Involvement of Hypoxia-inducing Factor-1α-dependent Plasminogen Activator Inhibitor-1 Up-regulation in Cyr61/CCN1-induced Gastric Cancer Cell Invasion

Abstract

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1α (HIF-1α) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1α protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1α protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl2 treatment, both well known for induction of HIF-1α. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1α up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1α protein accumulation. Blockage of HIF-1α activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1α strongly inhibited their invasion ability, suggesting that elevation in HIF-1α protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1α-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1α to block HIF-1α activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1α-dependent up-regulation of PAI-1. Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1α (HIF-1α) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1α protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1α protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl2 treatment, both well known for induction of HIF-1α. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1α up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1α protein accumulation. Blockage of HIF-1α activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1α strongly inhibited their invasion ability, suggesting that elevation in HIF-1α protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1α-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1α to block HIF-1α activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1α-dependent up-regulation of PAI-1. An emerging family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth is named the CCN family. Cysteine-rich 61 (Cyr61) 2The abbreviations used are: Cyr61, cysteine-rich 61; rCyr61, recombinant Cyr61; HIF-1, hypoxia-inducing factor-1; DN, dominant negative; PAI-1, plasminogen activator inhibitor-1; CHX, cycloheximide; IGF-1, insulin-like growth factor-1; HRE, hypoxia response element; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; RT, reverse transcription; eIF4E, eukaryotic initiator factor 4E. belongs to the CCN family of angiogenic regulators, which consist of Cyr61/CCN1, connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (Nov/CCN3), Wisp-1/elm1 (CCN4), Wisp-2/rCop1 (CCN5), and Wisp-3 (CCN6) (1Kang Y. Siegel P.M. Shu W. Drobnjak M. Kakonen S.M. Cordon-Cardo C. Guise T.A. Massague J. Cancer Cell. 2003; 3: 537-549Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar, 2Mo F.E. Muntean A.G. Chen C.C. Stolz D.B. Watkins S.C. Lau L.F. Mol. Cell Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (349) Google Scholar, 3Tsai, M. S., Bogart, D. F., Castaneda, J. M., Li, P., and Lupu, R. (2002) Oncogene, 21, 8178-8185Google Scholar). The CCN family shares a uniform modular structure, which mediates various cellular functions such as angiogenesis, cell proliferation, cell migration, and neoplastic transformation (4Ristimaki A. Honkanen N. Jankala H. Sipponen P. Harkonen M. Cancer Res. 1997; 57: 1276-1280PubMed Google Scholar, 5Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath Jr., C.W. Cancer Res. 1993; 53: 1322-1327PubMed Google Scholar, 6Uefuji K. Ichikura T. Mochizuki H. J. Surg. Oncol. 2001; 76: 26-30Crossref PubMed Scopus (77) Google Scholar). Cyr61 is a ligand that is encoded by an early response gene responsible for proangiogenesis. It regulates cell adhesion, ameliorates growth factor-stimulated DNA synthesis, and promotes neovascularization and tumor growth (7Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (433) Google Scholar, 8Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar, 9Lau L.F. Nathans D. EMBO J. 1985; 4: 3145-3151Crossref PubMed Scopus (367) Google Scholar). Results from our own studies and investigations of other have demonstrated that Cyr61 expression is associated with advanced breast adenocarcinoma, pancreatic cancer, gliomas, and gastric adenocarcinoma (3Tsai, M. S., Bogart, D. F., Castaneda, J. M., Li, P., and Lupu, R. (2002) Oncogene, 21, 8178-8185Google Scholar, 10Lin M.T. Chang C.C. Chen S.T. Chang H.L. Su J.L. Chau Y.P. Kuo M.L. J. Biol. Chem. 2004; 279: 24015-24023Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Lin M.T. Zuon C.Y. Chang C.C. Chen S.T. Chen C.P. Lin B.R. Wang M.Y. Jeng Y.M. Chang K.J. Lee P.H. Chen W.J. Kuo M.L. Clin. Cancer Res. 2005; 11: 5809-5820Crossref PubMed Scopus (85) Google Scholar, 12Menendez J.A. Mehmi I. Griggs D.W. Lupu R. Endocr. Relat. Cancer. 2003; 10: 141-152Crossref PubMed Scopus (109) Google Scholar, 13Tsai M.S. Bogart D.F. Li P. Mehmi I. Lupu R. Oncogene. 2002; 21: 964-973Crossref PubMed Scopus (69) Google Scholar, 14Xie D. Yin D. Tong X. O'Kelly J. Mori A. Miller C. Black K. Gui D. Said J.W. Koeffler H.P. Cancer Res. 2004; 64: 1987-1996Crossref PubMed Scopus (157) Google Scholar). Tumor invasion and metastasis are critical steps in the aggressive phenotype of human cancers. In a clinical study, Cyr61 is overexpressed in 39% of primary human breast cancers (14Xie D. Yin D. Tong X. O'Kelly J. Mori A. Miller C. Black K. Gui D. Said J.W. Koeffler H.P. Cancer Res. 2004; 64: 1987-1996Crossref PubMed Scopus (157) Google Scholar). Overexpression of Cyr61 in breast cancer and glioma cells resulted in enhancement of anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice (14Xie D. Yin D. Tong X. O'Kelly J. Mori A. Miller C. Black K. Gui D. Said J.W. Koeffler H.P. Cancer Res. 2004; 64: 1987-1996Crossref PubMed Scopus (157) Google Scholar). Our previous observation has also pointed out that the level of Cyr61 expression is positively correlated with cancer stage, tumor size and lymph node metastasis in gastric cancer patients (11Lin M.T. Zuon C.Y. Chang C.C. Chen S.T. Chen C.P. Lin B.R. Wang M.Y. Jeng Y.M. Chang K.J. Lee P.H. Chen W.J. Kuo M.L. Clin. Cancer Res. 2005; 11: 5809-5820Crossref PubMed Scopus (85) Google Scholar). These findings strongly support that the Cyr61 expression is associated with an aggressive phenotype and may play a vital role during tumor invasion. Insight into the mechanism underlying Cyr61-mediated cancer cell invasion is critical and as yet unexplored. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that is composed of two subunits, HIF-1α and HIF-1β. HIF-1β, also known as the arylhydrocarbon nuclear transcription translocator, is constitutively expressed, whereas HIF-1α expression is increased upon hypoxia (16Semenza G.L. Biochem. Pharmacol. 2000; 59: 47-53Crossref PubMed Scopus (450) Google Scholar). Under hypoxic conditions, HIF-1α is stabilized, dimerizes with HIF-1β, translocates into the nucleus, and transactivates a broad array of downstream genes, including activation mitogenic, pro-invasive, pro-angiogenic, and pro-metastatic genes (17Lewis C. Murdoch C. Am. J. Pathol. 2005; 167: 627-635Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Wenger R.H. J. Exp. Biol. 2000; 203: 1253-1263Crossref PubMed Google Scholar). Interestingly, it has been demonstrated that HIF-1α is also regulated by oxygen-independent mechanisms. Oncogenic genes (e.g. Ha-ras, myc, or src), tumor suppressor genes (e.g. p53, PTEN, or VHL), and a variety of growth factors (EGF, IGF-1, insulin, interleukin-1β, HGF, TNF-α, thrombin) are associated with HIF-1-mediated tumor progression (19Long L. Rubin R. Brodt P. Exp. Cell Res. 1998; 238: 116-121Crossref PubMed Scopus (69) Google Scholar). To date, numerous reports have proven that HIF-1α is overexpressed in many human cancers. HIF-1α-transactivated genes, such as iNOS, IGF, and VEGF, playing important roles in tumor metastasis and invasion (20Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google are overexpressed in a of tumors and cell R. T.V. C. M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). the detailed role of HIF-1α in tumor invasion is in of the tumor cells. In study, we the of HIF-1α in invasion ability in gastric cancer cells. found that HIF-1α protein was elevated and constitutively in Cyr61-overexpressing gastric cancer cells. (DN)-HIF-1α could the invasive ability of Cyr61-overexpressing suggesting a critical role of HIF-1α in such an invasion activator inhibitor-1 a HIF-1α-dependent invasion was significantly in Cyr61-overexpressing cells. Our a mechanism for HIF-1α-dependent up-regulation of PAI-1, which to the invasion activity of Cyr61. and human Cyr61 was from cycloheximide and were from was from or or ERK1/2 and were from or or and or antibodies were from Cell was from PAI-1 was from The Cell gastric cells and were from the and and was from and gastric cell were in with and and from Cell were in a cells were from the with and are as and are as and nuclear and protein the protein were from cells or cells a as M.L. Su J.L. C.Y. Chen Chang C.C. Kuo M.L. Mol. Pharmacol. 2005; PubMed Scopus Google Scholar). HIF-1α are as are as of the are as PAI-1 of the PAI-1-specific antisense oligonucleotides was and that of oligonucleotides was PAI-1 gastric cancer cells were to and PAI-1 antisense oligonucleotides were transfected into tumor cells. of the cells were used for invasion or for cells were to a of in and with or rCyr61 for and DNA were to the in R. T.V. C. M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google of the PAI-1 was by the and were by a and with Cyr61 expression was associated with HIF-1α expression in a of human gastric adenocarcinoma cell these cell we found a the expression levels of Cyr61 and HIF-1α protein but not mRNA levels expression of Cyr61 by transfected with of Cyr61 expression significantly HIF-1α protein level in and cells with a In addition, cells were transfected with Cyr61 expression resulted in significantly HIF-1α protein level Cyr61 a that is to a To we the Cyr61 protein in from the gastric adenocarcinoma cells. demonstrated a Cyr61 protein in from cells. of Cyr61 proteins were also from Cyr61 transfected cells To the Cyr61 has an HIF-1α we Cyr61 protein by (7Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (433) Google Scholar, M.L. F.E. Lau L.F. Mol. Cell Biol. PubMed Scopus Google Scholar, Lau L.F. Cell Google by Lau of to the from and Cyr61 transfected and both cells significant of Cyr61 Cyr61 protein in significantly the endogenous HIF-1α protein level to the induction of HIF-1α Cyr61, we recombinant Cyr61 (rCyr61) into the to the downstream of human gastric adenocarcinoma cells to rCyr61 for resulted in a induction of HIF-1α protein expression and rCyr61 were with gastric adenocarcinoma cell we used a hypoxia and as a comparison, are well known of HIF-1α that of cells to rCyr61, or not significantly HIF-1α mRNA levels the rCyr61 to cells resulted in HIF-1α protein expression The of CoCl2 was also found that CoCl2 is a of HIF-1α expression with Cyr61. These that Cyr61 and CoCl2 play a distinct role in signaling to increase the HIF-1α protein in the of HIF-1α protein level increased suggesting the Cyr61 and growth factors in HIF-1α induction mechanism Cyr61 HIF-1α protein in human gastric adenocarcinoma cells with a induction as with CoCl2 and HIF-1α is a transcription factor that nuclear for the nuclear of HIF-1α protein in cells with and significant levels of HIF-1α in nuclear of cells with rCyr61 but not in cells also nuclear for HIF-1α in cells but not in the cells were with the Cyr61 transfected cells and Our implicated that Cyr61 expression and nuclear of HIF-1α. we HIF-1α is correlated with the transcription activity of cells were transfected with a Treatment with and rCyr61 resulted in and in activity with cells the induction was also in Cyr61 transfected cells induction with that of we transfected with a a of HIF-1α that effectively with endogenous HIF-1α for with that in of the of with target and the activation M.L. Su J.L. C.Y. Chen Chang C.C. Kuo M.L. Mol. Pharmacol. 2005; PubMed Scopus Google Scholar, J.A. Semenza G.L. Mol. Cell Biol. PubMed Scopus Google Scholar, E. Wang G.L. R. Semenza G.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The showed that the activity was significantly by the in cells. Our data that Cyr61 not only the expression of HIF-1α protein but also the transcription of HIF-1α mRNA that was significant increase in HIF-1α mRNA expression by rCyr61 in human gastric cells that Cyr61 was to HIF-1α mRNA These the of increased of HIF-1α protein were the of reduced of the protein in response to recombinant Cyr61 or an increase in of the To the of Cyr61 HIF-1α protein synthesis, we a of HIF-1α in the of the protein to the and the of data by from the of HIF-1α was than in cells but in recombinant cells. observation is with previous studies that CoCl2 HIF-1α but also that was significant in HIF-1α protein upon in the or of recombinant Cyr61 rCyr61 to increase HIF-1α protein levels by rather than by To these of rCyr61 for in cells and with or the for HIF-1α protein was highly elevated in cells than in cells and HIF-1α protein promoted by MG132, was significantly inhibited by with both and and Cyr61 HIF-1α expression by of the protein, it to have an with which by the of the of cells to rCyr61 with CoCl2 resulted in a of HIF-1α protein The that HIF-1α protein by Cyr61 is through de novo protein To the the of Cyr61 HIF-1α protein cells were with and which are of MEK, PI3K, and kinase these inhibited the induction of HIF-1α protein expression in cells To the and were or in the of and were also The increased of that was by rCyr61 was by but not by or The increased of which was by the rCyr61 treatment, was by but not by or the induction of HIF-1α by rCyr61 was inhibited in a by and both and are for induction of HIF-1α protein In and significant the expression of HIF-1α in cells These that Cyr61 and CoCl2 as a distinct mechanisms. The PI3K, and has been to protein via of and G.L. Cancer. 2003; 3: PubMed Scopus Google Scholar). we Cyr61 HIF-1α expression through a and we also the of and in response of In HIF-1α protein and the of both and which were by rCyr61 could by in a Our that the Cyr61 HIF-1α through and the found that Cyr61 was highly in advanced gastric adenocarcinoma, and of Cyr61 in human gastric cancer cell significantly increased their invasion (11Lin M.T. Zuon C.Y. Chang C.C. Chen S.T. Chen C.P. Lin B.R. Wang M.Y. Jeng Y.M. Chang K.J. Lee P.H. Chen W.J. Kuo M.L. Clin. Cancer Res. 2005; 11: 5809-5820Crossref PubMed Scopus (85) Google Scholar). To the we HIF-1α in gastric cancer cell invasion. of and expression to cells significantly the invasion ability were also in cells In cells transfected with Cyr61 expression significantly cell invasion and when with expression the invasion ability in gastric cells is to the increase in HIF-1α The as to which gene is the downstream that to the Cyr61-mediated HIF-1α-dependent cell invasion. It has been known that several genes such as PAI-1, and are the of HIF-1α G.L. Cancer. 2003; 3: PubMed Scopus Google Scholar). To we the expression of these genes in cells by It that only the mRNA of PAI-1 was increased in a in cells with cells of PAI-1 protein expression were also in Cyr61-overexpressing cells In the previous (11Lin M.T. Zuon C.Y. Chang C.C. Chen S.T. Chen C.P. Lin B.R. Wang M.Y. Jeng Y.M. Chang K.J. Lee P.H. Chen W.J. Kuo M.L. Clin. Cancer Res. 2005; 11: 5809-5820Crossref PubMed Scopus (85) Google we have found that Cyr61 is in invasion in human gastric cancer cell In study, we have demonstrated that Cyr61 HIF-1α-dependent invasion protein PAI-1. to PAI-1 is in Cyr61-mediated gastric cancer cell invasion, gastric cancer cell including and were for their expression levels of PAI-1 protein and in invasion Our that the level of PAI-1 protein was highly elevated in cells but was in cells. cells an level of PAI-1 protein with other gastric cancer cells In a cells the invasive ability these gastric cancer cell In which of PAI-1, invasive These to that PAI-1 could in the progression of human gastric cancer cells. To the role of PAI-1 in Cyr61-mediated cell invasive of PAI-1 expression was with antisense the PAI-1-specific antisense oligonucleotides used significantly the invasion ability by in cells with the was used to the PAI-1-specific antisense oligonucleotides activity with PAI-1-specific antisense oligonucleotides in Cyr61-overexpressing cells also abolished cell invasion in a Supportively, of Cyr61-overexpressing cells with effectively inhibited gastric cancer cell invasion The studies and demonstrated a critical role of PAI-1 in Cyr61-mediated up-regulation of the gastric cancer cell invasion. of HIF-1α by with significantly reduced the of PAI-1 mRNA expression in suggesting that the HIF-1α is for the PAI-1 mRNA expression that the increase of PAI-1 protein in cells was also effectively by with we HIF-1α to the response the PAI-1 gene by in were and of the PAI-1 Treatment of cells with rCyr61 or by showed that HIF-1α was constitutively to of the PAI-1 In the Cyr61 PAI-1 expression is by the HIF-1α-dependent of the CCN family are growth and the of their to the cellular clinical that Cyr61, one of CCN family is in the and progression of human malignancies. The significant of Cyr61 in human by of tumor Cyr61 of and cells. N. A. L. Cancer Res. PubMed Scopus Google have demonstrated that Cyr61 expression tumor cell and invasion in breast cancer and their that gene of Cyr61 in breast cancer cells induction in the detailed mechanism by which Cyr61 tumor invasiveness has not yet been In the study, we a of Cyr61 in gastric cancer cell invasion by for the that Cyr61 is to HIF-1α through de novo protein up-regulation of HIF-1α by Cyr61 in a variety of gastric cancer cell PI3K/AKT/mTOR and ERK1/2 signaling we the mechanism for cell invasion, which is by HIF-1α-dependent PAI-1 The role of HIF-1α in tumor has been in cell P. M. A. C. Cancer Res. 2000; Google Scholar, M. M. H. T. S. K. T. M. T. Exp. Mol. Pathol. 2005; PubMed Scopus Google Scholar, A. C. A. E. Clin. Exp. 2005; 22: PubMed Scopus Google Scholar, S. D. N. J. P. Semenza G.L. Cancer Res. 2003; Google Scholar, E. M. Exp. Cell Res. 2005; PubMed Scopus Google Scholar, D. H. Laughner E. 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PAI-1 is to plasminogen and the has a highly reduced for and the to In conclusion, we have that Cyr61 the transcription factor HIF-1α protein through PI3K/AKT/mTOR and ERK1/2 signaling in cells. Cyr61 expression is correlated with HIF-1α and downstream gene PAI-1. Blockage of HIF-1α activation to reduced PAI-1 and in cell invasion. a detailed mechanism underlying the role of Cyr61 in gastric cancer ability via an HIF-1α-dependent up-regulation of PAI-1. with