Reduction of tumorigenesis in androgen receptor overexpressed Huh7 cells through TGF-β induced cellular senescence phenotypes

Abstract

PURPOSE: Hepatocellular carcinoma (HCC) is a primary malignancy (cancer) of the liver. HCC has been known as androgen-dependent tumor with incidence of five times higher and worse prognosis in male than female. Steroid hormones and their receptors play a major role in the development of many types of human and animal cancers. It has been known that androgen and its receptor (AR) are significantly related with hepatocarcinogenesis both in human and animals, however, the level of AR in hepatoma is variable and their exact effects are still poorly explained. Therefore, we investigated the role of androgen receptor (AR) in HCC development. METHOD: To investigate the potential role of AR in hepatoma, Huh7-AR and Huh7-V cell lines were established by transfection of Huh7 hepatocelluar carcinoma (HCC) cells with human androgen receptor (AR) in pCMV5 and its empty vector, respectively. And their in vivo and in vitro tumorigenesis were evaluated in nude mice as well as in cell culture systems. RESULTS: AR overexpressing Huh7 cells, Huh7-AR, and its control, Huh7-V cells, were established. AR expression was measured by RT-PCR and immunoblot analyses. Huh7-AR cells, which were responsive to 5 alpha-dihydrotestosterone (DHT), revealed a decreased tumorigenecity in nude mice in addition to reduced clonogenicity in vitro, as compared with those of Huh7-V cells; Tumor volume and multiplicity of the HCC development in nude mice and invasiveness of the Huh7-AR cells, measured by Matrigel chamber analysis, were significantly reduced than those of Huh7-V cells. Interestingly, when cultured in the presence of DHT, Huh7-AR cells induced cellular senescence phenotypes, such as SA-beta-galactosidase activity, nuclear actin translocation, cytoplasmic p-Erk1/2 sequestration and reduced telomerase activity. When underlying mechanisms were studied in more detail in the cell culture system with or without DHT, TGF-beta1 mRNA expression was increased in the Huh7-AR cells and HCC tumors in the presence of DHT. At the same time, secretion of TGF-beta 1 was significantly higher in the Huh7-AR cells than that in Huh7-V cells. The effect of TGF-beta 1 on the induction of senescence of Huh7-AR cells was studied by infection of dominant negative TGFbeta-RII adenovirus (TGFbeta RII DN-Ad); The reduced growth rate and other cellular senescence phenotypes were significantly recovered after TGFbeta RII DN-Ad infection, compared with the infection of its control viruses. The above data strongly indicate that androgen receptor inhibits progression of HCC formation by Huh7-AR cells through the induction of senescence phenotypes via TGF-beta 1 expression in the presence of DHT.

안드로젠과 안드로젠 수용체(AR)는 사람과 동물에서 간암 발생 과정과 의미있게 관련되어 있음이 알려져 있으나, 간 암 및 주변조직에서 안드로젠 수용체의 발현수준이 다양하여 정확한 효과를 알 수 없었다. 간암 발생과정에 있어 안드로젠과 안드로젠 수용체의 가능한 역할을 알아보기 위해서 사람의 간암 세포주인 Huh7 세포에 pCMV5-hAR과 pCMV5 empty vector를 안정하게 주입하여 G418로 선별하였다. 그 결과 vector만 주입된 clone (이 후로는 Huh7-V라 함)에서는 hAR의 발현이 되지 않았고, hAR이 주입된 clone (이 후로는 Huh7-AR이라 함)에서는 hAR의 발현됨을 확인하였다. Huh7-AR 세포는 5α-dihydrotestosterone (DHT)에 반응성을 나타내었으며, Huh7-V 세포보다 성장속도가 떨어짐을 알 수 있었다. Huh7-AR 세포는 in vivo나 in vitro 상태에서 모두 종양형성 정도가 감소하였고, 암세포 전이능력도 Huh7-V 세포보다 감소하였다. 흥미로운 사실은 DHT를 포함한 스테로이드가 있는 상태로 Huh7-AR 세포를 오래 배양하다 보면 SA-β-galactosidase 염색이 되며, 핵 내의 actin 이동, 세포질내의 인산화된 p-Erk1/2 축적과 telomerase activity가 감소되는 등의 노화 현상을 나타내었다. 이런 현상이 나타나는 기전을 알아보기 위해 Huh7-AR 세포를 DHT가 있을 때와 없을 때 세포를 각각 배양한 경우 DHT가 있을 때 TGF-β1 발현과 분비가 증가되었다. 또한, Huh7-AR 세포를 주입하여 생긴 종양조직에서도 Huh7-V 세포를 주입하여 생긴 종양보다 TGF-β1 발현이 증가되었다. AR 과발현이 종양세포에 미치는 영향들이 TGF-β1발현으로 인한 것임을 확인하기 위하여 TGF-β1 신호를 차단해 주는 dominant negative TGF-β 수용체 바이러스를 세포에 감염시켰더니 AR 발현으로 암세포의 성장속도와 노화 현상들이 극복되었다. 따라서 안드로젠 수용체를 과발현하는 Huh7-AR 세포주의 종양 진행 저해현상은 TGF-β1 발현촉진을 통한 세포노화 현상유도에 의한 것이라고 할 수 있겠다.