From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

Targeting of copper-trafficking chaperones causes gene-specific systemic pathology in Drosophila melanogaster: prospective expansion of mutational landscapes that regulate tumor resistance to cisplatin.

Copper, a transition metal, is an essential component for normal growth and development. It acts as a critical co-factor of many enzymes that play key roles in diverse cellular processes. The present study attempts to investigate the regulatory functions decisively controlling copper trafficking during development and aging of the Drosophila model system. Hence, through engagement of the GAL4/UAS genetic platform and RNAi technology, we herein examined the in vivo significance of Atox1 and CCS genes, products of which pivotally govern cellular copper trafficking in fly tissue pathophysiology. Specifically, we analyzed the systemic effects of their targeted downregulation on the eye, wing, neuronal cell populations and whole-body tissues of the fly. Our results reveal that, in contrast to the eye, suppression of their expression in the wing leads to a notable increase in the percentage of malformed organs observed. Furthermore, we show that Atox1 or CCS gene silencing in either neuronal or whole-body tissues can critically affect the viability and climbing capacity of transgenic flies, while their double-genetic targeting suggests a rather synergistic mode of action of the cognate protein products. Interestingly, pharmacological intervention with the anti-cancer drug cisplatin indicates the major contribution of CCS copper chaperone to cisplatin's cellular trafficking, and presumably to tumor resistance often acquired during chemotherapy. Altogether, it seems that Atox1 and CCS proteins serve as tissue/organ-specific principal regulators of physiological Drosophila development and aging, while their tissue-dependent downregulation can provide important insights for Atox1 and CCS potential exploitation as predictive gene biomarkers of cancer-cell chemotherapy responses.

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer.

Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. "Acetyl-chromatin" homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation.

Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients.

BACKGROUND: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. METHODS: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. RESULTS: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. CONCLUSIONS: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

Unraveling the human protein atlas of metastatic melanoma in the course of ultraviolet radiation-derived photo-therapy.

To explore the photo-therapeutic capacity of UV radiation in solid tumors, we herein employed an nLC-MS/MS technology to profile the proteomic landscape of irradiated WM-266-4 human metastatic-melanoma cells. Obtained data resulted in proteomic catalogues of 5982 and 7280 proteins for UVB- and UVC-radiation conditions, respectively, and indicated the ability of UVB/C-radiation forms to eliminate metastatic-melanoma cells through induction of synergistically operating programs of apoptosis and necroptosis. However, it seems that one or more WM-266-4 cell sub-populations may escape from UV-radiation's photo-damaging activity, acquiring, besides apoptosis tolerance, an EMT phenotype that likely offers them the advantage of developing resistance to certain chemotherapeutic drugs. Low levels of autophagy may also critically contribute to the selective survival and growth of UV-irradiated melanoma-cell escapers. These are the cells that must be systemically targeted with novel therapeutic schemes, like the one of UV radiation and Irinotecan herein suggested to be holding strong promise for the effective treatment of metastatic-melanoma patients. Given the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic agent, all individuals being subjected to risk factors for melanoma development have to be appropriately informed and educated, in order to integrate the innovative PPPM concept in their healthcare-sector management. SIGNIFICANCE: This study reports the application of nLC-MS/MS technology to deeply map the proteomic landscape of UV-irradiated human metastatic-melanoma cells. Data bioinformatics processing led to molecular-network reconstructions that unearthed the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic factor in metastatic-melanoma cellular environments. Our UV radiation-derived "photo-proteomic" atlas may prove valuable for the identification of new biomarkers and development of novel therapies for the disease. Given that UV radiation represents a major risk factor causing melanoma, a PPPM-based life style and clinical practice must be embraced by all individuals being prone to disease's appearance and expansion.

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

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.