Zhenghe Wang

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Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. If p21 is disrupted via gene targeting, the cells die through apoptosis. If the PUMA gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X(L) and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of BAX makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.

Tyrosine phosphorylation, regulated by protein tyrosine phosphatases (PTPs) and kinases (PTKs), is important in signaling pathways underlying tumorigenesis. A mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs (PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, PTPN14), affecting 26% of colorectal cancers and a smaller fraction of lung, breast, and gastric cancers. Fifteen mutations were nonsense, frameshift, or splice-site alterations predicted to result in truncated proteins lacking phosphatase activity. Five missense mutations in the most commonly altered PTP (PTPRT) were biochemically examined and found to reduce phosphatase activity. Expression of wild-type but not a mutant PTPRT in human cancer cells inhibited cell growth. These observations suggest that the mutated tyrosine phosphatases are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.

DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation. We describe a previously unknown mode of regulation of DNMT1 protein stability through the coordinated action of an array of DNMT1-associated proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60, which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. In contrast, DNMT1 was stabilized by histone deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus-associated ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as well as the association between HAUSP and DNMT1, suggested that during the cell cycle the initiation of DNMT1 degradation was coordinated with the end of DNA replication and the need for DNMT activity. In human colon cancers, the abundance of DNMT1 correlated with that of HAUSP. HAUSP knockdown rendered colon cancer cells more sensitive to killing by HDAC inhibitors both in tissue culture and in tumor xenograft models. Thus, these studies provide a mechanism-based rationale for the development of HDAC and HAUSP inhibitors for combined use in cancer therapy.

Following its tyrosine phosphorylation, STAT3 is methylated on K140 by the histone methyl transferase SET9 and demethylated by LSD1 when it is bound to a subset of the promoters that it activates. Methylation of K140 is a negative regulatory event, because its blockade greatly increases the steady-state amount of activated STAT3 and the expression of many (i.e., SOCS3) but not all (i.e., CD14) STAT3 target genes. Biological relevance is shown by the observation that overexpression of SOCS3 when K140 cannot be methylated blocks the ability of cells to activate STAT3 in response to IL-6. K140 methylation does not occur with mutants of STAT3 that do not enter nuclei or bind to DNA. Following treatment with IL-6, events at the SOCS3 promoter occur in an ordered sequence, as shown by chromatin immunoprecipitations. Y705-phosphoryl-STAT3 binds first and S727 is then phosphorylated, followed by the coincident binding of SET9 and dimethylation of K140, and lastly by the binding of LSD1. We conclude that the lysine methylation of promoter-bound STAT3 leads to biologically important down-regulation of the dependent responses and that SET9, which is known to help provide an activating methylation mark to H3K4, is recruited to the newly activated SOCS3 promoter by STAT3.