| Hepatocellular carcinoma (HCC) is an often lethal malignancy with limited treatment options. Sexual dimorphism in cancer etiology and prognosis is one of its important feathers. Commonly accepted, sex hormones, i.e., estrogens and androgens, are the drivers of sexual dimorphism in disease phenotypes. It was well known that, in prostate cancer and breast cancer, which are also sexually dimorophic, FOXA1, forkhead box protein A1, plays an dominant role in AR- or ER-dependent pathway. It was recently reported that the effects of estrogens and androgens on HCC development are dependent upon Foxal and the sexual dimorphic HCC is completely reversed in Foxal- and Foxa2-deficient mice after diethylnitrosamine-induced hepatocarcinogenesis, pointing FOXA1 as a key determinant of HCC development and sexual dimorphism. Here we explored the functional significance and relevance of FOXA1 sequence variation and dysregulation in HCC development and metastasis.Chaper 1:HCC risk associated nonsynonymous SNP (rs7144658) of FOXA1 significantly affects the expression of its target genes involved in cell proliferation, cell cycling and xenobiotic metabolism.1. Common nonsynonymous SNP of FOXA1 is associated with HCC risk. In a parallel HCC molecular epidemiology study with large sample size, we analyzed genetic association of FOXA1 sequence variants and susceptibility of liver cancer, and found that one common nonsynonymous SNP (rs7144658, A247G, Thr83Ala) was significantly associated with risk of HCC.2. The FOXA1/247G variant has lower binding activity to the cis-elements of its target genes than the FOXA1/247A variant. We performed transient transfection assays in HepG2 cells with the expression plasmids pcDNA3.1-FOXA/247A, pcDNA3.1-FOXAl/247G and pcDNA3.1 as negative control. After collecting the cells, we performed chromatin immunoprecipitation (ChIP) assays followed by quantitative real time PCR. The results showed that, the +247G-containing expression plasmid had lower binding activity than the +247A-containing expression plasmid, suggesting that the base +247 mutation from A to G significantly reduces FOXA1 binding activity.3. The FOXA1/247G variant has different transcriptional activity from the FOXA1/247A variant. We further performed luciferase reporter assays to determine whether the different binding activity result in different transcriptional activity in HepG2 cells. The results showed that FOXA1/247G had lower transcriptional activity than FOXA1/247A to DIO1 (p<0.01) and UGT2B17 (p<0.01), in contrast, higher to p27Kipl (p<0.05), and no significant difference in NTCP.4. ER differentially influences FOXA1/247A and FOXA1/247G binding activity due to its different binding affinity to them. HepG2 cells were transfected with FOXA1/247A or FOXA1/247G, plus pcDNA3.1-ER respectively, and ChIP-qPCR was performed. It was clarified that ER could influence FOXA1 binding to binding sites, and this influence sometimes is difference between FOXA1/247A and FOXA1/247G. As to DIO1, no matter +247A or +247G of FOXA1, their binding activity after co-expressing ER were 3-fold higher than before, but the ratio changed slightly (42-38%). Unlike DIO1, ER sharply reduced FOXA1 binding activity to UGT2B17 and p27Kip, especially FOXA1/247A, resulting in no difference. To NTCP, ER slightly enhanced FOXA1-247A binding activity but slightly reduced FOXA1-247G binding activity, resulting in.enlarged disparity between 247A and 247G (from 43-25%).5. FOXA1/247G had lower binding affinity (approximately 50%) to ER than FOXA1/247A. To find out the role of ER in FOXA1 binding events, we conducted co-immunoprecipation (co-IP). HepG2 cells were planted in 100mm cell-culture plates and transfected with pcDNA3.1, Myc-FOXA1/247G and Myc-FOXA1/247A, plus pcDNA3.1-ER respectively. The cellular extracts were used for co-IP with agarose conjugated Myc-Tag Antibody. The immunoprecipitated complexes were resolved by gel electrophoresis and western blot analysis using antibodies to Myc-tag and ER. The WB results showed that FOXA1/247G had lower binding affinity (approximately 50%) to ER than FOXA1/247A.Chapter 2:WT1 promotes HCC metastasis via FOXA1 pathwayHCC is one of the most common and aggressive malignant tumor worldwide. High rate of recurrence and metastasis of HCC remains one of the major obstacles for treatment, but the molecular basis of HCC me-tastasis is still poorly understood, with few effectual diagnostic biomarkers and therapeutic targets thus far. Therefore to find the HCC metastasis-related genes and clarify the metastasis mechanism would be crucial for the treatment of HCC.In this study, we found FOXA1 is regulated by WT1. The overexpression ofWT1 results in dysregulation of FOXA1 and promotes HCC metastasis.1. WT1, FOXA1 and AGR2 were up-regulated and co-expressed in metastatic cell lines. The protein expression of WT1, FOXA1 and AGR2 were evaluated in a panel of HCC cell lines (L02, Chang’s, HepG2, Hep3B, Huh7, MHCC97L and MHCC97H). These three proteins was all up-regulated in metastasis HCC cell lines MHCC97L, MHCC97H.2. FOXA1 was up-regulated by WT1. In the HepG2 cells transfected with WT1 four isoforms respectively, Western Blot analysis showed that WT1-1 didn’t influence FOXA1 expression, but WT1-4 could up-regulate FOXA1 expression. With the up-regulation of FOXA1, the expression of AGR2 was also increased.3. WT1 did not influence HCC cells growth. WT1 expression was knocked down in MHCC97H cells and MTT array was performed. Compared to si-NC, MHCC97H proliferation didn’t change. Interestingly, the proliferation of HepG2 transfected with WT1-4 was increased, suggesting that although WT1 can’t influence HCC cells growth, its isoform WT1-4 had the ability to increase cell proliferation.4. WT1 promoted HCC cells migration and invasion in vitro. Endogenous expression of WT1 in MHCC97H was disrupted by transiently transfected with siRNA, and WT1 disruption caused significant decrease in the migration and invasion of MHCC97H. Overexpression of WT1 in HepG2 cells with WT1-4 promoted HepG2 cells migration and invasion, nor did WT1-1. There results demonstrated that WT1 promoted HCC cells migration and invasion in vitro via its isoform WT1-4.5. The pro-metastatic effect of WT1 on HCC is likely mediated by FOXA1. As a transcriptional factor, WT1 promotes HCC metastasis through the regulation of its target genes. We found that WT1 isoform WT1-4 but not WT1-1 could regulate FOXA1 expression. Of a coincidence, WT1-4 but not WT1-1 had the ability to promote HCC metastasis. So it was reasonable that pro-metastatic effect of WT1 on HCC is likely via FOXA1. |
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