Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, University La Sapienza, Rome, Italy.
J Hepatol. 2013 Jan;58(1):65-72. doi: 10.1016/j.jhep.2012.08.023. Epub 2012 Sep 4.
BACKGROUND & AIMS: The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity.
Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression.
We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity.
Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.
肿瘤命运取决于细胞自主特性和龛位微环境线索。转化生长因子β（TGFβ）是影响肝细胞癌（HCC）肿瘤去分化、诱导上皮-间充质转化（EMT）和获得转移特性的主要微环境因素。转录因子 HNF4α 的丢失是 HCC 向更具侵袭性表型进展的主要机制；其重新表达，减少肿瘤形成并抑制 EMT 程序，已被提议作为 HCC 基因治疗的治疗工具。我们研究了 TGFβ 对 EMT 抑制和肿瘤抑制因子 HNF4α 活性的影响。
通过划痕愈合和侵袭试验分析细胞迁移和侵袭。通过 RT-qPCR 和免疫荧光评估 EMT。利用 ChIP 和 EMSA 测定法研究 HNF4α 的 DNA 结合活性。通过 2-DE 分析评估 HNF4α 的翻译后修饰（PTMs）。通过化学抑制和组成型活性突变体表达来调节 GSK3β 活性。
我们证明了 TGFβ 的存在会损害 HNF4α 作为肿瘤抑制因子的效率。我们发现 TGFβ 诱导 HNF4α 的 PTMs，这与靶基因启动子上 HNF4α DNA 结合活性的早期丧失相关。此外，我们确定了 GSK3β 激酶是介导 HNF4α 功能失活的 TGFβ 靶标之一：GSK3β 化学抑制导致 HNF4α DNA 结合受损，而组成型活性 GSK3β 突变体则会损害 TGFβ 诱导的对 HNF4α 肿瘤抑制活性的抑制作用。
我们的数据表明，在 TGFβ 的主导下，HNF4α 介导的 HCC 基因治疗存在局限性。
