Interaction of HIF-1α and Notch3 Is Required for the Expression of Carbonic Anhydrase 9 in Breast Carcinoma Cells

Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carc...

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Published inGenes & cancer Vol. 4; no. 11-12; pp. 513 - 523
Main Authors Shareef, Mohammed M, Udayakumar, Thirupandiyur S, Sinha, Vishal K, Saleem, Shahid M, Griggs, Wendy W
Format Journal Article
LanguageEnglish
Published United States SAGE Publications 01.11.2013
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Abstract Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.
AbstractList Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP 2191 suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.
Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.
Author Udayakumar, Thirupandiyur S
Saleem, Shahid M
Shareef, Mohammed M
Sinha, Vishal K
Griggs, Wendy W
AuthorAffiliation 1 Department of Radiation Oncology, University of Miami, Miami, FL, USA
2 Miller School of Medicine, University of Miami, Miami, FL, USA
4 Department of Computer Sciences, University of Kentucky, Lexington, KY, USA
3 Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA
5 Weis Center for Research, Geisinger Clinic, Danville, PA, USA
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Title Interaction of HIF-1α and Notch3 Is Required for the Expression of Carbonic Anhydrase 9 in Breast Carcinoma Cells
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