Regulation of vascular endothelial growth factor expression ... - Nature

Oncogene (2001) 20, 3751 ± 3756 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Regulation of vascular endothelial growth factor expression by acidosis in human cancer cells Qian Shi1, Xiangdong Le1, Bailiang Wang1, James L Abbruzzese1, Qinghua Xiong1, Yanjuan He1 and Keping Xie*,1,2 1

Department of Gastrointestinal Medical Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, TX 77030, USA; 2Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, TX 77030, USA

The in¯uence of acidosis on the expression of the vascular endothelial growth factor (VEGF) gene was determined. FG human pancreatic adenocarcinoma cells were incubated for various time periods in media at a physiologically relevant pH level (6.7 ± 7.4). The expression of VEGF mRNA and protein secretion was inversely correlated with pH in a pH- and time-dependent manner. Transient acidosis also activated the VEGF promoter/ enhancer luciferase reporter, which was consistent with an increased VEGF gene transcription rate and VEGF mRNA half-life. These data indicated that acidosis transcriptionally and posttranscriptionally regulates VEGF expression, suggesting that an acidic tumor microenvironment contributes to tumor angiogenesis and progression. Oncogene (2001) 20, 3751 ± 3756. Keywords: angiogenesis; VEGF; regulation; acidosis; pancreatic cancer Tumor growth and metastasis are dependent on angiogenesis, which requires coupled interactions among malignant cells, cells resident in the surrounding tissue, and cells recruited from the circulation. The induction of angiogenesis is mediated by several molecules released by both tumor cells and host cells (Folkman, 1996). Among the numerous biologically active compounds, vascular endothelial growth factor (VEGF) also known as vascular permeability factor has been shown to play a key role in angiogenesis (Leung et al., 1989; Senger et al., 1983). Despite the intensive angiogenesis characteristic of some tumors, the overall vasculature of most tumors is highly defective and marginally functional, leading to heterogeneous perfusion of tumor tissues (Wike-Hooley et al., 1984; Kallinowski and Vaupel, 1988; Rotin et al., 1989; Vaupel et al., 1989; Tannock, 1996). Measurements using pH microelectrodes have shown that the extracellular pH of solid tumors is generally lower or more acidic than that of corresponding normal tissues

*Correspondence: K Xie Received 23 October 2000; revised 22 March 2001; accepted 2 April 2001

(Wike-Hooley et al., 1984; Tannock, 1996). This low extracellular pH results mainly from elevated glycolysis and defective vascularization (Boucher et al., 1990; Intaglietta, 1977; Shubik, 1982; Vaupel et al., 1989). The extracellular pH is expected to be particularly low in the hypoxic regions of tumors due to upregulated anaerobic glycolysis in viable hypoxic tumor cells and retarded removal of metabolic wastes (Intaglietta, 1977; Shubik, 1982; Vaupel et al., 1989). In addition, the coexistence of hypoxia and acidosis may profoundly in¯uence many aspects of cancer biology (Blancher and Harris, 1998). While hypoxia has been shown to regulate tumor angiogenesis via induction of VEGF (Shweiki et al., 1992; Blancher and Harris, 1998), it remains unclear whether tumor acidosis also regulates VEGF expression and tumor angiogenesis. VEGF is a heparin-binding glycoprotein having potent angiogenic, mitogenic, and vascular permeability-enhancing activities speci®c for endothelial cells (Senger et al., 1983; Leung et al., 1989). It is expressed by various normal tissue cells and overexpressed by numerous tumor cells. Although it is constitutively expressed by many tumor cells and transformed cell lines, including human pancreatic cancer cells (Itakura et al., 1997), the expression of VEGF is still subject to regulation by various factors both in vitro and in vivo in the tumor microenvironment (Senger et al., 1983; Leung et al., 1989). Recent studies have shown that although VEGF protein can be detected in tumor cells distributed throughout proliferating tumors, the most signi®cant elevation in VEGF expression has been found predominantly in tumor cells surrounding necrotic areas, where the tumor cells are believed to undergo severe hypoxic and acidic stress (Shweiki et al., 1992; Shi et al., 1999, 2000). Whether acidic pH regulates VEGF expression is not clear, however. To determine the role of low tumor pH in the expression and regulation of VEGF, FG human pancreatic adenocarcinoma cells (Vezeridis et al., 1989; Shi et al., 1999, 2000) were plated in 15-cm culture dishes at 90% con¯uence. Twenty-four hours later, the cells were given fresh medium at pH 7.4, 7.1, 6.9, or 6.7 and incubated for 6 and 12 h. Also cellular mRNA was harvested for VEGF mRNA analysis using Northern blotting. As shown in Figure 1a, the

Regulation of VEGF by acidosis Q Shi et al

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Figure 1 In¯uence of acidosis on VEGF expression. The FG cells were incubated in medium at dierent pH for 6 and 12 h. Cellular mRNA was isolated using the FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA), and VEGF expression was determined using standard Northern blot analysis and equal loading was monitored by hybridizing the same membrane ®lter with b-actin (a). The quantitation of VEGF secretion into the culture supernatants was carried out using the VEGF ELISA kit (R & D Systems Inc., Minneapolis, MN, USA) (b). FG human pancreatic adenocarcinoma cells were incubated for 12 h in medium at dierent pH and VEGF, IL-8, and bFGF mRNA expression was determined using Northern blot analysis (c). SKOV-3 human ovarian cancer, PC-3 human prostate cancer, and SW 620 human colon cancer cells were also incubated in medium at dierent pH levels. VEGF expression was determined using Northern blot analysis (d, e, and f). Note that a low pH induced VEGF expression at mRNA and protein levels (*P50.01)

expression of VEGF mRNA was increased when cells were treated for 6 h in culture medium at pH 7.1, 6.9 or 6.7. Longer incubation led to further elevation of VEGF mRNA expression in the cells in the medium at pH 7.1 or 6.9, but signi®cantly decreased expression in the cells in the medium at pH 6.7. Additionally, culture supernatants were harvested for measurement of VEGF secretion using ELISA. Consistent with the elevation of VEGF mRNA expression, the acidic culture medium led to increased VEGF secretion (Figure 1b). These data indicated that short exposure to acidosis upregulates VEGF expression; however, extensive exposure to acidosis or intensive acidosis inhibited VEGF expression. Therefore, depending on the extent of exposure and cell type, a low pH may lead to diverse expression of VEGF (Jang and Hill, 1997; Scott et al., 1998; Brooks et al., 1998; D'Arcangelo et al., 2000). To determine whether the expression of other angiogenic molecules was also aected, the FG cells were incubated for 6 h in medium at pH 7.4, 7.1, 6.9 or 6.7. Cellular mRNA was isolated, and the expression of VEGF, interleukin-8 (IL-8), and basic ®broblast Oncogene

growth factor (bFGF) was determined using Northern blot analysis. As shown in Figure 1c, VEGF expression was upregulated, as was IL-8 expression, which was consistent with our recent reports (Shi et al., 1999, 2000). However, the expression of bFGF was not signi®cantly aected. Therefore, the regulation of gene expression by acidosis was gene-speci®c. To determine whether low extracellular pH induced VEGF expression in other human cell lines, SK-OV-3 human ovarian cancer (ATCC #HTB77), SW620 human colon cancer (ATCC #CCL227), and PC-3 human prostate cancer cells (ATCC #CRL1435) were incubated in media at dierent pH for 6 h. VEGF expression was determined as described above. As shown clearly in Figure 1d, e and f, a low extracellular pH level induced VEGF expression in these human tumor cell lines to slightly dierent extents. Moreover, the increased VEGF secretion correlated with an increase in relative angiogenic activity as determined by in vitro endothelial cell proliferation assay (data not shown). Therefore, like platelet-derived endothelial cell growth factor/thymidine phosphorylase (Griths et al., 1997), inducible nitric oxide synthase (Bellocq et al.,


1998), and IL-8 (Shi et al., 1999, 2000; Xie, 2001), VEGF can be upregulated by a low tumor pH, suggesting that the tumor pH regulates angiogenesisrelated molecules and contributes to tumor progression. VEGF expression is dynamically regulated by many factors at both the transcriptional and posttranscriptional level (Levy et al., 1996; Liu et al., 1995; Stein et al., 1995; Damert et al., 1997; Claey et al., 1998). The steady-state level of VEGF mRNA expression is determined by both the transcription rate and transcript degradation rate (mRNA stability). To investigate whether altered VEGF transcript stability

Figure 2 In¯uence of acidosis on VEGF mRNA stability. (a) FG cells were incubated in medium at pH 7.4 or 6.9 in the presence of 5 mg/ml actinomycin D. Cellular mRNA was isolated 0, 1, 2, 4, 8 and 16 h after the addition of actinomycin D for Northern blot analyses and equal loading was monitored by bactin. The amount of VEGF and b-actin mRNA at each time point was quanti®ed by densitometry after Northern blot analysis using the ImageQuant software sample measurement system. (b) The half-life of VEGF mRNA was calculated by drawing the best-®t linear curve on a plot of VEGF versus time; the time at half-maximal VEGF expression was taken to be the half-life. Note that the VEGF mRNA half-life was signi®cantly increased in FG cells under low pH conditions

contributes to the increased steady-state level of VEGF mRNA expression, we measured the VEGF mRNA half-life in FG cells following exposure to medium at dierent pH. Actinomycin D (5 mg/ml) was added to the culture to block further gene transcription; the mRNA was isolated 0, 1, 2, 4, 8 and 16 h after the addition of actinomycin D. The amount of VEGF mRNA and b-actin mRNA at each time point was quanti®ed after Northern blot analysis (Figure 2a), and the VEGF mRNA half-life was calculated by drawing the best-®t linear curve on a plot of VEGF mRNA versus time (Figure 2b). The VEGF mRNA half-life of cells at a pH of 6.9 was much longer than that of cells at pH 7.4. The VEGF mRNA half-life of cells at pH 7.1 was also increased but to a lesser extent compared with that of cells at a pH of 6.9. These data indicated that the stability of VEGF mRNA was signi®cantly increased in the tumor cells incubated in a medium at low pH. Also, the increased VEGF mRNA stability was consistent with reports showing that there are stability-sensitive elements in the 3'-UTR of the VEGF transcript (Stein et al., 1995; Damert et al., 1997; Claey et al., 1998). However, it remains to be determined whether acidosis acts upon these elements through the same factors. To determine whether increased transcription also contributed to increased VEGF expression at a low pH, we measured VEGF transcription rates in FG cells grown in media having dierent pH levels using a

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Figure 3 In¯uence of acidosis on VEGF gene transcription. FG cells were incubated in media at pH 7.4, 6.9 or 6.7 for 6 h. Nucleus isolation and in vitro transcription were performed. Nuclear RNA labeled with a-32P-UTP was then hybridized with VEGF and b-actin cDNA immobilized on a ®lter. (a) The ®lter was then washed and exposed to X-ray ®lm. (b) Quantitative results were obtained by densitometry analysis, standardized to bactin, and expressed as fold change (numbers in italic). This is one representative experiment of two Oncogene


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standard nuclear run-on assay. The cells were incubated for 6 h in a medium at pH 7.4 ± 6.7. The VEGF transcription rate was determined and normalized according to the transcription rate of the housekeeping gene b-actin. The rate of VEGF transcription increased 3.3-fold in FG cells at pH 7.1 compared with that in cells at pH 7.4; however, more extensive acidosis, e.g., pH 6.9 or 6.7, inhibited VEGF transcription (Figure 3a,b). These data demonstrated that only transient exposure to acidosis at a pH near the neutral level activates VEGF gene transcription, whereas persistent exposure to a low pH does the opposite. In contrast, a low pH, e.g., pH 6.9 that did not activate but rather inhibited transcription did enhance transcript stability. Therefore, pH regulated VEGF expression at both the transcriptional and posttranscriptional level. At a near-neutral pH level, VEGF expression was elevated due to both activation of transcription and increased transcript stability, whereas at an acidic pH level, increased VEGF expression was mostly due to increased VEGF transcript stability and, to a much less extent, increased gene transcription.

To further con®rm the in¯uence of acidosis on VEGF transcription and stability and characterize the DNA sequences involved in VEGF gene transcriptional activation and transcript stabilization caused by low pH, we constructed a luciferase reporter plasmid (pVEGF) in which the luciferase gene was ¯anked by VEGF 5'-¯anking sequences and the VEGF 3'-UTR. The VEGF 5'-¯anking sequences spanning 72274 to +50 relative to the transcription initiation site (Tischer et al., 1991) were fused to the 5' end of the ®re¯y luciferase gene and the VEGF 3'-UTR spanning +1 to +1923 relative to the stop codon (Levy et al., 1996) was fused to the 3' end of the ®re¯y luciferase gene right after its stop codon. The pVEGF reporter was cotransfected into FG cells with pBA-RL (Huang et al., 1998, 2000), which was used as an internal control to monitor transfection eciency (Figure 4a). The transfected cells, which were more than 90% con¯uent, were incubated in media at dierent pH (from 7.4 to 6.7) for 6 h, and the activity of both luciferases was measured using the Dual Luciferase Assay kit (Promega, Madison, WI, USA). The speci®c pVEGF activity was then determined. Although pH 6.7 did not

Figure 4 In¯uence of acidosis on VEGF promoter/enhancer activity. VEGF promoter/enhancer and internal control pBA-RL luciferase constructs were generated as follows. (a) The 5'-¯anking region of the human VEGF gene (+50 to 72274 bp) was subcloned into the KpnI and NdeI sites of pGL2-basic (Promega) which contains ®re¯y luciferase coding sequences, and its simian virus 40 intron and polyadenylation signal elements were replaced by the full-length VEGF 3'-UTR. The ®nal pVEGF reporter contained the luciferase sequences ¯anked by the 5'-¯anking and 3'-UTR sequences. The FG cells were plated into 10-cm culture dishes and transfected with the pVEGF and pBA-RL luciferase constructs using a stable transfection kit (Stratagene, La Jolla, CA, USA). Twenty-four hours after transfection, the cells were incubated in media at dierent pH for 6 h (b) or in media at pH 7.4 or 6.9 for 3, 6, 12 or 24 h (c). Both ®re¯y and renilla luciferase activity was measured using the Dual Luciferase Assay kit (Promega) according to the manufacturer's instructions, and VEGF promoter/enhancer activity was calculated. The relative VEGF promoter/ enhancer activity at pH 7.4 was given a value of 1. Note that the VEGF promoter activity was signi®cantly increased in cells at pH 7.1 and 6.9 as compared with that in cells at pH 7.4 (*P50.01) Oncogene


increase pVEGF activity, transient exposure to pH 7.1 and 6.9 did increase pVEGF activity as compared with that to pH 7.4; also activation of pVEGF by the pH level peaked at 6 h, whereas longer exposure, e.g., more than 24 h, decreased pVEGF activity (Figure 4b). Therefore, the sequences of the 5'-¯anking region and/ or the 3'-UTR conferred the responsiveness of the reporter gene to acidosis. Because the reporter may re¯ect both promoter/enhancer and stabilizing activity, we are currently investigating the element or elements responsive to pH changes. VEGF expression in many types of normal and malignant cells is regulated by a plethora of external factors, e.g., cytokines, growth factors, gonadotropins, and many other extracellular molecules. Additionally major stimulators of VEGF expression include hypoxia and hypoglycemia (Shweiki et al., 1992). Also, initial analysis of the VEGF promoter region revealed several potential transcription factor binding sites, such as HIF-1, AP-1, AP-2, Egr-1, Sp1 (Tischer et al., 1991) and many others (Ema et al., 1997; Flamme et al., 1997; Stein et al., 1995; Akiri et al., 1998), which may be involved in VEGF transcription regulation. For example, hypoxia-mediated VEGF induction involves the transactivation of a VEGF promoter by hypoxiainducible factor 1 (Levy et al., 1996; Liu et al., 1995) as well as the stabilization of VEGF mRNA by proteins that bind to sequences located in the 3'-UTR of VEGF mRNA (Stein et al., 1995; Damert et al., 1997; Claey et al., 1998). It remains to be determined whether the same mechanisms may be involved in VEGF regulation by acidosis. In addition, there are three putative AP-1like binding sites in the VEGF promoter (Tischer et al., 1991; Ladoux and Frelin, 1994; Finkenzeller et al., 1995; Forsythe et al., 1996; Damert et al., 1997). We have also recently identi®ed two NF-kB binding sites on the VEGF 3'-UTR, 5'-AGGAGCCTC-3' (+15 to +23) and 5'-GGGGATTCCC-3' (+1171 to +1180). It is known that acidic pH activates both AP-1 and NFkB, which are important to the upregulation of IL-8 by acidosis (Watanabe et al., 1998; Shi et al., 1999, 2000; Xie, 2001). Bellocq et al. (1998) have also shown that these factors are involved in extracellular pH-mediated upregulation of inducible nitric oxide synthase. To that end, NF-kB binding activity was determined in the cells exposed to dierent pH. As shown in Figure 5, transient exposure to pH 7.1 and 6.9 increased NF-kB binding activity to the NF-kB site of VEGF gene, whereas prolonged exposure did the opposite. This ®nding was consistent with a report showing that dominant negative IkBa down-regulated VEGF expression (Huang et al., 2000). Whether acidosis also activates AP-1 binding activity to AP-1 sites of VEGF gene is currently under investigation. Therefore, it is highly possible that the presence and cooperation of the NF-kB and AP-1 binding sites may contribute to the activation of VEGF transcription by low extracellular pH. In addition, only transient but not prolonged acidic exposure activated transcription factor activity, which may explain why prolonged acidosis or severe acidosis inhibits VEGF expression

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Figure 5 In¯uence of acidosis on NF-kB binding activity. The FG cells were plated into 15-cm culture dishes. Twenty-four hours later, the cells were incubated in media at dierent pH for 3 or 6 h. Nuclear protein extracts were prepared and electrophoretic mobility shift assay was performed according to our published procedure (Shi et al., 1999). The sequence of putative NF-kB binding site of VEGF gene was 5'-gctttggggattccctccac-3' and the sequence of consensus NF-kB probe is 5'-agttgaggggactttcccagg-3' (Promega, Madison, WI, USA)

as described in a previous report (Scott et al., 1998) as well as the current study (Figure 1). In summary, we found that transient exposure to a low extracellular pH (6.7 ± 7.1) induced an increased steady-state level of VEGF mRNA expression, which correlated with prolonged VEGF mRNa stability and transactivation of the VEGF gene. Because most solid tumors contain regions of pH lower than that in their corresponding normal tissues, we expect that heterogeneous tumor pH may lead to similar patterns of heterogeneous VEGF expression. A further understanding of the mechanisms of regulation of VEGF expression by acidosis and possible interaction with hypoxia (Blancher and Harris, 1998) may represent another direction to facilitate exploitation of acidic tumor environments as potential selective targets for development of novel therapeutic strategies.

Acknowledgments We thank Don Norwood for editorial comments and Judy King for assistance in the preparation of this manuscript. This work was supported in part by the Multidisciplinary Pancreatic Program Research Grant, by Research Project Grant #RPG-00-054-01-CMS from the American Cancer Society, and by Cancer Center Support Core Grant CA16672-23 from the National Cancer Institute (to K Xie). Oncogene


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