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);LLPLAP2191 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 513-523 |
| Number of pages | 11 |
| Journal | Genes and Cancer |
| Volume | 4 |
| Issue number | 11-12 |
| DOIs | |
| State | Published - Nov 2013 |
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Keywords
- CA9
- hypoxia
- Notch3
- PBX1
- VHL
ASJC Scopus subject areas
- Cancer Research
- Genetics
Cite this
Interaction of HIF-1α and Notch3 Is Required for the Expression of Carbonic Anhydrase 9 in Breast Carcinoma Cells. / Shareef, Mohammed M.; Udayakumar, Thirupandiyur; Sinha, Vishal K.; Saleem, Shahid M.; Griggs, Wendy W.
In: Genes and Cancer, Vol. 4, No. 11-12, 11.2013, p. 513-523.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Interaction of HIF-1α and Notch3 Is Required for the Expression of Carbonic Anhydrase 9 in Breast Carcinoma Cells
AU - Shareef, Mohammed M.
AU - Udayakumar, Thirupandiyur
AU - Sinha, Vishal K.
AU - Saleem, Shahid M.
AU - Griggs, Wendy W.
PY - 2013/11
Y1 - 2013/11
N2 - 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);LLPLAP2191 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.
AB - 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);LLPLAP2191 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.
KW - CA9
KW - hypoxia
KW - Notch3
KW - PBX1
KW - VHL
UR - http://www.scopus.com/inward/record.url?scp=84891551077&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84891551077&partnerID=8YFLogxK
U2 - 10.1177/1947601913481670
DO - 10.1177/1947601913481670
M3 - Article
AN - SCOPUS:84891551077
VL - 4
SP - 513
EP - 523
JO - Genes and Cancer
JF - Genes and Cancer
SN - 1947-6019
IS - 11-12
ER -