Activation of Hypoxia-Inducible Factor-1α Is Necessary for Lysophosphatidic Acid–Induced Vascular Endothelial Growth Factor Expression

Abstract

Purpose: Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). In the present study, we investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF. Experimental Design: Immunoblotting, reverse transcription-PCR, ELISA, immunofluorescence, and chromatin immunoprecipitation assay were used to examine the expression of VEGF and HIF-1α in various cancer cells. Specific HIF-1α small interfering RNA was transfected to various cancer cells to determine the role of HIF-1α in LPA-induced VEGF expression. Results: LPA induced expressions of VEGF and HIF-1α in OVCAR-3, CAOV-3, PC-3, and SK-Hep1 cells but not in SKOV-3 and Hep-3B cells. In OVCAR-3 and PC-3 cells, the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin/p70S6K and p42/p44 mitogen-activated protein kinase pathways were required for LPA-induced HIF-1α and VEGF expressions, whereas only the phosphoinositide 3-kinase/mammalian target of rapamycin/p70S6K pathway was important in SK-Hep1 cells. Immunofluorescence microscopy assay showed translocation of HIF-1α to nucleus by LPA, and chromatin immunoprecipitation assay revealed the binding of HIF-1α to the promoter of VEGF by LPA. Importantly, we found that small interfering RNA–induced reduction of HIF-1α expression significantly attenuated VEGF expression by LPA. Conclusions: Our results show for the first time that LPA induces VEGF via HIF-1α activation and reveal a critical role of HIF-1α in LPA-induced cancer cell proliferation and angiogenesis.

Regulation of Hypoxia-inducible Factor 1α (HIF-1α) by Lysophosphatidic Acid Is Dependent on Interplay between p53 and Krüppel-like Factor 5

“…Additional factors such as PI3KCA or PTEN status might contribute to HIF-1␣ regulation. In keeping with this idea, LPA-induced HIF-1␣ in ovarian cancer OVACAR-3 and pancreatic cancer PC-3 cells was PI3K-dependent, whereas we found that LY290042 does not attenuate HIF-1␣ induction in colon cancer cells (41). Alternatively, HIF-1␣ induction by LPA could be controlled by the relative level of mutp53 (see below).…”

“…Consistently, LPA induces HIF-1␣ and VEGF expression in SK-Hep1 cells with wtp53 but not in p53-null Hep-3b cells (41,42). On the other hand, it is reported that LPA stimulated HIF-1␣ expression in ovarian OVCAR-3 and CAVOV-3 cells, despite the presence of mutp53 (41,43). The basis for the differential cellular responses by the different cell types is not known.…”

The Rho/ROCK pathway for lysophosphatidic acid-induced proteolytic enzyme expression and ovarian cancer cell invasion

“…Immunoblotting Cells lysates were prepared as described (Lee et al, 2006). Antibodies for Rho and glyceraldehyde-3-phosphate dehydrogenase were from Upstate Biotechnology (Lake Placid, NY, USA) and Santa Cruz Biotechnology Inc., respectively.…”

Enhancement of invasion of hepatocellular carcinoma cells through lysophosphatidic acid receptor

“…LPA activates multiple intracellular signalling pathways including the mitogen-activated protein (MAP) kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, promoting growth factors and protease production, and induction of tumour cell invasion. 29 LPAR-mediated downstream molecular signalling pathways have yet to be fully investigated, however. LPA upregulates the hypoxia-inducible factor-1a subunit by activating PI3K and MAPK signalling in SK-Hep1 HCC cells, 28 and MMP9 expression is induced through activation of extracellular signal-regulated kinase (ERK) and PI-3K/Akt pathways.…”