Autocrine Vascular Endothelial Growth Factor Signaling Promotes Cell Proliferation and Modulates Sorafenib Treatment Efficacy in Hepatocellular Carcinoma Sui Peng,1* Ye Wang,2,3* Hong Peng,2* Dong Chen,2 Shunli Shen,3 Baogang Peng,2 Minhu Chen,1 Riccardo Lencioni,4 and Ming Kuang2,3 Tumor cells express vascular endothelial growth factor (VEGF) that can activate VEGF receptors (VEGFRs) on or within tumor cells to promote growth in an angiogenesisindependent fashion; however, this autocrine VEGF pathway has not been reported in hepatocellular carcinoma (HCC). Sorafenib, an angiogenic inhibitor, is the only drug approved for use in advanced HCC patients. Yet the treatment efficacy is diverse and the mechanism behind it remains undetermined. Our aims were to study the molecular mechanisms underlying autocrine VEGF signaling in HCC cells and evaluate the critical role of autocrine VEGF signaling on sorafenib treatment efficacy. By immunohistochemistry, we found robust nuclear and cytoplasmic staining for active, phosphorylated VEGF receptor 1 (pVEGFR1) and phosphorylated VEGF receptor 2 (pVEGFR2), and by western blotting we found that membrane VEGFR1 and VEGFR2 increased in HCC tissues. We showed that autocrine VEGF promoted phosphorylation of VEGFR1 and VEGFR2 and internalization of pVEGFR2 in HCC cells, which was both pro-proliferative through a protein lipase Cextracellular kinase pathway and self-sustaining through increasing VEGF, VEGFR1, and VEGFR2 mRNA expressions. In high VEGFR1/2-expressing HepG2 cells, sorafenib treatment inhibited cell proliferation, reduced VEGFR2 mRNA expression in vitro, and delayed xenograft tumor growth in vivo. These results were not found in low VEGFR1/2-expressing Hep3B cells. In an advanced HCC population on sorafenib treatment for postoperative recurrence, we found that the absence of VEGFR1 or VEGFR2 expression in resected tumor tissues before sorafenib treatment was associated with poorer overall survival. Conclusion: Autocrine VEGF signaling directly promotes HCC cell proliferation and affects the sorafenib treatment outcome in vitro and in vivo, which may enable better stratification for clinical treatment decisions. (HEPATOLOGY 2014;60:1264-1277)
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epatocellular carcinoma (HCC) is the sixth most frequent malignancy, and the third most prevalent cause of tumor-related death globally.1 Although curative treatments such as liver resection, liver transplantation, and local ablation have
improved the outcome in early-stage HCC, most patients are not considered candidates for these therapies because of an advanced tumor stage or inadequate liver function at the time of diagnosis.2 This limits their treatment to fewer options, such as target-
Abbreviations: ERK, extracellular kinase; HCC, hepatocellular carcinoma; PLC, protein lipase C; pVEGFR1, phosphorylated vascular endothelial growth factor receptor 1; pVEGFR2, phosphorylated vascular endothelial growth factor receptor 2; rhVEGF, recombinant human vascular endothelial growth factor; tVEGFR1, total vascular endothelial growth factor receptor 1; tVEGFR2, total vascular endothelial growth factor receptor 2; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. From the 1Department of Gastroenterology and Hepatology; 2Department of Hepatobiliary Surgery; 3Division of Interventional Ultrasound, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China; 4Division of Diagnostic and Interventional Radiology, Department of Oncology, Transplant and Advanced Technologies in Medicine, University of Pisa, Pisa, Italy. Received September 23, 2013; accepted May 19, 2014. *These authors contributed equally to the study Supported in part by the National Natural Science Foundation of China (81272312) and by the Science and Technology Planning Project of Guangdong Province, China (2010B031600209). 1264
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oriented chemotherapeutic methods. Even so, the survival rate has increased only a little over recent decades.3-5 Clearly, better chemotherapeutic strategies are needed. Angiogenesis inhibitors have been described as the beginning of a new era in cancer therapy. Sorafenib is the only certified angiogenesis inhibitor for treating advanced HCC.6 The SHARP and ORIENTAL trials have shown the survival benefits of sorafenib and made it the new standard therapy for patients with advanced HCC.7,8 However, survival was improved only for a few months, with considerable differences in the sensitivity to the drug treatment among individuals. Patients may develop resistance to treatment by activation of alternative pathways that bypass the growth-suppressive effects of sorafenib.9 Thus, identification of predictors through analysis of additional sorafenib-targeted pathways will enable a clinician to make more effective treatment decisions for advanced HCC and to develop better methods for prediction of treatment responses. Angiogenesis is regulated largely by signaling through the vascular endothelial growth factor (VEGF) and its receptors, VEGF receptor 1 (VEGFR1) (Flt-1) and VEGF receptor 2 (VEGFR2) (Flk-1),10 which are the major targets of sorafenib. In addition to the well-known effects of VEGF on angiogenesis, recent data suggest that autocrine VEGF signaling in tumor cells plays an important role in promoting their proliferation and inhibiting apoptosis. Autocrine VEGF signaling occurs when VEGF secreted by tumor cells binds VEGF receptors on the tumor cell surface. Many different human tumor cell types have been found to coexpress VEGF and its receptors.11,12 In lung cancer cells, VEGF stimulation initiates the VEGFR2-dependent mammalian target of rapamycin (mTOR) pathway and enhances VEGF secretion to promote angiogenesis.13 The VEGF-VEGFR2 interaction is essential for the anchorage of xenograft tumors and inhibition of VEGFR2 activities has resulted in complete suppression of tumor growth.13 The angiogenesis-independent cell proliferation effect has been well demonstrated in the K5-SOS conditional knockout mice. When VEGFR1 is genetically knocked out in the epidermal cells but not in the endothelial cells of these mice, they exhibit a decrease in cell proliferation
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and in the development of skin tumors.14 Therefore, autocrine VEGF signaling may be an additional target of angiogenesis inhibitors such as sorafenib. Despite the well-known effects of VEGF on angiogenesis, autocrine VEGF signaling on HCC cell proliferation has not been investigated. In the present study we determined how autocrine VEGF signaling affects HCC cell proliferation and evaluated the contribution of VEGFR1 and VEGFR2 to VEGF-mediated cell proliferation. Moreover, we determined if autocrine VEGF signaling plays an important role in the sorafenib treatment for HCC both in vitro and in vivo.
Materials and Methods Patients and Tissues. HCC samples including tumor, peritumor, and tumor-free liver tissues were obtained from 81 HCC patients who had undergone curative hepatectomy at the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, from 2010 to 2013. Ethical approval for research on human subjects was obtained from the Institutional Review Board of the First Affiliated Hospital of Sun Yat-Sen University and written consent was obtained from each patient. Among the 81 patients, 35 received sorafenib treatment for postoperative recurrent BarcelonaClinic Liver Cancer stage C tumors. The relationship between phosphorylated VEGFR1 (pVEGFR1) or phosphorylated VEGFR2 (pVEGFR2) expression was evaluated by immunohistochemistry and the overall survival was determined by survival analysis (Supporting Materials). Reagents and Cell Lines. Sorafenib tablets used in animal experiments were acquired from Bayer (Leverkusen, Germany). Hep3B and HepG2 cells were purchased from ATCC (Manassas, VA), Huh7 from Life Technologies (Grand Island, NY), and QGY-7703 (or 7703) was a gift from Dr. Li Jiang of the Cancer Center of Sun Yat-Sen University. To establish cell lines with stable knockdown of VEGFR1 or VEGFR2, we purchased custom short hairpin RNA (shRNA) against human VEGFR1 and VEGFR2 from OriGene Technologies (Rockville, MD). Retroviral particles were generated using a previously established method (Supporting Materials).15
Address reprint requests to: Ming Kuang, M.D., Ph.D., Department of Hepatobiliary Surgery, Division of Interventional Ultrasound, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China. E-mail: [email protected]; fax: 86-20-87335577. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27236 Potential conflict of interest: Nothing to report.
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Inhibition of VEGF/VEGFR1/VEFGR2 Signaling. Collection of Conditioned Medium. HepG2 and Hep3B cells were grown in 15-cm Petri dishes to 80% The cells were treated with a VEGF neutralizing anticonfluence. The medium was aspirated off and the body, a VEGFR1 neutralizing antibody, a VEGFR2 monolayer was washed thrice with phosphate-buffered neutralizing antibody, or sorafenib before exposing to saline, once with serum-free Dulbecco’s modified Eagle’s rhVEGF. For RNAi experiments, the cells were transmedium (DMEM), and then replenished with serum- fected with nontargeting control siRNA or specific free DMEM supplied with 1% bovine serum albumin. VEGFR1 and VEGFR2 siRNA using a published After a 48-hour incubation the medium was collected, method (Supporting Materials).15 filtered, and stored at 280 C until use. RNA Extraction and Real-Time Reverse TranAnimals. Nonobese diabetic/severe combined scription Polymerase Chain Reaction. Total RNA immunodeficiency mice were purchased from Vital was isolated with acid guanidinium thiocyanate-phenolRiver Laboratory Animal Technology (Beijing, China) chloroform extraction for patient tissues and for cultured and housed under standard conditions. The animal cells with the RNeasy Mini kit. Reverse transcription was study was conducted under protocols approved by the performed using M-MLV reverse transcriptase per the Animal Care and Use Committee of Sun Yat-Sen Uni- manufacturer’s instructions. Real-time polymerase chain versity (Supporting Materials). reactions (PCR) for VEGF, VEGFR1, and VEGFR2 Immunohistochemistry. HCC tumors with matched mRNA were carried out using a published protocol peritumor and tumor-free liver tissues and tumor masses (Supporting Materials and Supporting Table 2).17 from xenograft experiments were processed using standard Immunofluorescent Staining. The cells were histological procedures. For Ki67 and VEGF staining, seeded at a density of 1 3 105 cells per well onto glass only viable areas of the tumor were evaluated. The results coverslips and incubated with primary antibodies were analyzed by a semiquantitative scoring method against pVEGFR2 (Supporting Materials). (Supporting Materials and Supporting Table 1).16 Sorafenib Treatment of Xenograft Tumors. Membrane, Cytoplasmic, and Nuclear Protein HepG2 or Hep3B cells were inoculated at the right Extraction. Tumor and control tissues were minced flank of the mice. After developing a palpable mass, on ice and homogenized by polytron. Proteins from the mice were randomized to either the sorafenib treatmembrane, cytoplasm, and nuclei were extracted and ment group or control group (n 5 7 per group). The fractionated by centrifugation (Supporting Materials). mice were administered a daily oral gavage with Total Protein Extraction. Total protein was 30 mg/kg sorafenib or vehicle-only solution. Tumor extracted using 13 cell lysis buffer for cultured cells or size (length and width) was measured every 3 days and 13 radioimmunoprecipitation assay buffer for tissues, the tumor volume was calculated based on an estabaccording to the manufacturer’s instruction (Support- lished methods (Supporting Materials).18 ing Materials). Data Analyses. Quantitative data are expressed as Immunoblots. Membrane, cytoplasmic, nuclear, or the mean 6 standard error of the mean. Unless inditotal protein was analyzed using western blot and cated otherwise, statistical analyses were performed quantified by densitometry (Supporting Materials and using an unpaired Student t test for individual comSupporting Table 1). parisons and one-way analysis of variance (ANOVA) Cell Proliferation and Enzyme-Linked Immuno- and the Student-Newman-Keuls for multiplesorbent Assay (ELISA). Cells were equally seeded comparisons. The Kaplan-Meier estimate was used for and treated with conditioned medium, recombinant survival analysis and the log-rank test for group comhuman VEGF (rhVEGF), neutralizing antibody, or parison. P 0.05 was considered statistically significant pharmacologic inhibitors. Cell proliferation was then for all analyses (Supporting Materials). determined using a BrdU Cell Proliferation Assay kit and the secreted form of VEGF was quantified using a Results VEGF ELISA kit (Supporting Materials). Gradually Increased Expression of VEGF, Activation of VEGF Pathway Signaling. Equally seeded cells were exposed to conditioned medium in pVEGFR1, Total VEGFR1 (tVEGFR1), pVEGFR2, the presence or absence of VEGF neutralizing anti- and Total VEGFR2 (tVEGFR2) From Liver, Peritubody for 0, 60, or 180 minutes. For verification, the mor to Tumor Tissues. Earlier studies have shown cells were treated with 30 ng/mL of rhVEGF for 0, that expression of VEGF and receptors increased in 15, 30, 60, 180, or 360 minutes. Total proteins were HCC tissues,19 and serum levels of VEGF and soluble VEGFR1 are found to be of prognostic value in HCC harvested for analysis (Supporting Materials).
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Fig. 1. Immunohistochemical staining of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 in tumor-free liver (L), peritumoral (P), tumor tissues (T) from 81 HCC patients. (A) Representative photomicrographs (4003) showing the locations of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 and with gradient increased staining intensity from liver, peritumor to tumor. (B) The staining intensity was scored as 0 5 no cell labeling; 1 5 rare cell labeling; 2 5 less than 50% cell labeling; 3 5 more than 50% cell labeling; 4 5 100% cell labeling. The results were compared by ANOVA and the StudentNewman-Keuls multiple-comparisons test using PASW Statistics 18.0 for Windows (IBM, Chicago, IL). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to liver control.
development.20 To determine if an autocrine VEGF signaling axis is associated with HCC progression, we performed immunohistochemical staining for VEGF, tVEGFR1, and tVEGFR2 on HCC tumor tissues with matched liver and peritumor tissues from 81 HCC patients. We found that VEGF staining was positive in the cytoplasm of liver cells (Fig. 1A), and the staining intensity significantly increased in peritumor (P < 0.01) and tumor tissues (P < 0.001) (Fig. 1A,B). tVEGFR1 was stained mainly in the cytoplasm of liver tissues, and the staining intensity increased in peritumor (P < 0.05) and tumor tissues (P < 0.001) (Fig. 1A,B). tVEGFR2 was stained weakly in liver tissues but increased in peritumor (P < 0.05) and tumor tissues (P < 0.001) (Fig. 1A,B). Stewart et al.21 reported that when activated by VEGF, VEGFR became phosphorylated and moved from the surface membrane to the cytoplasm and nucleus in endothelial cells. To determine if VEGFRs present in HCC cells are activated, we further stained for pVEGFR1 and pVEGFR2. pVEGFR1 was clearly located in the nuclei, and also in cytoplasms of liver cells with an increased nuclear staining in cancer cells (P < 0.01) (Fig. 1A,B). pVEGFR2 was stained weakly in cytoplasm of liver and peritumor tissues; however, in tumor tissues, pVEGFR2 stained mainly in the nucleus with a significantly increased intensity (P < 0.01) (Fig. 1A,B). To confirm the findings by immunohistochemistry, we performed western blotting with frozen tissues
from 46 patients. VEGF protein expression gradually increased in liver and peritumor tissues (1.4-fold), and was significantly higher in tumor tissues (2.3-fold) (P < 0.001) (Fig. 2A,B). The expression of pVEGFR1 and tVEGFR1 in liver, peritumor, and tumor tissues increased, whereas the ratio of pVEGFR1 to tVEGFR1 was not significantly different among the three tissues (Fig. 2A,B). The expression of pVEGFR2 and tVEGFR2 in liver and tumor tissues increased, respectively (Fig. 2A), whereas the ratio of pVEGFR2 to tVEGFR2 was significantly higher in tumor tissues (P < 0.05) (Fig. 2B). To quantitate the expression levels of VEGF receptors in the membrane, cytoplasm, and nuclei we performed western blotting with fractionated membrane, cytoplasm, and nuclear proteins from the HCC patients. The membrane fractions of tVEGFR1, tVEGFR2, and pVEGFR2, and nuclear fractions of pVEGFR1, tVEGFR1, and pVEGFR2 greatly increased in tumor cells as compared with those of liver cells (Fig. 2C). Most strikingly, the membrane and nuclear pVEGFR2 were undetectable in liver and peritumor tissues but were clearly present in the membrane and nuclei of tumor tissue (Fig. 2C). These results suggested that activation and increased nuclear accumulation of pVEGFR2 were associated with HCC progression. Autocrine VEGF Signaling Promoted Cell Proliferation in HCC. To determine if the increased expression and activity of VEGF and VEGFR were
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Fig. 2. Western blotting analysis of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression in tumor-free liver (L), peritumor (P), and tumor tissues (T) from 46 hepatocellular carcinoma patients. (A) Representative western blots for quantitative analysis of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression. GAPDH was included as a loading control. (B) Scatterplots of the densitometric data depict the distribution of VEGF, ratio of pVEGFR1 to tVEGFR1, and ratio of pVEGFR2 to tVEGFR2 among liver, peritumor, and tumor tissues. Mean values were compared by ANOVA. (C) Representative western blot showed the relative expression levels of pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 in membrane, cytoplasmic, and nuclear location among liver, peritumor, and tumor tissues. Numbers represent the relative quantity of protein with respect to the loading control.
positively associated with cell proliferation in HCC, we selected four HCC cell lines, Hep3B, HepG2, Huh7, and 7703, to characterize the VEGF-mediated cell proliferation. The four cell lines all expressed and secreted VEGF (Fig. 3A, Supporting Fig. 1), whereas the activated VEGF receptors were differentially expressed, with the highest level in HepG2, medium level in 7703 and Huh7, and the lowest level in Hep3B (Fig. 3B). By treating cells with recombinant human VEGF (rhVEGF), the cell proliferation significantly increased in HepG2 (113.6%), Huh7 (117.4%), and 7703 (114.0%) (P < 0.05), but not in Hep3B (Fig. 3B). Treatment with VEGF
neutralizing antibody significantly reduced cell proliferation in HepG2 (212.4%), Huh7 (213.5%), and 7703 (210.3%) (P < 0.05), but not in Hep3B (Fig. 3C). Knowing that HCC cells expressing high-level VEGFR were responsive to rhVEGF treatment, we next determined if HCC cell proliferation could indeed be promoted by its own secreted VEGF. We treated the high- (HepG2) and low- (Hep3B) VEGFR-expressing cells with their corresponding conditioned medium in the presence or absence of VEGF neutralizing antibody. The cell proliferation of HepG2 in its conditioned medium was significantly higher than those in basal medium (P < 0.01), which was
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Fig. 3. Autocrine VEGF signaling enhanced cell proliferation in cultured HCC cells. (A) Western blotting analysis of the basal expression of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression in Hep3B, HepG2, Huh7, and 7703 cells. GAPDH was included as a loading control. Numbers represent the relative quantity of protein with respect to the loading control. (B) The HCC cell proliferation in response to recombinant human VEGF (rhVEGF) treatment. (C) The HCC cell proliferation after depriving of free medium VEGF by incubating with VEGF neutralizing antibodies (VEGF-NA). The HepG2 (D) and Hep3B (E) cell proliferation under conditioned medium (CM) or basal medium (BM), with or without VEGF-NA. Mean 6 standard error of the mean, t test, *P < 0.05, ** P < 0.01.
significantly reduced by the VEGF neutralizing antibody (P < 0.05) (Fig. 3D). On the other hand, the cell proliferation of Hep3B was not enhanced by its conditioned medium (Fig. 3E). Autocrine VEGF Promotes Cell Proliferation Through VEGFR1/R2-Protein Lipase C (PLC)Extracellular Kinase (ERK) 1/2 Pathway. To determine if the autocrine VEGF-mediated cell proliferation was activated through VEGF receptor-dependent pathways, we treated the HepG2 and Hep3B cells with conditioned medium for 0, 60, and 180 minutes and examined receptor phosphorylation and pathway molecule expression. In HepG2 cells, pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 gradually increased from 0, 60, to 180 minutes after conditioned medium treatment (Fig. 4A), and these responses were completely blocked in the expression of pVEGFR2 and pPLC-c1 or greatly suppressed in the expression
of pVEGFR1 and pERK1/2 by simultaneously treating cells with VEGF neutralizing antibody (Fig. 4A). For verification, the HepG2 cells were treated with rhVEGF at different time intervals and the same set of pathway molecules were examined. The expression of pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 increased 15 minutes after treatment, and then continuously increased (pVEGFR2) or gradually declined (pVEGFR1, pPLC-c1, and pERK1/2) afterwards (Supporting Fig. 2A). As compared with HepG2, the expression levels of pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 proteins in Hep3B cell were not changed after conditioned medium (Fig. 4B) or rhVEGF treatment (Supporting Fig. 2B). To determine if activated VEGFR1 and VEGFR2 were responsible for activation of pathway molecules PLC-c1 and ERK1/2, we treated the HepG2 and Hep3B cells with VEGFR neutralizing antibody for 24
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Fig. 4. Pathway analysis of the autocrine VEGF-mediated cell proliferation in HepG2 (A) and Hep3B cells (B). Cells were incubated with conditioned medium in the presence or absence of VEGF neutralizing antibody and the phosphorylation of the pathway molecules VEGFR1, VEGFR2, PLC-c1, ERK1/2 were measured by western blotting. Numbers represent the relative quantity of protein with respect to the loading control GAPDH.
hours followed by a 15-minute rhVEGF treatment. In HepG2 cells, after blocking with VEGFR1 or VEGFR2 neutralizing antibody, the phosphorylation of PLC and ERK1/2 no longer increased following the rhVEGF treatment (Fig. 5A, left panel). In contrast, neither rhVEGF treatment nor blocking VEGFR1 or VEGFR2 had changed PLC and ERK1/2 phosphorylation in Hep3B cells (Fig. 5B, left panel). Next, we determined the functional significance of VEGFR1 and VEGFR2 neutralization. In HepG2 cells, treatment with VEGFR1 or VEGFR2 neutralizing antibody significantly reduced VEGF secretion (Fig. 5A, middle panel), and diminished cell proliferation derived from rhVEGF treatment (Fig. 5A, right panel). In contrast, none of these effects was observed in Hep3B cells (Fig. 5B, middle, right panels). VEGFR1 and VEGFR2 Were Interdependent on Autocrine VEGF Signaling. As depicted earlier, VEGFR1 and VEGFR2 were both responsive to VEGF treatment and were both essential to PLC-ERK signaling. To further understand the role of each VEGFR in autocrine signaling, we generated stable VEGFR1-knockdown and VEGFR2-knockdown HepG2 cells and examined the pathway molecule expression in comparison with that of the parental HepG2 cells. Under basal conditions the VEGFR1-
knockdown cell expressed a reduced level of VEGFR2 and, likewise, VEGFR2-knockdown cell expressed much less VEGFR1 protein (Fig. 5C). Under the rhVEGF-treated condition, neither VEGFR1knockdown nor VEGFR2-knockdown significantly increased the phosphorylation of VEGFR1, VEGFR2, PLC-c1, or ERK1/2 (Fig. 5C). Nuclear Accumulation of pVEGFR2 Was Associated With the Increased VEGF and VEGFR mRNA Expression. One major effect of autocrine VEGF signaling is to produce ligand and receptors to generate reproducible growth signals. To determine if VEGF and VEGFR mRNA expression increased with the progression of HCC, we investigated the HCC tissues by real-time PCR. VEGF, VEGFR1, and VEGFR2 receptor mRNA were all significantly higher in tumor tissue as compared with liver and peritumor tissues (Supporting Fig. 3). VEGFR phosphorylation and nuclear translocation play an essential role in its own RNA synthesis in endothelial cells.22 To determine if the nuclear level of pVEGFRs was related to the transcriptional activity of VEGF and VEGFRs in HCC cells, we treated the HepG2 and Hep3B cells with VEGFR1, VEGFR2 siRNA, or with scramble siRNA included as control. After verifying the knockdown efficiency of VEGFR1
Fig. 5. The essential role of VEGFR1 and VEGFR2 in the autocrine VEGF signaling. (A,B, left panels): The pPLC-c1 and pERK1/2 protein expressions in HepG2 and Hep3B cells in response to rhVEGF treatment after preincubation with neutralizing antibodies (NA) against VEGFR1 (R1-NA) and VEGFR2 (R2-NA). The protein levels were measured by western blotting. (A,B, middle panels): The VEGF secretion in HepG2 and Hep3B cells upon treating with neutralizing antibodies against VEGF receptors. The medium VEGF was measured by ELISA. (A,B, right panels): Cell proliferation of HepG2 and Hep3B cells in response to rhVEGF treatment after blocking the VEGF receptors. Mean 6 SEM, t test, *P < 0.05. (C) VEGFR1 and VEGFR2 were interdependent of their basal expression and on activation of downstream PLC-ERK signaling. Parental HepG2 cells (P) and HepG2 cells with stable knockdown of VEGFR1 (VR1) or VEGFR2 (VR2) were treated with or without rhVEGF, and the protein level was measured by western blotting. Numbers represent the relative quantity of protein with respect to the loading control GAPDH.
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Fig. 6. Autocrine VEGF signaling promoted VEGFR2 nuclear translocation and enhanced VEGF, VEGFR1 and VEGFR2 mRNA expression in HepG2 cells. (A) Transient knockdown of VEGFR1 and VEGFR2 using siRNA. Representative western blots showed total VEGFR1 and VEGFR2 protein expression, and GAPDH was included as loading control. (B,C) In the cells transfected with control (ctl) siRNA, the recombinant human VEGF (rhVEGF) promoted the accumulation of pVEGFR2 in the cytoplasm and nuclear location that was inhibited by treatment with VEGFR1 (R1) or VEGFR2 (R2) siRNA. (B) The cytoplasmic and nuclear proteins were measured by western blotting, with GAPDH and TFIID included as loading control for cytoplasmic and nuclear protein, respectively. Numbers on western blots represent the relative quantity of protein with respect to the loading control. (C) The location of pVEGFR2 was determined by immunofluorescent staining. The pVEGFR2 (pY951) was stained turquoise and nucleus was counterstained with DAPI (blue). (D) Quantitation of VEGF, VEGFR1, and VEGFRs mRNA expression by real-time PCR. Mean 6 SEM, t test, **P < 0.01 compared to control siRNA without rhVEGF treatment; 11P < 0.01 compared to control siRNA with rhVEGF treatment.
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Fig. 7. Differential effects of sorafenib treatment on HCC cell proliferation. (A,B) Sorafenib inhibited cell proliferation in HepG2 and Hep3B cells, and the susceptibility to sorafenib treatment was higher in HepG2 than that of Hep3B cells. Mean 6 SEM, t test, **P < 0.01 versus control. (C,D) Sorafenib inhibited VEGFR1 and VEGFR2 mRNA expression in HepG2 cells but not in Hep3B cells. Mean 6 SEM, t test, *P < 0.05 versus control. (E,F) Differential effects of sorafenib treatment on xenograft tumor growth. Sorafenib delayed HepG2 tumor growth with insignificant effects on Hep3B tumors. Mean 6 SEM, one-way ANOVA, *P < 0.05 versus vehicle control.
and VEGFR2 siRNAs by western blotting (Fig. 6A), the siRNA-transfected cells were then exposed to rhVEGF either for 1 hour to examine cytoplasmic and nuclear protein expression, or overnight to examine mRNA expression. In HepG2 cells, the baseline pVEGFR1 protein was undetectable in cytoplasm and was at a very low level in nuclei. On the one hand, the rhVEGF treatment had modest enhancement of nuclear pVEGFR1 expression (data not shown).
On the other hand, the cytoplasmic and nuclear pVEGFR2 were each detectable at the baseline and increased upon rhVEGF exposure (Fig. 6B). Knockdown of VEGFR1 or VEGFR2 significantly reduced the cytoplasmic and nuclear pVEGFR2 levels (Fig. 6B). To confirm these observations, we stained cells for pVEGFR2 using anti-pY951 antibody.21 Prior to rhVEGF treatment, the pVEGFR2 was stained weakly on membrane and cytoplasmic regions (Fig. 6C). After
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Fig. 8. Kaplan-Meier overall survival curves of sorafenib-treated advanced HCC patients (N 5 35). Patients with negative expression of pVEGFR1 (A) or pVEGFR2 (B) had a significantly reduced the overall survival than patients with positive expression.
rhVEGF exposure, we observed increased accumulation of nuclear pVEGFR2 (Fig. 6C) that was reduced by RNAi against VEGFR1 and abolished by RNAi against VEGFR2 (Fig. 6C). Compared to HepG2 cells, neither pVEGFR1 nor pVEGFR2 was expressed at detectable levels in the nuclei of Hep3B cells under the basal or rhVEGF treatment condition (data not shown). We then determined if the expression of nuclear VEGFR was associated with VEGF and VEGFR mRNA expression by real-time PCR. We found that rhVEGF significantly enhanced the VEGF (1.9-fold) mRNA level in HepG2 cells (P < 0.05), which was suppressed by RNAi against VEGFR1 or VEGFR2 (Fig. 6D, bottom panel). The rhVEGF treatment significantly enhanced the RNA expression level of VEGFR1 (1.6-fold, P < 0.05), which was modestly reduced by RNAi against VEGFR2 (Fig. 6D, top panel). The rhVEGF treatment also significantly increased VEGFR2 (about 50-fold, P < 0.05) mRNA expression, which was diminished by RNAi against VEGFR1 (Fig. 6D, middle panel). In contrast, rhVEGF treatment did not significantly change VEGF, VEGFR1, and VEGFR2 mRNA expression in Hep3B cells (data not shown). Taken together, these data suggested that the nuclear accumulation of pVEGFR2 was enhanced by VEGF stimulation and mediated by the interaction of VEGFR1 and VEGFR2, and that the nuclear level of pVEGFR2 was positively associated with VEGF and VEGFR mRNA expression in autocrine VEGF-active HCC cells. Efficacy of Sorafenib Treatment Was Contingent on Autocrine VEGF Signaling. It is well established that sorafenib inhibits HCC cell proliferation. To determine if down-regulation of autocrine VEGF
signaling played a role in the inhibitory effect of sorafenib, we again chose HepG2 (with active autocrine loop) and Hep3B (with inactive autocrine loop) to evaluate the effect of sorafenib on VEGF secretion, cell proliferation, and mRNA synthesis. Treatment with 5 lM of sorafenib significantly reduced VEGF secretion in HepG2 and Hep3B cells (P < 0.05) (Supporting Fig. 5). At the same concentration, sorafenib inhibited HepG2 cell proliferation significantly (255.1%) (P < 0.01) (Fig. 7A), but only had a modest inhibitory effect on Hep3B cells (210.9%) (Fig. 7B). By real-time PCR, we found that sorafenib had no significant effect on VEGF and VEGFR1 but significantly inhibited VEGFR2 mRNA expression in HepG2 cells (P < 0.05) (Fig. 7C). In Hep3B cells sorafenib had no suppressive effect on VEGF, VEGR1, and VEGFR2 mRNA expression (Fig. 7D). We next evaluated the effect of sorafenib on xenograft tumors. HepG2 and Hep3B were injected subcutaneously into the mice. When the mice first developed a palpable (0.5 cm) mass, they were treated with either sorafenib (30 mg/kg/day) or vehicle solution daily. Sorafenib-treated HepG2 tumor displayed a substantial delayed growth after a 1-week treatment (Fig. 7E). The mean tumor volumes between the vehicle- and sorafenib-treated groups were significantly different after 21=2 weeks of treatment (P < 0.05) (Fig. 7E). On the other hand, the suppressive effect was not observed until 2 weeks of treatment with Hep3B tumors (Fig. 7F), and the mean tumor volumes between the vehicle- and sorafenib-treated groups were not different throughout the experiment (Fig. 7F). By immunohistochemistry for cell proliferating antigen Ki-67, we found that the sorafenib treatment reduced Ki67-positive cells in HepG2 tumors
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(Supporting Fig. 6A) but not in the Hep3B tumors (Supporting Fig. 6A). To determine if the growthsuppressive effect was associated with the inhibition of autocrine VEGF signaling in xenograft tumors, we stained the HepG2 tumor sections using bevacizumab (Avastin, Genentech, South San Francisco, CA), a specific antibody against human cell-derived VEGF. We found that sorafenib treatment dramatically reduced the VEGF expression (and secretion) in HepG2 tumors (Supporting Fig. 6B). pVEGFR1 and pVEGFR2 Expression Levels Were Correlated With the Overall Survival of SorafenibTreated HCC Patients. Among the tumor tissues from 35 advanced HCC patients on sorafenib treatment, positive staining of pVEGFR1 and pVEGFR2 was found in 15 (42.9%) and 13 cases (37.1%), respectively. Seven cases were positive for both pVEGFR1 and pVEGFR2 (20.0%). We found no correlation between pVEGFR1 or pVEGFR2 expression and the clinical parameters such as sex, age, and alphafetoprotein level (data not shown), suggesting that these parameters were independent of the expression of the VEGF receptors. In the 35 advanced HCC patients on sorafenib treatment for postoperative recurrence, we found that the absence of VEGFR1 or VEGFR2 expression in resected tumor tissues before sorafenib treatment was associated with poorer overall survival (P < 0.05) (Fig. 8A,B).
Discussion In this study, we found robust nuclear and cytoplasmic expression for active, phosphorylated VEGFR2 and VEGFR1, and through protein fractionation we found that membranes VEGFR1 and VEGFR2 increased in HCC tissues. In combination with the HCC patient tissues and cell culture system, we showed that autocrine VEGF promoted HCC cell proliferation in a PLC-ERK-dependent pathway. We demonstrated that VEGFR1 and VEGFR2 were interdependent in the activation of the VEGFR-PLCERK pathway that was both pro-proliferative and selfsustaining because it caused the production of more VEGF, VEGFR1, and VEGFR2 mRNA. We showed that sorafenib inhibited cell proliferation, decreased VEGF secretion, and reduced VEGF and VEGFR2 mRNA expression in HCC cells. We further showed that HCC cells with higher levels of VEGFR1 and VEGFR2 were more sensitive to the growth-suppressive effects of sorafenib. Treatment by sorafenib inhibited xenograft tumor growth, which was more effective on high VEGFR-expressing tumors.
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Finally, the overall survival analysis suggested that in advanced HCC patients with sorafenib treatment, poorer overall survival was significantly correlated with a low expression of active VEGF receptors. It is well known that tumor cells secrete VEGF and induce angiogenesis to promote tumor growth.23,24 However, recent studies suggest that VEGF produced by tumor cells can also activate receptors on or within the tumor cells themselves to sustain growth in an angiogenesis-independent manner.14 Previous studies have confirmed the expression of VEGF in HCC tumor cells,19 but the role of VEGF was suggested to be angiogenesis-related since the VEGF level was found positively associated with microvascular vessel density.24 However, VEGFR1 and VEGFR2 were then found expressed in cultured HCC cells as well as in HCC tissues by different research groups.25,26 In this study, we found that VEGFR1 and VEGFR2 were expressed on the membrane of HCC cells at a substantially higher level as compared with matched liver and peritumor controls. The results suggest that autocrine VEGF signaling exists in normal liver tissue that is upregulated in the progression of HCC carcinogenesis. Autocrine VEGF signaling promotes tumor cell proliferation and viability through angiogenesisindependent pathways in a variety of tumor types.12,14 In HCC, knockdown of intracellular VEGF reduced cell proliferation, migration, survival, and adhesion ability in the HCC cell line BEL7402.27 However, the direct interaction between VEGF and VEGF receptors on HCC cell surface has never been tested in an experimental model. In this study, we found that VEGF enhanced HCC proliferation through interaction with VEGFR1 and VEGFR2 in a PLC-ERK-dependent pathway. These findings substantiated our observation in HCC patients that up-regulation of VEGF and VEGFR was associated with a higher level of cell proliferation marker proteins pERK1/2 and CCND1 (data not shown), and suggested that our selected HCC cells were appropriate models for the study of VEGF signaling in HCC. Although both VEGFR1 and VEGFR2 were found significantly higher in HCC tissues, it remained unknown if there is a distinctive role of these two receptors in HCC cell proliferation. In this study, we found that both VEGFR1 and VEGFR2 contributed an essential role to the autocrine VEGF signaling in HCC. Blocking VEGFR1 by neutralized antibody, siRNA, or shRNA (stable cell line) inhibited the response of VEGFR2 to conditioned medium or VEGF treatment and vice versa, implying that VEGFR1 and VEGFR2 may phosphorylate each other
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once activated by VEGF. As a result, the downstream PLC-ERK signaling was suppressed, followed by decreased VEGF secretion, VEGFR mRNA and protein expression, and cell proliferation. These findings were consistent with the earlier reports on a murine HCC model in which both VEGFR1 and VGFR2 blocking antibody suppressed the VEGF-mediated tumor growth associated with a reduction of tumoral neovascularization.28 In endothelial cells, internalization and nuclear translocation of VEGFR2, guided by VEGFR1 and activated PI3K/Akt signaling, are essential processes for cellular function such as wound healing.29 However, the importance of nuclear translocation of VEGF receptor on the autocrine signaling is not well understood. Domingues et al.22 recently found that the nuclear VEGFR2 participated in the transcriptional regulation of its own RNA synthesis and amplified the angiogenic response in endothelial cells. It is not known if a similar regulatory mechanism exists in human cancer cells. In this study, we first reported that significantly higher levels of nuclear pVEGFR1 and pVEGFR2 proteins were observed in clinical HCC specimens as compared with matched liver and peritumor controls. Using a cell culture system, we further showed that VEGF-induced accumulation of pVEGFR2 in cytoplasm and nucleus was directly associated with the VEGF, VEGFR1, and VEGFR2 mRNA expression in HepG2 cells. Overall, our results suggested that nuclear translocation of activated VEGF receptor played an essential role in maintaining autocrine VEGF signaling in HCC cancer cells. Recognized as a drug with multiple targets, sorafenib inhibits angiogenesis through blocking the functions of VEGF receptors on endothelial cells, blocks Raf/MEK/ ERK signaling cascade to inhibit cell proliferation, and down-regulates the translation of antiapoptotic molecule Mcl-1 to increase apoptosis of cancer cells.6 However, little attention has been paid to its inhibitory effects through down-regulating autocrine VEGF signaling on cancer cells. In this study, we showed that sorafenib treatment suppressed VEGF secretion and inhibited VEGFR2 mRNA expression. Our results suggested that sorafenib might have substantial angiogenesisindependent effects directly through blocking autocrine VEGF signaling on HCC cancer cells. More interestingly, by using the cell culture system and in vivo xenograft tumors, we found that the efficacy of sorafenib treatment partly relied on an active autocrine transduction system. Our results indicated that the interaction between VEGFR1 and VEGFR2 has contributed the full activity of autocrine VEGF signaling in HCC cells
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and the deficiency of either VEGFR1 or VEGFR2 will significantly affect the VEGF signaling activity, and reduce sorafenib treatment efficacy. The critical role of autocrine VEGF on sorafenib treatment efficacy was further evidenced by the survival analysis with advanced HCC patients. Clinical studies have reported that angiogenic molecules such as VEGF, angiopoietin 2, or the proto-oncogene c-Kit can refine prognostic prediction within statistical modeling,30 but none of these molecules have been incorporated in the assessment of an individual patient. Our data shows that in advanced HCC patients on sorafenib treatment for postsurgical recurrences, the absence of VEGFR1 or VEGFR2 expression in resected HCC tissue is associated with an adverse prognosis. These results indicate that sorafenib is more effective in patients with tumor tissues that have higher autocrine VEGF signaling activity. Thus, the results of this study may offer an insight into identification of novel predictors and therapeutic strategies for advanced HCC and provide better methods for prediction of sorafenib treatment outcome. Our overall survival analysis is limited in that it is a small retrospective study, which raises a concern of whether the correlation between low pVEGFR expression and poor post-sorafenib treatment survival represents the general advanced HCC patient population. Nevertheless, earlier studies have shown that serum levels of VEGF,20 VEGFR1,26 and VEGFR225 are of prognostic value in HCC development; we therefore believe that the current exploratory study does reflect, to some extent, the situation in the advanced HCC patients of a Chinese population, although further validation of the results is needed by a prospective study with a larger cohort of patients. Acknowledgment: We thank Dr. Lihua Duan and Professor Zhengkai Cao for reviewing and scoring our immunohistochemical staining data.
References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108. 2. Dawe RS. Hepatocellular carcinoma. Lancet 2004;363:899. 3. Abou-Alfa GK, Huitzil-Melendez FD, O’Reilly EM, Saltz LB. Current management of advanced hepatocellular carcinoma. Gastrointest Cancer Res 2008;2:64-70. 4. Mendizabal M, Reddy KR. Current management of hepatocellular carcinoma. Med Clin North Am 2009;93:885-900, viii. 5. Yuen MF, Hou JL, Chutaputti A, Asia Pacific Working Party on Prevention of Hepatocellular Carcinoma. Hepatocellular carcinoma in the Asia pacific region. J Gastroenterol Hepatol 2009;24:346-353. 6. Gauthier A, Ho M. Role of sorafenib in the treatment of advanced hepatocellular carcinoma: an update. Hepatol Res 2013;43:147-154.
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7. Faivre S, Raymond E, Boucher E, Douillard J, Lim HY, Kim JS, et al. Safety and efficacy of sunitinib in patients with advanced hepatocellular carcinoma: an open-label, multicentre, phase II study. Lancet Oncol 2009;10:794-800. 8. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359:378-390. 9. Huang XY, Ke AW, Shi GM, Zhang X, Zhang C, Shi YH, et al. alphaB-Crystallin complexes with 14-3-3zeta to induce epithelialmesenchymal transition and resistance to sorafenib in hepatocellular carcinoma. HEPATOLOGY 2013;57:2235-2247. 10. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-676. 11. Guo S, Colbert LS, Fuller M, Zhang Y, Gonzalez-Perez RR. Vascular endothelial growth factor receptor-2 in breast cancer. Biochim Biophys Acta 2010;1806:108-121. 12. Hamerlik P, Lathia JD, Rasmussen R, Wu Q, Bartkova J, Lee M, et al. Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J Exp Med 2012;209:507520. 13. Chatterjee S, Heukamp LC, Siobal M, Schottle J, Wieczorek C, Peifer M, et al. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. J Clin Invest 2013;123:3183. 14. Lichtenberger BM, Tan PK, Niederleithner H, Ferrara N, Petzelbauer P, Sibilia M. Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development. Cell 2010;140: 268-279. 15. Zhang X, Yu C, Wilson K, Zhang HY, Melton SD, Huo X, et al. Malignant transformation of non-neoplastic Barrett’s epithelial cells through well-defined genetic manipulations. PLoS One 2010;5. 16. Oudar O, Moreau A, Feldmann G, Scoazec JY. Expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver tissue. J Hepatol 1998;29:901-909. 17. Zhang Q, Moe OW, Garcia JA, Hsia CC. Regulated expression of hypoxia-inducible factors during postnatal and postpneumonectomy lung growth. Am J Physiol Lung Cell Mol Physiol 2006;290:L880-889. 18. Holloway SE, Beck AW, Shivakumar L, Shih J, Fleming JB, Brekken RA. Selective blockade of vascular endothelial growth factor receptor 2 with an antibody against tumor-derived vascular endothelial growth factor controls the growth of human pancreatic adenocarcinoma xenografts. Ann Surg Oncol 2006;13:1145-1155. 19. Yamaguchi R, Yano H, Nakashima Y, Ogasawara S, Higaki K, Akiba J, et al. Expression and localization of vascular endothelial growth factor
Author names in bold designate shared co-first authorship.
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receptors in human hepatocellular carcinoma and non-HCC tissues. Oncol Rep 2000;7:725-729. Kemik O, Sumer A, Kemik SA, Purisa S, Tuzun S. Circulating levels of VEGF family and their receptors in hepatocellular carcinoma. Bratisl Lek Listy 2010;111:485-488. Stewart M, Turley H, Cook N, Pezzella F, Pillai G, Ogilvie D, et al. The angiogenic receptor KDR is widely distributed in human tissues and tumours and relocates intracellularly on phosphorylation. An immunohistochemical study. Histopathology 2003;43:33-39. Domingues I, Rino J, Demmers JA, de Lanerolle P, Santos SC. VEGFR2 translocates to the nucleus to regulate its own transcription. PLoS One 2011;6:e25668. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-674. Chen ZB, Shen SQ, Ding YM, Wang WX, Tao JP, Liang LJ, et al. The angiogenic and prognostic implications of VEGF, Ang-1, Ang-2, and MMP-9 for hepatocellular carcinoma with background of hepatitis B virus. Med Oncol 2009;26:365-371. Huang J, Zhang X, Tang Q, Zhang F, Li Y, Feng Z, et al. Prognostic significance and potential therapeutic target of VEGFR2 in hepatocellular carcinoma. J Clin Pathol 2011;64:343-348. Li T, Zhu Y, Qin CY, Yang Z, Fang A, Xu S, et al. Expression and prognostic significance of vascular endothelial growth factor receptor 1 in hepatocellular carcinoma. J Clin Pathol 2012;65:808-814. Zhang L, Wang JN, Tang JM, Kong X, Yang JY, Zheng F, et al. VEGF is essential for the growth and migration of human hepatocellular carcinoma cells. Mol Biol Rep 2012;39:5085-5093. Yoshiji H, Noguchi R, Kuriyama S, Yoshii J, Ikenaka Y, Yanase K, et al. Different cascades in the signaling pathway of two vascular endothelial growth factor (VEGF) receptors for the VEGF-mediated murine hepatocellular carcinoma development. Oncol Rep 2005;13:853-857. Santos SC, Miguel C, Domingues I, Calado A, Zhu Z, Wu Y, et al. VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing. Exp Cell Res 2007;313:1561-1574. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012; 379:1245-1255.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website.
H
epatocellular carcinoma (HCC) is the sixth most frequent malignancy, and the third most prevalent cause of tumor-related death globally.1 Although curative treatments such as liver resection, liver transplantation, and local ablation have
improved the outcome in early-stage HCC, most patients are not considered candidates for these therapies because of an advanced tumor stage or inadequate liver function at the time of diagnosis.2 This limits their treatment to fewer options, such as target-
Abbreviations: ERK, extracellular kinase; HCC, hepatocellular carcinoma; PLC, protein lipase C; pVEGFR1, phosphorylated vascular endothelial growth factor receptor 1; pVEGFR2, phosphorylated vascular endothelial growth factor receptor 2; rhVEGF, recombinant human vascular endothelial growth factor; tVEGFR1, total vascular endothelial growth factor receptor 1; tVEGFR2, total vascular endothelial growth factor receptor 2; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. From the 1Department of Gastroenterology and Hepatology; 2Department of Hepatobiliary Surgery; 3Division of Interventional Ultrasound, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China; 4Division of Diagnostic and Interventional Radiology, Department of Oncology, Transplant and Advanced Technologies in Medicine, University of Pisa, Pisa, Italy. Received September 23, 2013; accepted May 19, 2014. *These authors contributed equally to the study Supported in part by the National Natural Science Foundation of China (81272312) and by the Science and Technology Planning Project of Guangdong Province, China (2010B031600209). 1264
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oriented chemotherapeutic methods. Even so, the survival rate has increased only a little over recent decades.3-5 Clearly, better chemotherapeutic strategies are needed. Angiogenesis inhibitors have been described as the beginning of a new era in cancer therapy. Sorafenib is the only certified angiogenesis inhibitor for treating advanced HCC.6 The SHARP and ORIENTAL trials have shown the survival benefits of sorafenib and made it the new standard therapy for patients with advanced HCC.7,8 However, survival was improved only for a few months, with considerable differences in the sensitivity to the drug treatment among individuals. Patients may develop resistance to treatment by activation of alternative pathways that bypass the growth-suppressive effects of sorafenib.9 Thus, identification of predictors through analysis of additional sorafenib-targeted pathways will enable a clinician to make more effective treatment decisions for advanced HCC and to develop better methods for prediction of treatment responses. Angiogenesis is regulated largely by signaling through the vascular endothelial growth factor (VEGF) and its receptors, VEGF receptor 1 (VEGFR1) (Flt-1) and VEGF receptor 2 (VEGFR2) (Flk-1),10 which are the major targets of sorafenib. In addition to the well-known effects of VEGF on angiogenesis, recent data suggest that autocrine VEGF signaling in tumor cells plays an important role in promoting their proliferation and inhibiting apoptosis. Autocrine VEGF signaling occurs when VEGF secreted by tumor cells binds VEGF receptors on the tumor cell surface. Many different human tumor cell types have been found to coexpress VEGF and its receptors.11,12 In lung cancer cells, VEGF stimulation initiates the VEGFR2-dependent mammalian target of rapamycin (mTOR) pathway and enhances VEGF secretion to promote angiogenesis.13 The VEGF-VEGFR2 interaction is essential for the anchorage of xenograft tumors and inhibition of VEGFR2 activities has resulted in complete suppression of tumor growth.13 The angiogenesis-independent cell proliferation effect has been well demonstrated in the K5-SOS conditional knockout mice. When VEGFR1 is genetically knocked out in the epidermal cells but not in the endothelial cells of these mice, they exhibit a decrease in cell proliferation
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and in the development of skin tumors.14 Therefore, autocrine VEGF signaling may be an additional target of angiogenesis inhibitors such as sorafenib. Despite the well-known effects of VEGF on angiogenesis, autocrine VEGF signaling on HCC cell proliferation has not been investigated. In the present study we determined how autocrine VEGF signaling affects HCC cell proliferation and evaluated the contribution of VEGFR1 and VEGFR2 to VEGF-mediated cell proliferation. Moreover, we determined if autocrine VEGF signaling plays an important role in the sorafenib treatment for HCC both in vitro and in vivo.
Materials and Methods Patients and Tissues. HCC samples including tumor, peritumor, and tumor-free liver tissues were obtained from 81 HCC patients who had undergone curative hepatectomy at the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, from 2010 to 2013. Ethical approval for research on human subjects was obtained from the Institutional Review Board of the First Affiliated Hospital of Sun Yat-Sen University and written consent was obtained from each patient. Among the 81 patients, 35 received sorafenib treatment for postoperative recurrent BarcelonaClinic Liver Cancer stage C tumors. The relationship between phosphorylated VEGFR1 (pVEGFR1) or phosphorylated VEGFR2 (pVEGFR2) expression was evaluated by immunohistochemistry and the overall survival was determined by survival analysis (Supporting Materials). Reagents and Cell Lines. Sorafenib tablets used in animal experiments were acquired from Bayer (Leverkusen, Germany). Hep3B and HepG2 cells were purchased from ATCC (Manassas, VA), Huh7 from Life Technologies (Grand Island, NY), and QGY-7703 (or 7703) was a gift from Dr. Li Jiang of the Cancer Center of Sun Yat-Sen University. To establish cell lines with stable knockdown of VEGFR1 or VEGFR2, we purchased custom short hairpin RNA (shRNA) against human VEGFR1 and VEGFR2 from OriGene Technologies (Rockville, MD). Retroviral particles were generated using a previously established method (Supporting Materials).15
Address reprint requests to: Ming Kuang, M.D., Ph.D., Department of Hepatobiliary Surgery, Division of Interventional Ultrasound, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China. E-mail: [email protected]; fax: 86-20-87335577. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27236 Potential conflict of interest: Nothing to report.
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Inhibition of VEGF/VEGFR1/VEFGR2 Signaling. Collection of Conditioned Medium. HepG2 and Hep3B cells were grown in 15-cm Petri dishes to 80% The cells were treated with a VEGF neutralizing anticonfluence. The medium was aspirated off and the body, a VEGFR1 neutralizing antibody, a VEGFR2 monolayer was washed thrice with phosphate-buffered neutralizing antibody, or sorafenib before exposing to saline, once with serum-free Dulbecco’s modified Eagle’s rhVEGF. For RNAi experiments, the cells were transmedium (DMEM), and then replenished with serum- fected with nontargeting control siRNA or specific free DMEM supplied with 1% bovine serum albumin. VEGFR1 and VEGFR2 siRNA using a published After a 48-hour incubation the medium was collected, method (Supporting Materials).15 filtered, and stored at 280 C until use. RNA Extraction and Real-Time Reverse TranAnimals. Nonobese diabetic/severe combined scription Polymerase Chain Reaction. Total RNA immunodeficiency mice were purchased from Vital was isolated with acid guanidinium thiocyanate-phenolRiver Laboratory Animal Technology (Beijing, China) chloroform extraction for patient tissues and for cultured and housed under standard conditions. The animal cells with the RNeasy Mini kit. Reverse transcription was study was conducted under protocols approved by the performed using M-MLV reverse transcriptase per the Animal Care and Use Committee of Sun Yat-Sen Uni- manufacturer’s instructions. Real-time polymerase chain versity (Supporting Materials). reactions (PCR) for VEGF, VEGFR1, and VEGFR2 Immunohistochemistry. HCC tumors with matched mRNA were carried out using a published protocol peritumor and tumor-free liver tissues and tumor masses (Supporting Materials and Supporting Table 2).17 from xenograft experiments were processed using standard Immunofluorescent Staining. The cells were histological procedures. For Ki67 and VEGF staining, seeded at a density of 1 3 105 cells per well onto glass only viable areas of the tumor were evaluated. The results coverslips and incubated with primary antibodies were analyzed by a semiquantitative scoring method against pVEGFR2 (Supporting Materials). (Supporting Materials and Supporting Table 1).16 Sorafenib Treatment of Xenograft Tumors. Membrane, Cytoplasmic, and Nuclear Protein HepG2 or Hep3B cells were inoculated at the right Extraction. Tumor and control tissues were minced flank of the mice. After developing a palpable mass, on ice and homogenized by polytron. Proteins from the mice were randomized to either the sorafenib treatmembrane, cytoplasm, and nuclei were extracted and ment group or control group (n 5 7 per group). The fractionated by centrifugation (Supporting Materials). mice were administered a daily oral gavage with Total Protein Extraction. Total protein was 30 mg/kg sorafenib or vehicle-only solution. Tumor extracted using 13 cell lysis buffer for cultured cells or size (length and width) was measured every 3 days and 13 radioimmunoprecipitation assay buffer for tissues, the tumor volume was calculated based on an estabaccording to the manufacturer’s instruction (Support- lished methods (Supporting Materials).18 ing Materials). Data Analyses. Quantitative data are expressed as Immunoblots. Membrane, cytoplasmic, nuclear, or the mean 6 standard error of the mean. Unless inditotal protein was analyzed using western blot and cated otherwise, statistical analyses were performed quantified by densitometry (Supporting Materials and using an unpaired Student t test for individual comSupporting Table 1). parisons and one-way analysis of variance (ANOVA) Cell Proliferation and Enzyme-Linked Immuno- and the Student-Newman-Keuls for multiplesorbent Assay (ELISA). Cells were equally seeded comparisons. The Kaplan-Meier estimate was used for and treated with conditioned medium, recombinant survival analysis and the log-rank test for group comhuman VEGF (rhVEGF), neutralizing antibody, or parison. P 0.05 was considered statistically significant pharmacologic inhibitors. Cell proliferation was then for all analyses (Supporting Materials). determined using a BrdU Cell Proliferation Assay kit and the secreted form of VEGF was quantified using a Results VEGF ELISA kit (Supporting Materials). Gradually Increased Expression of VEGF, Activation of VEGF Pathway Signaling. Equally seeded cells were exposed to conditioned medium in pVEGFR1, Total VEGFR1 (tVEGFR1), pVEGFR2, the presence or absence of VEGF neutralizing anti- and Total VEGFR2 (tVEGFR2) From Liver, Peritubody for 0, 60, or 180 minutes. For verification, the mor to Tumor Tissues. Earlier studies have shown cells were treated with 30 ng/mL of rhVEGF for 0, that expression of VEGF and receptors increased in 15, 30, 60, 180, or 360 minutes. Total proteins were HCC tissues,19 and serum levels of VEGF and soluble VEGFR1 are found to be of prognostic value in HCC harvested for analysis (Supporting Materials).
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Fig. 1. Immunohistochemical staining of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 in tumor-free liver (L), peritumoral (P), tumor tissues (T) from 81 HCC patients. (A) Representative photomicrographs (4003) showing the locations of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 and with gradient increased staining intensity from liver, peritumor to tumor. (B) The staining intensity was scored as 0 5 no cell labeling; 1 5 rare cell labeling; 2 5 less than 50% cell labeling; 3 5 more than 50% cell labeling; 4 5 100% cell labeling. The results were compared by ANOVA and the StudentNewman-Keuls multiple-comparisons test using PASW Statistics 18.0 for Windows (IBM, Chicago, IL). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to liver control.
development.20 To determine if an autocrine VEGF signaling axis is associated with HCC progression, we performed immunohistochemical staining for VEGF, tVEGFR1, and tVEGFR2 on HCC tumor tissues with matched liver and peritumor tissues from 81 HCC patients. We found that VEGF staining was positive in the cytoplasm of liver cells (Fig. 1A), and the staining intensity significantly increased in peritumor (P < 0.01) and tumor tissues (P < 0.001) (Fig. 1A,B). tVEGFR1 was stained mainly in the cytoplasm of liver tissues, and the staining intensity increased in peritumor (P < 0.05) and tumor tissues (P < 0.001) (Fig. 1A,B). tVEGFR2 was stained weakly in liver tissues but increased in peritumor (P < 0.05) and tumor tissues (P < 0.001) (Fig. 1A,B). Stewart et al.21 reported that when activated by VEGF, VEGFR became phosphorylated and moved from the surface membrane to the cytoplasm and nucleus in endothelial cells. To determine if VEGFRs present in HCC cells are activated, we further stained for pVEGFR1 and pVEGFR2. pVEGFR1 was clearly located in the nuclei, and also in cytoplasms of liver cells with an increased nuclear staining in cancer cells (P < 0.01) (Fig. 1A,B). pVEGFR2 was stained weakly in cytoplasm of liver and peritumor tissues; however, in tumor tissues, pVEGFR2 stained mainly in the nucleus with a significantly increased intensity (P < 0.01) (Fig. 1A,B). To confirm the findings by immunohistochemistry, we performed western blotting with frozen tissues
from 46 patients. VEGF protein expression gradually increased in liver and peritumor tissues (1.4-fold), and was significantly higher in tumor tissues (2.3-fold) (P < 0.001) (Fig. 2A,B). The expression of pVEGFR1 and tVEGFR1 in liver, peritumor, and tumor tissues increased, whereas the ratio of pVEGFR1 to tVEGFR1 was not significantly different among the three tissues (Fig. 2A,B). The expression of pVEGFR2 and tVEGFR2 in liver and tumor tissues increased, respectively (Fig. 2A), whereas the ratio of pVEGFR2 to tVEGFR2 was significantly higher in tumor tissues (P < 0.05) (Fig. 2B). To quantitate the expression levels of VEGF receptors in the membrane, cytoplasm, and nuclei we performed western blotting with fractionated membrane, cytoplasm, and nuclear proteins from the HCC patients. The membrane fractions of tVEGFR1, tVEGFR2, and pVEGFR2, and nuclear fractions of pVEGFR1, tVEGFR1, and pVEGFR2 greatly increased in tumor cells as compared with those of liver cells (Fig. 2C). Most strikingly, the membrane and nuclear pVEGFR2 were undetectable in liver and peritumor tissues but were clearly present in the membrane and nuclei of tumor tissue (Fig. 2C). These results suggested that activation and increased nuclear accumulation of pVEGFR2 were associated with HCC progression. Autocrine VEGF Signaling Promoted Cell Proliferation in HCC. To determine if the increased expression and activity of VEGF and VEGFR were
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Fig. 2. Western blotting analysis of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression in tumor-free liver (L), peritumor (P), and tumor tissues (T) from 46 hepatocellular carcinoma patients. (A) Representative western blots for quantitative analysis of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression. GAPDH was included as a loading control. (B) Scatterplots of the densitometric data depict the distribution of VEGF, ratio of pVEGFR1 to tVEGFR1, and ratio of pVEGFR2 to tVEGFR2 among liver, peritumor, and tumor tissues. Mean values were compared by ANOVA. (C) Representative western blot showed the relative expression levels of pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 in membrane, cytoplasmic, and nuclear location among liver, peritumor, and tumor tissues. Numbers represent the relative quantity of protein with respect to the loading control.
positively associated with cell proliferation in HCC, we selected four HCC cell lines, Hep3B, HepG2, Huh7, and 7703, to characterize the VEGF-mediated cell proliferation. The four cell lines all expressed and secreted VEGF (Fig. 3A, Supporting Fig. 1), whereas the activated VEGF receptors were differentially expressed, with the highest level in HepG2, medium level in 7703 and Huh7, and the lowest level in Hep3B (Fig. 3B). By treating cells with recombinant human VEGF (rhVEGF), the cell proliferation significantly increased in HepG2 (113.6%), Huh7 (117.4%), and 7703 (114.0%) (P < 0.05), but not in Hep3B (Fig. 3B). Treatment with VEGF
neutralizing antibody significantly reduced cell proliferation in HepG2 (212.4%), Huh7 (213.5%), and 7703 (210.3%) (P < 0.05), but not in Hep3B (Fig. 3C). Knowing that HCC cells expressing high-level VEGFR were responsive to rhVEGF treatment, we next determined if HCC cell proliferation could indeed be promoted by its own secreted VEGF. We treated the high- (HepG2) and low- (Hep3B) VEGFR-expressing cells with their corresponding conditioned medium in the presence or absence of VEGF neutralizing antibody. The cell proliferation of HepG2 in its conditioned medium was significantly higher than those in basal medium (P < 0.01), which was
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Fig. 3. Autocrine VEGF signaling enhanced cell proliferation in cultured HCC cells. (A) Western blotting analysis of the basal expression of VEGF, pVEGFR1, tVEGFR1, pVEGFR2, and tVEGFR2 protein expression in Hep3B, HepG2, Huh7, and 7703 cells. GAPDH was included as a loading control. Numbers represent the relative quantity of protein with respect to the loading control. (B) The HCC cell proliferation in response to recombinant human VEGF (rhVEGF) treatment. (C) The HCC cell proliferation after depriving of free medium VEGF by incubating with VEGF neutralizing antibodies (VEGF-NA). The HepG2 (D) and Hep3B (E) cell proliferation under conditioned medium (CM) or basal medium (BM), with or without VEGF-NA. Mean 6 standard error of the mean, t test, *P < 0.05, ** P < 0.01.
significantly reduced by the VEGF neutralizing antibody (P < 0.05) (Fig. 3D). On the other hand, the cell proliferation of Hep3B was not enhanced by its conditioned medium (Fig. 3E). Autocrine VEGF Promotes Cell Proliferation Through VEGFR1/R2-Protein Lipase C (PLC)Extracellular Kinase (ERK) 1/2 Pathway. To determine if the autocrine VEGF-mediated cell proliferation was activated through VEGF receptor-dependent pathways, we treated the HepG2 and Hep3B cells with conditioned medium for 0, 60, and 180 minutes and examined receptor phosphorylation and pathway molecule expression. In HepG2 cells, pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 gradually increased from 0, 60, to 180 minutes after conditioned medium treatment (Fig. 4A), and these responses were completely blocked in the expression of pVEGFR2 and pPLC-c1 or greatly suppressed in the expression
of pVEGFR1 and pERK1/2 by simultaneously treating cells with VEGF neutralizing antibody (Fig. 4A). For verification, the HepG2 cells were treated with rhVEGF at different time intervals and the same set of pathway molecules were examined. The expression of pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 increased 15 minutes after treatment, and then continuously increased (pVEGFR2) or gradually declined (pVEGFR1, pPLC-c1, and pERK1/2) afterwards (Supporting Fig. 2A). As compared with HepG2, the expression levels of pVEGFR1, pVEGFR2, pPLC-c1, and pERK1/2 proteins in Hep3B cell were not changed after conditioned medium (Fig. 4B) or rhVEGF treatment (Supporting Fig. 2B). To determine if activated VEGFR1 and VEGFR2 were responsible for activation of pathway molecules PLC-c1 and ERK1/2, we treated the HepG2 and Hep3B cells with VEGFR neutralizing antibody for 24
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Fig. 4. Pathway analysis of the autocrine VEGF-mediated cell proliferation in HepG2 (A) and Hep3B cells (B). Cells were incubated with conditioned medium in the presence or absence of VEGF neutralizing antibody and the phosphorylation of the pathway molecules VEGFR1, VEGFR2, PLC-c1, ERK1/2 were measured by western blotting. Numbers represent the relative quantity of protein with respect to the loading control GAPDH.
hours followed by a 15-minute rhVEGF treatment. In HepG2 cells, after blocking with VEGFR1 or VEGFR2 neutralizing antibody, the phosphorylation of PLC and ERK1/2 no longer increased following the rhVEGF treatment (Fig. 5A, left panel). In contrast, neither rhVEGF treatment nor blocking VEGFR1 or VEGFR2 had changed PLC and ERK1/2 phosphorylation in Hep3B cells (Fig. 5B, left panel). Next, we determined the functional significance of VEGFR1 and VEGFR2 neutralization. In HepG2 cells, treatment with VEGFR1 or VEGFR2 neutralizing antibody significantly reduced VEGF secretion (Fig. 5A, middle panel), and diminished cell proliferation derived from rhVEGF treatment (Fig. 5A, right panel). In contrast, none of these effects was observed in Hep3B cells (Fig. 5B, middle, right panels). VEGFR1 and VEGFR2 Were Interdependent on Autocrine VEGF Signaling. As depicted earlier, VEGFR1 and VEGFR2 were both responsive to VEGF treatment and were both essential to PLC-ERK signaling. To further understand the role of each VEGFR in autocrine signaling, we generated stable VEGFR1-knockdown and VEGFR2-knockdown HepG2 cells and examined the pathway molecule expression in comparison with that of the parental HepG2 cells. Under basal conditions the VEGFR1-
knockdown cell expressed a reduced level of VEGFR2 and, likewise, VEGFR2-knockdown cell expressed much less VEGFR1 protein (Fig. 5C). Under the rhVEGF-treated condition, neither VEGFR1knockdown nor VEGFR2-knockdown significantly increased the phosphorylation of VEGFR1, VEGFR2, PLC-c1, or ERK1/2 (Fig. 5C). Nuclear Accumulation of pVEGFR2 Was Associated With the Increased VEGF and VEGFR mRNA Expression. One major effect of autocrine VEGF signaling is to produce ligand and receptors to generate reproducible growth signals. To determine if VEGF and VEGFR mRNA expression increased with the progression of HCC, we investigated the HCC tissues by real-time PCR. VEGF, VEGFR1, and VEGFR2 receptor mRNA were all significantly higher in tumor tissue as compared with liver and peritumor tissues (Supporting Fig. 3). VEGFR phosphorylation and nuclear translocation play an essential role in its own RNA synthesis in endothelial cells.22 To determine if the nuclear level of pVEGFRs was related to the transcriptional activity of VEGF and VEGFRs in HCC cells, we treated the HepG2 and Hep3B cells with VEGFR1, VEGFR2 siRNA, or with scramble siRNA included as control. After verifying the knockdown efficiency of VEGFR1
Fig. 5. The essential role of VEGFR1 and VEGFR2 in the autocrine VEGF signaling. (A,B, left panels): The pPLC-c1 and pERK1/2 protein expressions in HepG2 and Hep3B cells in response to rhVEGF treatment after preincubation with neutralizing antibodies (NA) against VEGFR1 (R1-NA) and VEGFR2 (R2-NA). The protein levels were measured by western blotting. (A,B, middle panels): The VEGF secretion in HepG2 and Hep3B cells upon treating with neutralizing antibodies against VEGF receptors. The medium VEGF was measured by ELISA. (A,B, right panels): Cell proliferation of HepG2 and Hep3B cells in response to rhVEGF treatment after blocking the VEGF receptors. Mean 6 SEM, t test, *P < 0.05. (C) VEGFR1 and VEGFR2 were interdependent of their basal expression and on activation of downstream PLC-ERK signaling. Parental HepG2 cells (P) and HepG2 cells with stable knockdown of VEGFR1 (VR1) or VEGFR2 (VR2) were treated with or without rhVEGF, and the protein level was measured by western blotting. Numbers represent the relative quantity of protein with respect to the loading control GAPDH.
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Fig. 6. Autocrine VEGF signaling promoted VEGFR2 nuclear translocation and enhanced VEGF, VEGFR1 and VEGFR2 mRNA expression in HepG2 cells. (A) Transient knockdown of VEGFR1 and VEGFR2 using siRNA. Representative western blots showed total VEGFR1 and VEGFR2 protein expression, and GAPDH was included as loading control. (B,C) In the cells transfected with control (ctl) siRNA, the recombinant human VEGF (rhVEGF) promoted the accumulation of pVEGFR2 in the cytoplasm and nuclear location that was inhibited by treatment with VEGFR1 (R1) or VEGFR2 (R2) siRNA. (B) The cytoplasmic and nuclear proteins were measured by western blotting, with GAPDH and TFIID included as loading control for cytoplasmic and nuclear protein, respectively. Numbers on western blots represent the relative quantity of protein with respect to the loading control. (C) The location of pVEGFR2 was determined by immunofluorescent staining. The pVEGFR2 (pY951) was stained turquoise and nucleus was counterstained with DAPI (blue). (D) Quantitation of VEGF, VEGFR1, and VEGFRs mRNA expression by real-time PCR. Mean 6 SEM, t test, **P < 0.01 compared to control siRNA without rhVEGF treatment; 11P < 0.01 compared to control siRNA with rhVEGF treatment.
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Fig. 7. Differential effects of sorafenib treatment on HCC cell proliferation. (A,B) Sorafenib inhibited cell proliferation in HepG2 and Hep3B cells, and the susceptibility to sorafenib treatment was higher in HepG2 than that of Hep3B cells. Mean 6 SEM, t test, **P < 0.01 versus control. (C,D) Sorafenib inhibited VEGFR1 and VEGFR2 mRNA expression in HepG2 cells but not in Hep3B cells. Mean 6 SEM, t test, *P < 0.05 versus control. (E,F) Differential effects of sorafenib treatment on xenograft tumor growth. Sorafenib delayed HepG2 tumor growth with insignificant effects on Hep3B tumors. Mean 6 SEM, one-way ANOVA, *P < 0.05 versus vehicle control.
and VEGFR2 siRNAs by western blotting (Fig. 6A), the siRNA-transfected cells were then exposed to rhVEGF either for 1 hour to examine cytoplasmic and nuclear protein expression, or overnight to examine mRNA expression. In HepG2 cells, the baseline pVEGFR1 protein was undetectable in cytoplasm and was at a very low level in nuclei. On the one hand, the rhVEGF treatment had modest enhancement of nuclear pVEGFR1 expression (data not shown).
On the other hand, the cytoplasmic and nuclear pVEGFR2 were each detectable at the baseline and increased upon rhVEGF exposure (Fig. 6B). Knockdown of VEGFR1 or VEGFR2 significantly reduced the cytoplasmic and nuclear pVEGFR2 levels (Fig. 6B). To confirm these observations, we stained cells for pVEGFR2 using anti-pY951 antibody.21 Prior to rhVEGF treatment, the pVEGFR2 was stained weakly on membrane and cytoplasmic regions (Fig. 6C). After
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Fig. 8. Kaplan-Meier overall survival curves of sorafenib-treated advanced HCC patients (N 5 35). Patients with negative expression of pVEGFR1 (A) or pVEGFR2 (B) had a significantly reduced the overall survival than patients with positive expression.
rhVEGF exposure, we observed increased accumulation of nuclear pVEGFR2 (Fig. 6C) that was reduced by RNAi against VEGFR1 and abolished by RNAi against VEGFR2 (Fig. 6C). Compared to HepG2 cells, neither pVEGFR1 nor pVEGFR2 was expressed at detectable levels in the nuclei of Hep3B cells under the basal or rhVEGF treatment condition (data not shown). We then determined if the expression of nuclear VEGFR was associated with VEGF and VEGFR mRNA expression by real-time PCR. We found that rhVEGF significantly enhanced the VEGF (1.9-fold) mRNA level in HepG2 cells (P < 0.05), which was suppressed by RNAi against VEGFR1 or VEGFR2 (Fig. 6D, bottom panel). The rhVEGF treatment significantly enhanced the RNA expression level of VEGFR1 (1.6-fold, P < 0.05), which was modestly reduced by RNAi against VEGFR2 (Fig. 6D, top panel). The rhVEGF treatment also significantly increased VEGFR2 (about 50-fold, P < 0.05) mRNA expression, which was diminished by RNAi against VEGFR1 (Fig. 6D, middle panel). In contrast, rhVEGF treatment did not significantly change VEGF, VEGFR1, and VEGFR2 mRNA expression in Hep3B cells (data not shown). Taken together, these data suggested that the nuclear accumulation of pVEGFR2 was enhanced by VEGF stimulation and mediated by the interaction of VEGFR1 and VEGFR2, and that the nuclear level of pVEGFR2 was positively associated with VEGF and VEGFR mRNA expression in autocrine VEGF-active HCC cells. Efficacy of Sorafenib Treatment Was Contingent on Autocrine VEGF Signaling. It is well established that sorafenib inhibits HCC cell proliferation. To determine if down-regulation of autocrine VEGF
signaling played a role in the inhibitory effect of sorafenib, we again chose HepG2 (with active autocrine loop) and Hep3B (with inactive autocrine loop) to evaluate the effect of sorafenib on VEGF secretion, cell proliferation, and mRNA synthesis. Treatment with 5 lM of sorafenib significantly reduced VEGF secretion in HepG2 and Hep3B cells (P < 0.05) (Supporting Fig. 5). At the same concentration, sorafenib inhibited HepG2 cell proliferation significantly (255.1%) (P < 0.01) (Fig. 7A), but only had a modest inhibitory effect on Hep3B cells (210.9%) (Fig. 7B). By real-time PCR, we found that sorafenib had no significant effect on VEGF and VEGFR1 but significantly inhibited VEGFR2 mRNA expression in HepG2 cells (P < 0.05) (Fig. 7C). In Hep3B cells sorafenib had no suppressive effect on VEGF, VEGR1, and VEGFR2 mRNA expression (Fig. 7D). We next evaluated the effect of sorafenib on xenograft tumors. HepG2 and Hep3B were injected subcutaneously into the mice. When the mice first developed a palpable (0.5 cm) mass, they were treated with either sorafenib (30 mg/kg/day) or vehicle solution daily. Sorafenib-treated HepG2 tumor displayed a substantial delayed growth after a 1-week treatment (Fig. 7E). The mean tumor volumes between the vehicle- and sorafenib-treated groups were significantly different after 21=2 weeks of treatment (P < 0.05) (Fig. 7E). On the other hand, the suppressive effect was not observed until 2 weeks of treatment with Hep3B tumors (Fig. 7F), and the mean tumor volumes between the vehicle- and sorafenib-treated groups were not different throughout the experiment (Fig. 7F). By immunohistochemistry for cell proliferating antigen Ki-67, we found that the sorafenib treatment reduced Ki67-positive cells in HepG2 tumors
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(Supporting Fig. 6A) but not in the Hep3B tumors (Supporting Fig. 6A). To determine if the growthsuppressive effect was associated with the inhibition of autocrine VEGF signaling in xenograft tumors, we stained the HepG2 tumor sections using bevacizumab (Avastin, Genentech, South San Francisco, CA), a specific antibody against human cell-derived VEGF. We found that sorafenib treatment dramatically reduced the VEGF expression (and secretion) in HepG2 tumors (Supporting Fig. 6B). pVEGFR1 and pVEGFR2 Expression Levels Were Correlated With the Overall Survival of SorafenibTreated HCC Patients. Among the tumor tissues from 35 advanced HCC patients on sorafenib treatment, positive staining of pVEGFR1 and pVEGFR2 was found in 15 (42.9%) and 13 cases (37.1%), respectively. Seven cases were positive for both pVEGFR1 and pVEGFR2 (20.0%). We found no correlation between pVEGFR1 or pVEGFR2 expression and the clinical parameters such as sex, age, and alphafetoprotein level (data not shown), suggesting that these parameters were independent of the expression of the VEGF receptors. In the 35 advanced HCC patients on sorafenib treatment for postoperative recurrence, we found that the absence of VEGFR1 or VEGFR2 expression in resected tumor tissues before sorafenib treatment was associated with poorer overall survival (P < 0.05) (Fig. 8A,B).
Discussion In this study, we found robust nuclear and cytoplasmic expression for active, phosphorylated VEGFR2 and VEGFR1, and through protein fractionation we found that membranes VEGFR1 and VEGFR2 increased in HCC tissues. In combination with the HCC patient tissues and cell culture system, we showed that autocrine VEGF promoted HCC cell proliferation in a PLC-ERK-dependent pathway. We demonstrated that VEGFR1 and VEGFR2 were interdependent in the activation of the VEGFR-PLCERK pathway that was both pro-proliferative and selfsustaining because it caused the production of more VEGF, VEGFR1, and VEGFR2 mRNA. We showed that sorafenib inhibited cell proliferation, decreased VEGF secretion, and reduced VEGF and VEGFR2 mRNA expression in HCC cells. We further showed that HCC cells with higher levels of VEGFR1 and VEGFR2 were more sensitive to the growth-suppressive effects of sorafenib. Treatment by sorafenib inhibited xenograft tumor growth, which was more effective on high VEGFR-expressing tumors.
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Finally, the overall survival analysis suggested that in advanced HCC patients with sorafenib treatment, poorer overall survival was significantly correlated with a low expression of active VEGF receptors. It is well known that tumor cells secrete VEGF and induce angiogenesis to promote tumor growth.23,24 However, recent studies suggest that VEGF produced by tumor cells can also activate receptors on or within the tumor cells themselves to sustain growth in an angiogenesis-independent manner.14 Previous studies have confirmed the expression of VEGF in HCC tumor cells,19 but the role of VEGF was suggested to be angiogenesis-related since the VEGF level was found positively associated with microvascular vessel density.24 However, VEGFR1 and VEGFR2 were then found expressed in cultured HCC cells as well as in HCC tissues by different research groups.25,26 In this study, we found that VEGFR1 and VEGFR2 were expressed on the membrane of HCC cells at a substantially higher level as compared with matched liver and peritumor controls. The results suggest that autocrine VEGF signaling exists in normal liver tissue that is upregulated in the progression of HCC carcinogenesis. Autocrine VEGF signaling promotes tumor cell proliferation and viability through angiogenesisindependent pathways in a variety of tumor types.12,14 In HCC, knockdown of intracellular VEGF reduced cell proliferation, migration, survival, and adhesion ability in the HCC cell line BEL7402.27 However, the direct interaction between VEGF and VEGF receptors on HCC cell surface has never been tested in an experimental model. In this study, we found that VEGF enhanced HCC proliferation through interaction with VEGFR1 and VEGFR2 in a PLC-ERK-dependent pathway. These findings substantiated our observation in HCC patients that up-regulation of VEGF and VEGFR was associated with a higher level of cell proliferation marker proteins pERK1/2 and CCND1 (data not shown), and suggested that our selected HCC cells were appropriate models for the study of VEGF signaling in HCC. Although both VEGFR1 and VEGFR2 were found significantly higher in HCC tissues, it remained unknown if there is a distinctive role of these two receptors in HCC cell proliferation. In this study, we found that both VEGFR1 and VEGFR2 contributed an essential role to the autocrine VEGF signaling in HCC. Blocking VEGFR1 by neutralized antibody, siRNA, or shRNA (stable cell line) inhibited the response of VEGFR2 to conditioned medium or VEGF treatment and vice versa, implying that VEGFR1 and VEGFR2 may phosphorylate each other
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once activated by VEGF. As a result, the downstream PLC-ERK signaling was suppressed, followed by decreased VEGF secretion, VEGFR mRNA and protein expression, and cell proliferation. These findings were consistent with the earlier reports on a murine HCC model in which both VEGFR1 and VGFR2 blocking antibody suppressed the VEGF-mediated tumor growth associated with a reduction of tumoral neovascularization.28 In endothelial cells, internalization and nuclear translocation of VEGFR2, guided by VEGFR1 and activated PI3K/Akt signaling, are essential processes for cellular function such as wound healing.29 However, the importance of nuclear translocation of VEGF receptor on the autocrine signaling is not well understood. Domingues et al.22 recently found that the nuclear VEGFR2 participated in the transcriptional regulation of its own RNA synthesis and amplified the angiogenic response in endothelial cells. It is not known if a similar regulatory mechanism exists in human cancer cells. In this study, we first reported that significantly higher levels of nuclear pVEGFR1 and pVEGFR2 proteins were observed in clinical HCC specimens as compared with matched liver and peritumor controls. Using a cell culture system, we further showed that VEGF-induced accumulation of pVEGFR2 in cytoplasm and nucleus was directly associated with the VEGF, VEGFR1, and VEGFR2 mRNA expression in HepG2 cells. Overall, our results suggested that nuclear translocation of activated VEGF receptor played an essential role in maintaining autocrine VEGF signaling in HCC cancer cells. Recognized as a drug with multiple targets, sorafenib inhibits angiogenesis through blocking the functions of VEGF receptors on endothelial cells, blocks Raf/MEK/ ERK signaling cascade to inhibit cell proliferation, and down-regulates the translation of antiapoptotic molecule Mcl-1 to increase apoptosis of cancer cells.6 However, little attention has been paid to its inhibitory effects through down-regulating autocrine VEGF signaling on cancer cells. In this study, we showed that sorafenib treatment suppressed VEGF secretion and inhibited VEGFR2 mRNA expression. Our results suggested that sorafenib might have substantial angiogenesisindependent effects directly through blocking autocrine VEGF signaling on HCC cancer cells. More interestingly, by using the cell culture system and in vivo xenograft tumors, we found that the efficacy of sorafenib treatment partly relied on an active autocrine transduction system. Our results indicated that the interaction between VEGFR1 and VEGFR2 has contributed the full activity of autocrine VEGF signaling in HCC cells
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and the deficiency of either VEGFR1 or VEGFR2 will significantly affect the VEGF signaling activity, and reduce sorafenib treatment efficacy. The critical role of autocrine VEGF on sorafenib treatment efficacy was further evidenced by the survival analysis with advanced HCC patients. Clinical studies have reported that angiogenic molecules such as VEGF, angiopoietin 2, or the proto-oncogene c-Kit can refine prognostic prediction within statistical modeling,30 but none of these molecules have been incorporated in the assessment of an individual patient. Our data shows that in advanced HCC patients on sorafenib treatment for postsurgical recurrences, the absence of VEGFR1 or VEGFR2 expression in resected HCC tissue is associated with an adverse prognosis. These results indicate that sorafenib is more effective in patients with tumor tissues that have higher autocrine VEGF signaling activity. Thus, the results of this study may offer an insight into identification of novel predictors and therapeutic strategies for advanced HCC and provide better methods for prediction of sorafenib treatment outcome. Our overall survival analysis is limited in that it is a small retrospective study, which raises a concern of whether the correlation between low pVEGFR expression and poor post-sorafenib treatment survival represents the general advanced HCC patient population. Nevertheless, earlier studies have shown that serum levels of VEGF,20 VEGFR1,26 and VEGFR225 are of prognostic value in HCC development; we therefore believe that the current exploratory study does reflect, to some extent, the situation in the advanced HCC patients of a Chinese population, although further validation of the results is needed by a prospective study with a larger cohort of patients. Acknowledgment: We thank Dr. Lihua Duan and Professor Zhengkai Cao for reviewing and scoring our immunohistochemical staining data.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website.
