|Author:||Mok, Ho Kit|
|Title:||Cholesterol biosynthesis : a critical determinant of cancer stemness and drug resistance in hepatocellular carcinoma?|
|Advisors:||Lee, Kin Wah Terence (ABCT)|
|Subject:||Liver -- Cancer -- Treatment|
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xix, 166 pages : color illustrations|
|Abstract:||Hepatocellular carcinoma (HCC), the major malignancy of liver cancers, accounts for the third leading cause of cancer related mortality worldwide in 2020. In spite of the vaccination and recent advances in screening and diagnosis, most HCC patients are diagnosed at their advanced stages. Unfortunately, the treatment options for the advanced HCC patients are limited. Sorafenib and lenvatinib, the molecular-targeted drugs against multiple kinases, are approved by FDA, yet with slight survival benefit due to the acquired drug resistance. The unsatisfactory clinical results have therefore prompted us to elucidate the drug resistance mechanism so as to develop a novel therapeutic strategy against HCC. To mimic the clinical situation, we have established sorafenib- and lenvatinib-resistant HCC patient-derived tumor xenografts (PDTXs) through several rounds of drug administration. RNA sequencing coupled with pathway analysis was performed to compare gene expression profiles between drug-resistant PDTXs and their mock counterparts. Strikingly, cholesterol biosynthesis was mostly and commonly upregulated in both drug-resistant PDTXs. Drug-resistant cells showed enriched liver CSC populations. Therefore, we examined whether cholesterol biosynthesis was augmented in enhanced liver CSC populations. Firstly, we found that this pathway was upregulated in enhanced liver CSC populations via in vitro passages of hepatospheres with administration of chemotherapeutic drugs. Furthermore, we found that this pathway was preferentially activated in liver CSCs compared to normal liver stem cells by comparing the genetic profiles between CD133+ and CD133- cells from liver regeneration and HCC mouse models. Using Upstream Regulatory Analysis, sterol regulatory element-binding protein 2 (SREBP2) was found to be the upstream regulator of the activated cholesterol biosynthesis in both drug-resistant PDTXs and CSCs-enriched hepatospheres.|
Using lentiviral-based CRISPR activation and knockdown approaches, SREBP2-mediated cholesterol biosynthesis was found to be crucial in the regulation of acquired drug resistance in HCC via augmentation of liver CSCs with clinical significance. Similarly, exogeneous cholesterol-treated and high cholesterol-utilizing HCC cells showed enhanced cancer stemness and drug resistance. Specifically, molecularly targeted drugs, including sorafenib and lenvatinib, induced the activation of caspase 3 (CASP3), which subsequently induced the nuclear translocation of SREBP2 from the endoplasmic reticulum, resulting in activation of the cholesterol biosynthesis-driven sonic hedgehog (SHH) signalling pathway, mediated via 25-hydroxycholesterol (25-OHC). The correlation among CASP3 activity, SREBP2 and SHH signalling was further reinforced by the positive correlation among cleaved CASP3, SREBP2 and GLI-1 in HCC clinical samples. Finally, the therapeutic efficacy of targeting cholesterol biosynthesis by using simvastatin, an FDA-approved drug in lowering cholesterol, was tested to override the drug resistance in HCC. The combined treatment of simvastatin and sorafenib/lenvatinib not only suppressed growth of patient-derived HCC organoids, but also exerted maximal growth suppression in two PDTX models by inhibiting liver CSC populations. In summary, our findings reveal that HCC cells expand CSC populations via CASP3- dependent, SREBP2-mediated cholesterol biosynthesis in combatting the tyrosine kinase inhibitor therapy and that targeting cholesterol biosynthesis can overcome the drug resistance in HCC cells.
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