Carcinogenesis Advance Access originally published online on March 13, 2008
Carcinogenesis 2008 29(10):1853-1861; doi:10.1093/carcin/bgn066
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Hypoxia-inducible factor (HIF)-1
directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF)
1 The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020, China
2 National Engineering Research Centers of Cell Products, AmCellGene Co. Ltd, Tianjin Economic and Development Area, Tianjin 300457, China
3 Tianjin Economic and Development Area Life and Technology Research Center, Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin Economic and Development Area, Tianjin 300457, China
* To whom correspondence should be addressed. Tel: +86 22 27317273; Fax: +86 22 27317273; Email: zchan{at}amcellgene.com
Stem cell factor (SCF) plays important roles in tumor growth and angiogenesis. However, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulated the expression of SCF in MCF-7 breast cancer cells in both messenger RNA and protein levels. When hypoxia-inducible factor (HIF)-1
expression was knocked down by RNA interference, the MCF-7 cell expression of SCF was decreased significantly. Furthermore, the SCF receptor, c-kit phosphorylation was significantly strengthened by the condition culture media from hypoxic MCF-7 and MCF-7-c cells. The survival of A549 cells was more dependent on SCF under hypoxia. Analysis of SCF promoter 5'-flanking region revealed a potential hypoxia-response element (HRE; 5'-GCGTG-3') located at –68 to –64 relative to the transcriptional start site. Chromatin immunoprecipitation assay demonstrated that HIF-1 directly bound to this region under normoxia, and this binding activity was significantly enhanced under hypoxia. Overexpression of HIF-1 significantly upregulated the expression of luciferase reporter gene under control of the SCF promoters in both MCF-7 cells and human embryonic kidney 293 cells, but mutation of the HRE site completely blocked this effect. Epidermal growth factor was also able to enhance the SCF expression under normoxia in MCF-7 cells, which was dependent on HIF-1. Taken together, our data demonstrated that HIF-1 was a key regulator of SCF expression in breast cancer cells. Hypoxia and epidermal growth factor receptor signal coexisted in the tumor microenvironment and might promote angiogenesis through HIF-1-mediated upregulation of SCF and other angiogenic factors.
Abbreviations: CCM, condition culture media; cDNA, complementary DNA; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; HEK293, human embryonic kidney; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; HUVEC, human umbilical vein endothelial cell; mRNA, messenger RNA; PI3K—AKT, phosphatidylinositol 3-kinase–serine-threonine protein kinase Akt; PCR, polymerase chain reaction; RT, reverse transcription; SCF, stem cell factor; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA; sSCF, secreted stem cell factor; VEGF, vascular endothelial growth factor
These authors contributed equally to this work. Received October 16, 2007; revised February 16, 2008; accepted February 29, 2008.