Dataset Information

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma.

ABSTRACT:

Background

Hepatocyte nuclear factor 4? (HNF4?) is a tissue-specific transcription factor that regulates the expression of numerous genes in hepatocytes and pancreatic ? cells. HNF4? has been reported to affect cell proliferation and chemoresistance in several cancers. However, the role of HNF4? in pancreatic adenocarcinoma (PDAC) has not been studied extensively and remains unclear.

Methods

By utilizing immunohistochemical (IHC) staining, we measured the expression of HNF4? in PDAC tissues. By silencing HNF4? in PDAC cell lines, we assessed the impact of HNF4? on pancreatic cancer cell proliferation and gemcitabine sensitivity. We used CCK8 and colony formation assays to examine the effect of HNF4? on cell proliferation. A flow cytometry assay was used to assess cell apoptosis. The expression of gemcitabine-related genes was detected by quantitative real?time PCR (qRT-PCR) and Western blotting. IHC was utilized to assess the correlation between HNF4? and human equilibrative nucleoside transporter 1 (hENT1) expression in PDAC patients. Chromatin immunoprecipitation (ChIP) and dual?luciferase reporter assays were used to confirm that hENT1 is a target gene of HNF4?.

Results

Increased HNF4? expression was detected in PDAC tissues; patients with higher HNF4? expression displayed worse prognosis. To elucidate the function of HNF4?, we examined its role in pancreatic cancer cell proliferation, apoptosis and gemcitabine resistance. In HNF4?-silenced Capan-1 and MiaPaCa-2 cells, we observed decreased cell proliferation and increased sensitivity to gemcitabine compared to those of controls. The mechanism of HNF4? in gemcitabine-related chemosensitivity was then explored. In response to HNF4? silencing, the expression levels of gemcitabine-related proteins, hENT1 and deoxycytidine kinase (dCK) were significantly increased. Additionally, hENT1 was negatively correlated with HNF4? in PDAC tissue samples. Moreover, we identified hENT1 as a downstream target of HNF4?.

Conclusion

HNF4? is a prognostic marker for overall survival, is required for pancreatic cancer cell proliferation and promotes resistance to gemcitabine by downregulating hENT1. Therefore, targeting HNF4? might reverse gemcitabine resistance and provide novel treatment strategies for PDAC.

SUBMITTER: Sun Q

PROVIDER: S-EPMC6398265 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma.

Sun Qiqing Q Xu Wenyan W Ji Shunrong S Qin Yi Y Liu Wensheng W Hu Qiangsheng Q Zhang Zheng Z Liu Mengqi M Yu Xianjun X Xu Xiaowu X

Cancer cell international 20190304

<h4>Background</h4>Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor that regulates the expression of numerous genes in hepatocytes and pancreatic β cells. HNF4α has been reported to affect cell proliferation and chemoresistance in several cancers. However, the role of HNF4α in pancreatic adenocarcinoma (PDAC) has not been studied extensively and remains unclear.<h4>Methods</h4>By utilizing immunohistochemical (IHC) staining, we measured the expression of HNF4α in PD ...[more]

PMID: 30867652

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Project description:IntroductionPDAC is an extremely aggressive tumor with a poor prognosis and remarkable therapeutic resistance. The dense extracellular matrix (ECM) which characterizes PDAC progression is considered a fundamental determinant of chemoresistance, with major contributions from mechanical factors. This study combined biomechanical and pharmacological approaches to evaluate the role of the cell-adhesion molecule ITGA2, a key regulator of ECM, in PDAC resistance to gemcitabine.MethodsThe prognostic value of ITGA2 was analysed in publicly available databases and tissue-microarrays of two cohorts of radically resected and metastatic patients treated with gemcitabine. PANC-1 and its gemcitabine-resistant clone (PANC-1R) were analysed by RNA-sequencing and label-free proteomics. The role of ITGA2 in migration, proliferation, and apoptosis was investigated using hydrogel-coated wells, siRNA-mediated knockdown and overexpression, while collagen-embedded spheroids assessed invasion and ECM remodeling.ResultsHigh ITGA2 expression correlated with shorter progression-free and overall survival, supporting its impact on prognosis and the lack of efficacy of gemcitabine treatment. These findings were corroborated by transcriptomic and proteomic analyses showing that ITGA2 was upregulated in the PANC-1R clone. The aggressive behavior of these cells was significantly reduced by ITGA2 silencing both in vitro and in vivo, while PANC-1 cells growing under conditions resembling PDAC stiffness acquired resistance to gemcitabine, associated to increased ITGA2 expression. Collagen-embedded spheroids of PANC-1R showed a significant matrix remodeling and spreading potential via increased expression of CXCR4 and MMP2. Additionally, overexpression of ITGA2 in MiaPaCa-2 cells triggered gemcitabine resistance and increased proliferation, both in vitro and in vivo, associated to upregulation of phospho-AKT.ConclusionsITGA2 emerged as a new prognostic factor, highlighting the relevance of stroma mechanical properties as potential therapeutic targets to counteract gemcitabine resistance in PDAC.

Project description:AIM:To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4?) in regulating hepatic expression of microRNAs. METHODS:Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4? expression in liver (Hnf4?-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4?, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4? on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3'UTR) of two genes in human hepatoma cells. RESULTS:Microarray data indicated that most microRNAs remained unaltered by Hnf4? deficiency in Hnf4?-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4?-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4?-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4? directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4? markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3'UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSION:HNF4? is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4? may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Dataset Information

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma.

Background

Methods

Results

Conclusion

Publications

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma.

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