Abstract
Using a retrovirus-mediated cDNA expression cloning approach, we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays, we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant negative GRHL2 proteins induces down-regulation of ERBB3 gene expression, a striking reduction in cell proliferation, and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying contradictory roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc finger enhancer-binding protein 1 (ZEB1), which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis.
Highlights
The role of the developmental transcription factor grainyhead-like 2 (GRHL2) in breast carcinogenesis is ill defined
We identified the GRHL2 transcription factor as a novel protooncogene capable of transforming NIH3T3 fibroblasts
Transforming Activities of the GRHL2 Transcription Factor— To identify genes possibly involved in breast carcinogenesis, a retrovirus-mediated cDNA expression cloning approach for the identification of protooncogenes, utilizing preneoplastic NIH3T3 fibroblasts as a recipient cell line, was employed
Summary
The role of the developmental transcription factor GRHL2 in breast carcinogenesis is ill defined. These and several other developmental studies [4, 7,8,9] clearly established a crucial role of GRHL2 in embryonic development, an implication of GRHL2 in other physiological processes, such as, for example, wound healing and cancer, is less well defined This is surprising because two members of the grainyhead family of transcription factors, namely dGrh and GRHL3, have attracted considerable interest in that these genes could be identified as important regulators in epithelial barrier formation and wound healing in flies and vertebrates, respec-. A comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers further demonstrates the pathophysiological relevance of GRHL2 in breast carcinogenesis
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