轉錄因子Prox1 是一個與果蠅prospero 基因同源的homeobox 基因，此蛋白
高度表現在成人肝細胞且與肝臟發育息息相關。相反的，在肝癌中Prox1 的低表
現與細胞分化差、預後差及病人低存活率密切相關，意味著Prox1 在肝癌中扮演
腫瘤抑制者的角色。然而Prox1 在肝癌中的腫瘤抑制功能之分子機制則尚未清
楚。在本研究中，我們發現在肝癌細胞株中Prox1 的表現與E-cadherin 為正相關，
但是與Twist1 及Vimentin 呈負相關。外源性Prox1 會抑制Twist1 表現而降低內
生性Prox1 則造成Twist1 表現增加。此外，我們以染色質免疫沉澱進一步證實了
Prox1 藉由結合到Twist1 啟動子-117/-111 區域而抑制Twist1 表現。在Prox1 抑制
Twist1 的狀況下會增加p53 表現並降低AKT2 表現，p53 在Prox1 抑制細胞生長
中扮演重要角色而AKT2 則與Prox1 抑制細胞侵襲力有關，而動物實驗也證實了
Prox1 會抑制腫瘤生長以及肺轉移。綜言之，Prox1 在肝癌細胞中藉由抑制Twist1
去引發p53依賴性細胞衰老與降低AKT2媒介的細胞侵襲力來達到腫瘤抑制的作
用。

Prospero-related homeobox 1 (Prox1) was cloned as homeobox gene which
homologous to the Drosophila prospero gene. As a transcription factor, Prox1 is
important for liver development and is highly expressed in adult hepatocytes. In
contrast, down-regulation of Prox1 in hepatocellular carcinoma (HCC) is associated
with poor differentiation, prognosis, and reduced overall survival which implying a
potential tumor suppressive role of Prox1 in HCC. However, the molecular
mechanisms of Prox1’s tumor suppressive function are still obscure. In this study, we
find that Prox1 expression is positively associated with E-cadherin and negatively
linked with Twist1 and vimentin in various HCC cell lines. Ectopic expression of
Prox1 reduces Twist1 while knockdown of Prox1 increased Twist1 expression. We
further identify a putative Prox1 binding site located at the -117/-111 bp of the Twist1
promoter which is critical for gene repression. Chromatin immunoprecipitation assays
also demonstrate the direct binding of Prox1 to human Twist1 promoter. In addition,
inhibition of Twist1 by Prox1 causes p53 up-regulation and AKT2 down-regulation.
Moreover, functional assays show that wild-type p53 induction is important for the
growth-inhibitory effect of Prox1 and AKT2 is involved in the inhibition of migration
and invasion by Prox1. In consistence with the results of cell-based study, animal
experiments demonstrate that Prox1 significantly attenuates tumor growth and lung
metastasis in vivo. Collectively, we conclude that Prox1 functions as a tumor
suppressor in HCC cells via inhibiting Twist1 to trigger p53-dependent
senescence-like phenotype and to reduce AKT2-mediated invasion.

Chapter 1. Introduction. 1
Section 1. Hepatocellular carcinoma (HCC) 2
Section 2. Prox1 4
Section 3. The emerging role of Prox1 in HCC 10
Section 4. Objectives of study 11
Chapter 2. Prox1 inhibits Twist1 expression via transcriptional repression 13
Chapter 3. Prox1 inhibits Twist1 to trigger p53-dependent senescence-like
phenotype in hepatocellular carcinoma cells 33
Chapter 4. Prox1 inhibits Twist1 to attenuate AKT2-mediated metastasis
in hepatocellular carcinoma cells 57
Chapter 5. Conclusion and prospect 73
Chapter 6. References 77
Supplementary information 88

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