Sp-1 and c-Myc mediate lysophosphatidic acid-induced expression of vascular endothelial growth factor in ovarian cancer cells via a hypoxia-inducible factor-1-independent mechanism.
OBJECTIVE
Lysophosphatidic acid (LPA), which is present in ascites of ovarian cancer patients, stimulates expression of vascular endothelial growth factor (VEGF). VEGF is essential for the development and abdominal dissemination of ovarian cancer. We examined how LPA drives VEGF expression to gain a better understanding of tumor angiogenesis under normoxic conditions.


METHODS
ELISA, Northern blotting, immunoblotting, quantitative PCR, and promoter reporter analysis in combination with small interfering RNA and pharmacologic inhibitors were used to examine LPA-induced VEGF expression and the underlying mechanisms.


RESULTS
LPA stimulated expression of multiple VEGF variants. A 123-bp fragment proximal to the transcriptional initiation site was identified to be functional promoter region responsible for the response to LPA. The fragment harbors consensus sites for several transcription factors including c-Myc and Sp-1 but not hypoxia-inducible factor-1. Blockade of Rho, ROCK, or c-Myc reduced LPA-dependent VEGF production and promoter activation, suggesting that the G12/13-Rho-ROCK-c-Myc cascade partially contributes to VEGF induction by LPA. More significantly, the multiple Sp-1 sites within the responsive region of the VEGF promoter were essential for LPA-mediated transcription. LPA induced Sp-1 phosphorylation and DNA-binding and transcriptional activities. The silencing of Sp-1 expression with small interfering RNA or inhibition of Sp-1 with pharmacologic inhibitors blocked VEGF production induced by LPA.


CONCLUSIONS
LPA stimulates hypoxia-inducible factor-1-independent VEGF expression to promote tumor angiogenesis through activation of the c-Myc and Sp-1 transcription factors.
Song
Yuanda
Y
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
Wu
Jinhua
J
Oyesanya
Regina A
RA
Lee
Zendra
Z
Mukherjee
Abir
A
Fang
Xianjun
X
D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis*; D008246:Lysophospholipids / Q000378:metabolism*; D008954:Models, Biological; D009389:Neovascularization, Pathologic; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000737:chemistry
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008246:Lysophospholipids; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; C032881:lysophosphatidic acid