Functional Studies of STK31: A Cell Fate Determinant in Spermatogonia and Cancer Development.

摘要

Spermatogenesis is a complicated process involving mitosis, meiosis and post-meiotic differentiation. Due to the lack of in vitro models, genes that are involved in mammalian spermatogenesis are largely unknown. Spermatogenesis and tumorigenesis share important biological similarities. This co-relation can be signified by a special group of genes called cancer/testis (CT) antigens, which are only expressed in the testes and cancer. Although cancer biology has been extensively studied for decades, promising therapeutic methods are not available for every type of cancer. Recent discovery of cancer stem cells and functional genomics studies have shed light on the development of new diagnostic and therapeutic approaches. This thesis describes the expression, cellular localization and function of a novel CT gene, STK31, in spermatogonia and cancer development.;In the first part of the experiment, the expression and cellular localization of STK31 were investigated. RT-PCR results showed that STK31 was reactivated in 47 -- 86% of multiple cancers. Immunofluorescent study and GFP tagging experiment showed that STK31 was localized in the cytoplasm and formed aggregated granules that divide asymmetrically during mitosis. Further study by co-staining with E-cadherin demonstrated that the mouse homolog, Stk31, was expressed in the transition state between undifferentiated and differentiated spermatogonia. These data suggest the possible involvement of STK31 in mouse spermatogonia and cancer development.;In the second part of the experiment, the function of Stk31 in mouse spermatogonia was investigated- A GSC culture on an STO feeder layer was established. Studies on growing properties, expression of molecular markers and germ cell transplantation showed that GSC culture maintained spermatogonial stem cell activity. Retinoic acid was then used to induce differentiation of GSC. The differentiation status was confirmed by monitoring the expression of molecular markers. RT-PCR and immunofluorescent study showed that the expression of Stk31 was induced in RA-induced differentiation and Stk31 proteins were asymmetrically distributed during GSC division. Overexpression of Stk31 in GSCs using retroviral transduction induced the differentiation phenotypes. These data indicate the involvement of Stk31 in mouse spermatogonia cell fate determination.;In the third part of the experiment, the function of STK31 in human colon cancer was investigated. A stable STK31 knock-down Caco2 cells were established by stably transfecting two miR RNAi designs with different efficiency into Caco2 cells. Flow cytometry analysis showed that knock-down of STK31 resulted in G1 phase arrest. Cell counts and MTS assays suggested that knock-down of STK31 decreased cell proliferation in confluent cultures. Knock-down of STK31 also enhanced cell attachment to several ECM proteins and decreases cell migration as suggested by attachment assays and migration assays. Moreover, knock-down of STK31 enhanced enterocytic differentiation and inhibited tumorigenicity both in vitro and in vivo as indicated by colony formation assays and xenograft assays. Date obtained from whole genome microarray studies indicate that STK31 regulates these "stemness" properties through altering the expression of key players in various pathways including KIT, SMAD1 and Cyclin D2. These results suggest the involvement of STK31 in colon cancer as a regulator of "sternness".;Further studies of Stk31 in spermatogenesis in vivo would allow the identification of the asymmetry machinery of GSCs and the signaling mechanism underlying cell fate determination. Further studies of STK31 in cancer stem cells would allow the development of new diagnostic and therapeutic approaches.