and (C) “small cell lung cancer” initiation and progression.

<p>Orange boxes: WM-266-4 melanoma proteins that have been identified in the present study. Green boxes: proteins that have been missed in this study (for either technical or biological reasons). Red fonts indicate protein products of oncogenes or tumor suppressor genes involved in each disease. The bioinformatics sub-routine employed was the KEGG pathway maps.</p

Reassembly of VIM/VIME- and NDRG1-specific interactomes in WM-266-4 melanoma cells.

<p>(A) VIM-based molecular interactome. (B) NDRG1-based molecular interactome. Green boxes indicate the EMT and MET critical determinants VIM and NDRG1, respectively. Yellow boxes denote the VIM- or NDRG1-interacting proteins identified in our WM-266-4 deep-proteome catalogue. Blue boxes point out the VIM- or NDRG1-interacting protein partners that have been missed in the present study. Orange boxes mark the two WM-266-4 proteins 1433G and ADT3 that are shared between the VIM- and NDRG1-specific interactomes. INTACT and CYTOSCAPE were the bioinformatics platforms engaged.</p

Classification of WM-266-4 deep-proteome components according to their: (A) “sub-cellular topology”, (B) “organelle compartmentalization”, (C) “general biological processes”, (D) “specific molecular functions”, (E) “signaling pathways” and (F) “oncoprotein resemblance to other tumors”.

<p>Due to their specific features, certain proteins can be categorized in more than one group. The bioinformatics sub-routines employed were: the (A and B) GO (<u>G</u>ene <u>O</u>ntology) “Cellular Components”, (C) GO “Biological Processes”, (D) GO “Molecular Functions”, (E) PANTHER pathways and (F) GAD. (A-D, F) analyzed through DAVID.</p

Reconstruction of molecular networks that control: (A) “melanogenesis”, (B) “pluripotency of stem cells” and (C) “melanoma”-formation processes.

<p>Orange boxes: WM-266-4 melanoma proteins identified in the present study. Green boxes: proteins that have been missed in this study (for either technical or biological reasons). Red fonts indicate protein products of oncogenes or tumor suppressor genes involved in the disease. The bioinformatics tool engaged was the KEGG pathway maps.</p

A characteristic image of WM-266-4 metastatic melanoma cells grown <i>in vitro</i> (upper panel).

<p>WM-266-4 representative melanoma tumor xenografts dissected from SCID mice revealing the heterogeneity of tumors’ (1–3) melanin content (lower panel). 1: melanin-free tumor, 2: melanin-enriched tumor (arrow) and 3: melanin-low tumor (arrow). Scale bars: 50 μm (upper panel) and 1 cm (lower panel).</p