|Title:||Functional characterization of adipocytes in regulation of liver cancer stem cells|
|Advisors:||Lee, Kin Wah Terence (ABCT)|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
Liver -- Cancer
|Department:||Department of Civil and Environmental Engineering|
|Pages:||xxii, 161 pages : color illustrations|
|Abstract:||Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world. An evident increase in HCC incidence is observed in both developing and developed countries as the consequence of rise in obesity, which is considered to be one of the major risk factors for development of non-alcoholic fatty liver disease (NAFLD). Given cancer stem cells (CSCs) are the root of tumor development, we hypothesize that adipocytes, one of the key cellular factors within the tumor microenvironment, may play a critical role in HCC pathogenesis via regulation of liver CSCs. Various reports on the interaction between adipocytes, abundant cellular factor in obese tumor microenvironment and cancer has been reported. However, the role of adipocytes in the regulation of liver CSCs has not been studied. In this study, we aim to dissect the crosstalk between adipocytes and HCC in regulation of liver CSC properties. For this purpose, we employed human visceral preadipocytes and differentiated it into functional adipocytes. Upon confirmation of the functionality of adipocytes, we examined the potential CSC enhancing effect of adipocytes on HCC cells in a co-culture system in which adipocytes and HCC cells were physically separated. Upon functional analysis, we first found that adipocytes enhanced self-renewal and tumorigenicity properties of HCC cells via paracrine secretion. Concurrently, condition medium (CM) of adipocytes enhanced liver CSC properties including self-renewal, tumorigenicity, migration, invasion, drug resistance and expression of liver CSC markers. Our results confirmed interaction between adipocytes and HCC aided in amelioration of liver CSC, and such CSC enhancing effect was further augmented by reciprocal interaction between adipocytes and HCC. To better understand the mechanistic mode of communication between adipocytes and HCC, we analyzed the adipocyte CM by orbitrap liquid mass spectrometry and identified fatty acid binding protein 4 (FABP4) to be critical player in this communication. Quantification of secreted FABP4 by enzyme-linked immunosorbent assay (ELISA) confirmed preferential secretion of FABP4 in adipocytes, while its secretion was further increased (~5-fold) upon stimulation by HCC cells. Recombinant FABP4 (rhFABP4) enhanced CSC properties of HCC cells; while FABP4 inhibitor (BMS309403) abolished the CSC enhancing effect of adipocytes CM. Clinically, we found that overexpression of FABP4 in non-tumor samples was significantly correlated with poor prognosis of HCC patients. We next deciphered the downstream mechanism by which adipocytes derived FABP4 regulates liver CSC properties. We carried out RNA sequencing and compared the genetic profiles between rhFABP4 treated PLC/PRF/5 cells and control counterparts, followed by pathway analysis using Database for annotation visualization and integrated discovery (DAVID). DAVID highlighted deregulation of genes involved in PI3K-Akt signaling pathway upon administration of rhFABP4. Western blot analysis confirmed the phosphorylation of Akt within 60 minutes of administration of rhFABP4. Further investigation on the downstream pathway of PI3K-Akt signaling identified the activation of wnt/β-catenin signaling pathway by rhFABP4. In conclusion, our study characterized the functional role of adipocytes on regulation of liver CSCs and elucidated a novel mechanism via FABP4/Akt/wnt-β-catenin signaling pathway. Targeting this signaling cascade may be a potential therapeutic strategy against NAFLD-induced HCC.|
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