Prox1 Suppressed Tumor Cell Growth by the Down Regulation of PKCβII Through the DNA Methylation

The homeobox gene Prox1 is a transcription factor related to the Drosophila prospero gene and known to be the master gene which controls the development of lymphatic vasculature. Recent studies have suggested that Prox1 is a strong candidate tumor suppressor gene in several kinds of malignancies. We previously demonstrated that the expression of Prox1 gene was frequently silenced by aberrant DNA methylation in about 60% of diffuse large B cell lymphoma (DLBCL). In this study, we showed that Prox1 regulated Protein Kinase C beta II (PKCβII) expression, which was overexpressed in poor risk DLBCL, and analyzed the inhibitory mechanism of tumor suppression by Prox1.

We established the Prox1 stable transformants of Hela cells which originally lacked Prox1 expression, and examined them in vitro cell proliferation and in vivo tumor formation using nude mice. Our experiments showed that Prox1 had inhibitory effect on both cell proliferation and tumor formation. cDNA microarray analysis indicated that the expression of PKCβII was highly suppressed by Prox1 overexpression. In Prox1-expressing Hela cells, mRNA expression of PKCβII was undetectable and protein was significantly down-regulated compared with Mock cells. The selective PKCβII inhibitor, enzastaurin was less effective on cell proliferation of Prox1-expressing cells compared with Mock cells. These results showed that PKCβII was required for tumor cell proliferation. We focused on the regulatory mechanism of PKCβII expression by Prox1. The 5'-promoter analysis of PKC-β gene revealed that the proximal region between 110bp of the promoter containing 2 Sp1 binding sites was responsible for the endogenous promoter activity, and Prox1 did not affect that. Mithramycin A, a relatively specific Sp1 inhibitor, and Sp family knockdown by siRNA on Hela cells decreased PKCβII expression, suggesting that Sp family was important for PKCβII transcription. Interestingly, 5-aza-deoxycytidine (5-Az) treatment restored PKCβII mRNA expression in Prox1-expressing Hela cells, suggesting that 5'-promoter region of PKCβII was methylated in these cells. Actually, bisulfite sequencing showed that the predicted CpG island of PKC-β gene, especially around these Sp1 binding sites, was highly methylated in Prox1-expressing Hela cells compared with Mock cells and 5-Az relieved DNA methylation.

Our study suggested that the decreased PKCβII expression suppressed tumor cell proliferation and Prox1 down-regulated PKCβII expression by enhancing DNA methylation of PKC-β 5'-promoter around Sp1 binding sites. This is the first to report the inverse relationship between Prox1 and PKCβII expression in cancer cells and the novel regulatory mechanism of PKC-β gene expression by Prox1 transcription factor.

No relevant conflicts of interest to declare.