Elucidating the Role of KLF4 in Hepatocyte Growth Factor-Induced Cell Scattering and Growth Inhibition of the Human Hepatocellular Carcinoma Cell Line HepG2
本三年期計劃的主要目標在於闡明Krüppel-like factor 4(KLF4)在肝臟生長因子(Hepatocytegrowth factor；HGF)誘發人類肝癌細胞HepG2 之細胞分散(cell scattering)與生長抑制效應中所扮演的角色，以及KLF4 如何影響這些現象的分子機制。HGF 與其他生長因子相較之下的特別之處在於其誘發細胞分散的能力。細胞分散與上皮細胞-間質細胞轉換(EMT)這兩種現象幾乎雷同：為了進行細胞分散或EMT，上皮細胞必須先行破壞細胞間E-cadherin 的聯結而掙脫細胞間彼此的牽絆，隨後獲取間質細胞之移行與侵襲能力的特性。EMT 已知是促進癌細胞侵襲與轉移的關鍵力量，而HGF 誘發EMT 的能力與HGF/c-Met (HGF 的接受器)訊號之過度活化促成癌細胞更加惡性的現象一致。因此，研究HGF 誘發細胞分散有助於從分子層次上瞭解細胞移行、侵襲與EMT 等細胞現象。KLF4 是一種轉錄因子，其在不同的癌症背景下分別具有致癌基因與抑癌基因的功能；近來KLF4 因為與胚胎幹細胞的自我更新與萬能性分化能力的維繫上具關鍵角色而備受矚目。然而，KLF4 是否在HGF 誘發細胞分散與EMT 扮演任何角色則仍然未知。我們先前有關KLF4 是否參與HGF 誘發細胞分散的研究中發現HGF 可以提昇KLF4 的mRNA 與蛋白量；更重要的是：抑制KLF4 之功能可以阻止HGF 誘發HepG2 之細胞分散，也同時阻止HGF 抑制E-cadherin 的蛋白量。有鑑於此，我們假設HGF 藉由提昇KLF4來壓抑E-cadherin 的表現，進而促使細胞分散現象的進行。除此之外，我們發現抑制KLF4 之功能也可以阻止HGF 提昇細胞週期抑制蛋白p21CIP1 的蛋白量。已知HGF 藉由提昇p21CIP1 來促使HepG2 之細胞週期停滯在G1 時期，但迄今對於HGF 如何提昇p21CIP1 則仍然未知。有鑑於p21CIP1 是KLF4 之典型的轉錄標的基因，我們假設HGF 藉由提昇KLF4 來促使p21CIP1 的表達，進而促成細胞週期的停滯。為了檢驗這些假設，我們在此提出這個三年期計劃。我們預期分年達成下列目標：(1) 確認KLF4 在HGF 誘發HepG2 細胞之細胞分散上所扮演的角色以及HGF 如何提昇KLF4 的分子機制 (第一年)(2) 闡明KLF4 促成HGF 誘發HepG2 細胞之細胞分散的分子機制 (第二年)(3) 釐清KLF4 在HGF 誘發HepG2 細胞之生長抑制上所扮演的角色 (第三年)本計劃的研究成果無疑地將提供KLF4 參與HGF 誘發HepG2 細胞之細胞分散與生長抑制效應的支持證據。更重要的是：本計劃的研究發現將幫助學界對於HGF 誘發之細胞分散、細胞侵襲、細胞生長與EMT 等細胞效應的分子基礎有更加深入的瞭解，而這些分子機制將有助於以HGF/c-Met 訊號路徑為標靶之抗癌藥物的開發提供紮實的理論基礎。The primary goal of this 3-year proposal is to elucidate the role of Krüppel-like factor 4 (KLF4)in hepatocyte growth factor (HGF)-induced cell scattering and cell cycle arrest of HepG2 cells, ahuman hepatocellular carcinoma cell line. HGF is distinct from other growth factors by its action toinduce epithelial cell scattering, a cellular process reminiscent of epithelial-mesenchymal transition(EMT). In both programs, epithelial cells start by disrupting E-cadherin-mediated intercellularadhesion and subsequently acquire a motile and invasive mesenchymal phenotype. Consistent withthe central role of EMT in promoting carcinoma invasion and metastasis, aberrant HGF/c-Met(HGF's receptor) signaling leads to an aggressive phenotype in human cancers. For that reason,HGF-induced scattering is useful for elucidating the molecular network responsible for cellmigration, invasion and EMT. Krüppel-like factor 4 (KLF4) is a C2H2 zinc finger transcription factorknown to mediate a cell context-dependent growth promoting or inhibitory effect as well as theself-renewal and pluripotency of embryonic stem cells. However, it's role in HGF-induced cellscattering and EMT remains elusive. Our preliminary study revealed that KLF4 is up-regulated byHGF; moreover, functional depletion of KLF4 abolishes HGF-induced cell scattering and restoresE-cadherin levels reduced by HGF. Accordingly, we hypothesize that HGF up-regulates KLF4 forE-cadherin repression to initiate cell scattering. Interestingly, functional blockade of KLF4 alsoprevents HGF-triggered increase in p21CIP1, whose up-regulation is central to HGF-induced G1 arrestof HepG2 cells. Given that the mechanism mediating HGF-enhanced p21CIP1 is currently unknownand p21CIP1 is a bona fide target of KLF4, we hypothesize that KLF4 is a key molecule responsiblefor HGF-enhanced p21CIP1. As a result, we here present this 3-year proposal to answer thesehypotheses by achieving the following specific aims:(1) To validate the role of KLF4 in HGF-induced cell scattering and the mechanisms underlyingHGF-mediated KLF4 up-regulation (the 1st year).(2) To elucidate the mechanims by which KLF4 promotes HGF-induced cell scattering (the 2nd year).(3) To explore the role of KLF4 in HGF-induced growth inhibition of HepG2 cells (the 3rd year).Findings of these studies will undoubtedly offer the first evidence to support the involvement ofKLF4 in HGF-induced cell scattering and growth inhibition of HepG2 cells. More importantly, thesefindings will provide a new insight into the molecular network modulated by HGF to exert itspleiotropic biological effects, including cell scattering, invasion, proliferation and EMT, offering themolecular basis for the rational design of novel therapeutics targeting the HGF/c-Met signaling
