Purpose: The mechanisms underlying frequent aberrant STAT3 activation in human cancers, including HNSCC, remain incompletely understood. While genomic activation of positive regulators of STAT3, especially protein tyrosine kinases, have been studied at length, genomic inactivation of negative STAT3 regulators, including promoter methylation of protein tyrosine phosphatases that target STAT3, have been understudied. Here we investigate the hypothesis that silencing of PTPRT by promoter methylation leads to increased STAT3 signaling in HNSCC.
Experimental Design: We have analyzed HM-450 and RNA-Seq data available from TCGA for 279 HNSCC tumors to determine the incidence of PTPRT promoter hypermethylation and its association with mRNA expression. We further analyzed an independent cohort of HNSCC tumors by methylation-specific PCR (MSP) to confirm the incidence found in TCGA samples. These tumor samples were also analyzed by immunohistochemistry to assess STAT3 activation. A panel of HNSCC cell lines was used to identify models for further mechanistic studies. Treatment of PTPRT-methylated cells with 5-azacytidine (5-aza), a non-specific DNA demethylating agent, and/or PTPRT-targeted shRNA was performed to determine the specific effects of PTPRT promoter methylation in HNSCC cells.
Results: TCGA data indicate that the incidence of aberrant PTPRT promoter hypermethylation in HNSCC is 59.5% (166/279 tumors analyzed). The observed hypermethylation of PTPRT is significantly associated with downregulation of PTPRT mRNA in these tumors (P<0.0001), suggesting a functional consequence of this event. Analysis of an independent cohort of HNSCC tumors by MSP demonstrates that PTPRT is promoter methylated in 56.7% (17/30) of tumors analyzed to date, strikingly consistent with TCGA data. 87.5% (7/8) of HNSCC cell lines analyzed to date are methylated at the PTPRT promoter. Treatment of a PTPRT promoter-methylated HNSCC cell line (Cal27) with 5-aza leads to increased PTPRT mRNA expression with a concomitant decrease in pSTAT3(Y705) expression, suggesting that PTPRT is functionally silenced by promoter methylation in these cells. Combination of 5-aza with PTPRT-targeted shRNA successfully blocks 5-aza-mediated PTPRT upregulation and pSTAT3(Y705) downregulation, suggesting that the functional effects of 5-aza with regard to STAT3 activation are PTPRT-specific in these cells.
Conclusion: The PTPRT promoter is frequently aberrantly hypermethylated in HNSCC in association with downregulation of PTPRT mRNA. Treatment of HNSCC cells that exhibit PTPRT promoter methylation and low PTPRT expression with 5-aza successfully and specifically leads to upregulation of PTPRT and downregulation of pSTAT3(Y705), suggesting that PTPRT promoter methylation may serve as a biomarker for response to STAT3 pathway inhibitors. The combination of such inhibitors with 5-aza in the context of PTPRT promoter methylation warrants further investigation.
Citation Format: Noah D. Peyser, Lin Wang, Jennifer R. Grandis. Promoter methylation of PTPRT provides a mechanism for STAT3 activation in HNSCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5286. doi:10.1158/1538-7445.AM2014-5286