The Study Of The Molecular Mechanism Underlying The Effect Of Lipid Metabolism Associated Gene CGI-58on The Development And Progression Of Cancer

Background：The etiology of cancer attributes to both genetic and environmental factors. Numerousevidence has been indicating the critical role of tumor microenvironment in regulatingcancer pathogenesis, but the underlying mechanism remains elusive. Cancer cells interactwith their extracellular matrix (ECM) during proliferation and migration. Fibronectin is animportant ECM glycoprotein in tumor microenvironment with several alternatively splicedvariants, including extra domain A (EDA). Previous studies have reported that theexpression levels of EDA are associated with the malignancy in several cancers, includingcolon cancer (CRC) as well as head and neck cancer, but the precise effect and molecularmechanism is still unknown.Metabolic reprogramming is a hallmark of malignant tumors. Many oncogenes andtumor suppressor genes have positioned at critical nodes of important metabolic networks,and their activities are hyper-responsive to metabolic perturbations. Additionally, inborn oracquired mutations of several metabolic enzymes are associated with development andprogression of several types of cancer, highlighting important roles of altered cellmetabolism in cancer etiology. A distinct metabolic phenotype possess during thedevelopment and progression of cancer, which is weakly associated with cancer subtype,but more strongly associated with tumor microenvironment-derived signature. Glycolyticbreakdown of glucose for ATP production in the presence of ample oxygen to fuelmitochondrial oxidative phosphorylation is a characteristic metabolic reprogramming inmultiple malignant tumors, and is known as “Warburg effect”. Although the Warburg effecthas been demonstrated to critically contribute to cancer pathogenesis, it is largely unknownhow cancer cells shift their energy metabolism to aerobic glycolysis.It has been revealed that tumor microenvironment is involved in regulating tumor metabolic reprogramming. Therefore, we presumed that EDA may play an important role infacilitating the development an progression of cancer via manipulating metabolicreprogramming. In this study, we established both full length form and shRNA formplasmid of EDA, transfected them into CRC cells, endothelial cells and nasopharyngealcarcinoma (NPC) cells, and the effect of EDA on the cancrt pathogenesis was investigated.In addition, we identified the lipid metabolism associated gene, CGI-58as a critical targetof EDA, and further used CGI-58intestine-specific knockout ApcMin/+mice,CGI-58-silenced and CGI-58-forced cells, and human CRC and melanoma tissues to revealthe novel role of CGI-58as a tumor suppressor in CRC and melanoma, and also as a switchof “Warburg effect”.Wnt signaling critically contributes to the development and progression of CRC, and isthe driven cause of the tumorigenesis of ApcMin/+mice. We further revealed the underlyingmechanism of the activation of Wnt signaling in the absence of CGI-58.To further investigate the casual role of CGI-58as a tumor suppressor, we checked thedatabase of Catalogue of Somatic Mutations in Cancer (COSMIC), and found that anotherstriking frequent loss is reported in tumors derived from nervous system. Melanoma is acommon type of nervous system-derived cancer displaying highly aggressive malignancy.Little is known about the mechanism underlying the highly aggressive behavior ofmelanoma. It was shown that another lipolytic enzyme, MAGL, promots invasion andmetastasis of multiple cancers, including melanoma. Due to the abundant expression ofCGI-58in normal skin and the striking phenotype of skin lesions in CGI-58deficientpatients, we then throw an appreciation in investigating the potential role of CGI-58inmelanoma pathogenesis.Objective：To investigate the molecular mechanism underlying the oncogenic role of EDA incancer pathogenesis; To reveal the novel tumor suppressor role of EDA associatedmetabolic gene CGI-58in the development and progression of CRC, and further identifythe casual role of CGI-58as a tumor suppressor in regulating the differentiation ofmelanoma.Materials and methods：1. Tissue chips consisting of human CRC and melanoma specimens and human normal colon tissues were used for statistical analysis of clinicopathological features;2. EDA or CGI-58manipulations in cells were established by lentiviral particles;3. Intestine-specific CGI-58knockout mice were generated by mating CGI-58-floxedmice with B6.SJL-Tg(Vil-cre)977Gum/J mice, followed by crossing CGI-58f/+/Vil-cremicewith CGI-58f/+mice to get homozygous CGI-58floxed mice with Vil-cre transgene,Results：1. EDA promotes the pathogenesis of CRC as well as NPCWe found that tumor tissue and serum EDA levels are substantially higher in advancedversus early stage human CRC, and EDA levels are significantly correlated with the clinicalfeatures of CRC patients. We further demonstrated that EDA sustains the properties ofCD133+/CD44+CRC cells via activating integrin/FAK/ERK pathway to driveWnt/β-catenin signaling. Furthermore, we assert that CRC cell-derived EDA significantlyfacilitates migration, sprouting and tubulogenesis of co-cultured lymphatic endothelial cells,indicating an important role of EDA in lymphangiogenesis of CRC. Intriguingly, we alsofound that endothelial cell-secreted-EDA promotes the metastatic capacity CRC cells viainducing an epithelial-mesenchymal-transition (EMT). In addition, our findingsdemonstrated that EDA levels are significantly correlated with the clinical features of NPCpatients. EDA substantially aggravates the radioresistance of NPC cells, and FAK/Akt/JNKsignaling was found to be a critical signaling mediating EDA function in regulating NPCradiosensitivity. Very impressively, we revealed that EDA suppresses a well establishedintracellular lipolytic activator, Comparative Gene Identification-58(CGI-58).2. CGI-58deficiency promotes CRC development and metastasis by inducingaerobic glycolysis.Extensive loss of CGI-58is a hallmark of CRCs and is significantly correlated with theclinical features of CRCs. Silencing of CGI-58in normal fibroblasts induces malignanttransformation. Intestine-specific knockout of CGI-58in ApcMin/+mice robustly increasestumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells,CGI-58deficiency induces epithelial-mesenchymal transition by suppressing theAMPKα-p53pathway, which is attributable to increased aerobic glycolysis. In humanCRCs, CGI-58expression falls substantially and correlates negatively with malignantfeatures. Our study is the first to link CGI-58to CRC pathogenesis. It suggests that cancer cells may develop aerobic glycolysis by suppressing CGI-58-mediated intracellularlipolysis.3. CGI-58deficiency activates Wnt signaling in CRC via inducing the nucleartranslocation of YAP/TAZ independent of β-catenin.The phenotype of CGI-58knockout in ApcMin/+mice indicates an activation of Wntsignaling, we then sought to explore the underlying molecular mechanism. Wnt/β-catenin isa classic signaling involved in CRC development and progression. We found that theexpression and the translocation of β-catenin showed no changes in CGI-58deficient cells,while the expression and the translocation of YAP/TAZ were substantially increased inCGI-58deficient cells. Furthermore, the protein-protein interaction between CGI-58/ADRPand YAP/TAZ was demonstrated to mediate the activation of Wnt signaling in CGI-58deficient CRC cells.4. Melanoma silences CGI-58to promote invasion capacity via inducing celldedifferentiationThe expression level of CGI-58is significantly correlated with the clinical features ofmelanoma. Aggressive melanoma cells showed substantially lower expression of CGI-58relative to non-aggressive melanoma cells. CGI-58deficiency induced reprogramming oflysophophatidic acid metabolism and the activation of Wnt5A signaling mediate thededifferentiation of melanoma cells, and therefore increased their metastatic capacity.Conclusions：1. Both CRC cell derived-EDA and endothelial cell derived-EDA significantlypromote the pathogenesis of CRC. EDA/integrin signaling are substantially attributable tothe malignancy of CRC, and may be potential specific targets in CRC therapy. EDA alsostrongly aggravates the radiosensitivity of NPC cells, and FAK/Akt/JNK signaling wasfound to critically mediates EDA effect on NPC radiosensitivity. EDA substantiallysuppresses the expression of a well-established cellular lipolytic activator, CGI-582. CGI-58deficiency robustly induces tumorigenesis and malignant transformation bypromoting aerobic glycolysis, highlighting a key role of blockade of cellular fat catabolismin driving cancer development and progression, and revealing the switch role of CGI-58inWarburg effect. The strong negative correlation between CGI-58expression levels andmalignant features of human colorectal carcinomas suggest that CGI-58and the associated metabolites may serve as biomarkers for colorectal cancer metastasis and prognosis.3.CGI-58deficiency induced Wnt activation is YAP/TAZ translocation dependentinstead of β-catenin dependent. Our findings showed a novel correlation between lipolyticgene and Wnt signaling.4. CGI-58plays a casual tumor suppressor role in melanoma. CGI-58deficiencyinduces dedifferentiation of melanoma cells via triggering LPA metabolic reprogramming toactivate Wnt5A signaling, and critically contributes to the invasion and metastasis capacityof melanoma.5.Collectively our results establish a regulatory network between tumormicrocnviroment and tumor metabolism to further explain the oncogenic mechanismunderlying the development and progression of cancer.