Cancer Letters 318 (2012) 221–225 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Blockade of VEGF-A suppresses tumor growth via inhibition of autocrine signaling through FAK and AKT Jungwhoi Lee a, Taeyun Ku a,b, Hana Yu a, Kyuha Chong a,b, Seung-Wook Ryu a,c, Kyungsun Choi a,c, Chulhee Choi a,b,c,⇑ a b c Cell Signaling and Bio Imaging Laboratory, Department of Bio and Brain Engineering, KAIST, Daejeon 305-701, Republic of Korea Graduate School of Medical Science and Engineering, KAIST, Daejeon 305-701, Republic of Korea KAIST Institute for the BioCentury, KAIST, Daejeon 305-701, Republic of Korea a r t i c l e i n f o Article history: Received 24 September 2011 Received in revised form 17 November 2011 Accepted 9 December 2011 Keywords: VEGF FAK AKT Apoptosis Autocrine signaling a b s t r a c t Blockade of VEGF signaling using RNA interferences, a neutralizing antibody, an antagonizing soluble VEGF receptor, and a receptor tyrosine kinase inhibitor induced anti-tumor effects in human astrocytoma U251-MG and ﬁbrosarcoma HT-1080 in vitro in a dose-dependent manner. Furthermore, blockade of VEGF-A using the doxycycline-inducible VEGF-A RNA interference system showed a signiﬁcant antitumor effect in a murine HT-1080-xenograft model. Anti-tumor effect through the blockade of VEGF signaling was mediated by FAK and AKT pathway in vitro and in vivo. These results collectively indicate that VEGF-A and its receptors can act as key inducer of tumor growth as well as angiogenesis. Ó 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Even though targeting the VEGF and VEGFRs pathway has been proven effective in the treatment of human cancers [1,2], the multitude of biological actions of VEGF blockade such as vessel disruption and normalization prompts us to exploit the alternative role of VEGF inhibition and its therapeutic potential . Traditional anti-angiogenic strategies attempt to inhibit new vessel formation and/or to destroy existing vessels to starve the tumor from its nutrients ; however, it is now becoming increasingly clear that normalizing, and not only pruning, the tumor vessels can be beneﬁcial . Moreover, the heterogeneity of clinical responsiveness and adverse effects has motivated new attraction in re-searching biological meanings of VEGF and its suppression in the complex tumor microenvironment [5,6]. We have previously shown that blockade of VEGF signaling induced a signiﬁcant anti-tumor effects in various human cancer cell types in vitro, especially human malignant glioblastoma and ﬁbrosarcoma cells . On the contrary, we had also shown that blockade of VEGF improved the perfusion of tumor-associated blood vessels as well as vascular permeability . In the present study, we tried to verify the effects of anti-VEGF treatment on the angiogenesis or tumor-growth, and investigate alternative mechanisms responsible for anti-tumor effects of VEGF blockade other than ablating tumor-associated vessels using in vitro and in vivo models. Our reports are the ﬁrst to suggest that pertinent inhibition of autocrine and paracrine VEGF signaling function may be effective to inhibit tumor growth in consort with anti-angiogenesis for VEGF-blockade dependent cancers. 2. Materials and methods 2.1. Cell culture and reagents Abbreviations: FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIF-1a, hypoxic inducible factor 1a; siRNA, small interfering RNA; shRNA, small hairpin RNA; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; Z-VAD, carbobenzoxy-valyl-alanyl -aspartyl-[O-methyl]-ﬂuoromethylketone. ⇑ Corresponding author at: Department of Brain and Bioengineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Tel.: +82 42 350 4321; fax: +82 42 350 4380. E-mail address: firstname.lastname@example.org (C. Choi). URL: http://csbi.kaist.ac.kr (C. Choi). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.12.014 HT-1080 (a human ﬁbrosarcoma cell line) and U251-MG (a human glioblastoma cell line) were obtained from the American Type Culture Collection (ATCC, Manassa, VA). The cells were grown in DMEM (Gibco BRL, Gaithersburg, MD, USA) medium supplemented with 10% Fetal Bovine Serum (Gibco) and 1 Â 105 unit/L Penicilin100 mg/L Streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidiﬁed atmosphere containing 5% CO2. AKT inhibitor (LY294002) and FAK inhibitor (SC203950) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for FLK-1 and FLT-1 were obtained from Santa Cruz Biotechnology and for phospho-FAK (Y397 and Y576/577), FAK, phospho-AKT (Ser473), AKT, 222 J. Lee et al. / Cancer Letters 318 (2012) 221–225 phospho-ERK (Thy202/204), ERK, NRP-1, caspase-3, cleaved caspase-3, CD31, PCNA, HIF-1a, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). Recombinant vascular endothelial growth factor-A was obtained from R&D Systems (Minneapolis, MN, USA). 2.2. siRNA transfection siRNA transfection was performed using an effectene (Qiagen, Hilden, Germany), according to the manufacturer’s protocol as described previously . The siRNA oligonucleotides that encode VEGF-A, FLT-1, FLK-1, NRP-1, and scrambled control were obtained from Bioneer (Daejeon, Korea). The sequences of the siRNAs were as follows: VEGF-A, 50 -AAAUGUGAAUGCAGACCAA-30 -dTdT, FLT-1: 50 -GACUC UCUUCUGGCUCCUA-30 -dTdT, FLK-1: 50 -CUCCUAAUGAGAGUUCCUU-30 -dTdT, NRP1: 50 -GUCCGAAUCAAGCCUGCAA-30 -dTdT and scrambled control, 50 -CCUACGCCAAUUUCGU-30 -dTdT. 2.3. Measurement of cell death To assess cell death, LDH (lactate dehydrogenase) assay (Promega, Madison, WI, USA) was carried out according to the manufacturer’s protocol. After 30 min incubation at room temperature, the absorbance was measured at 490 nm by using a microplate reader (Bio-Rad, Richmond, CA, USA). To evaluate cell viability, WST-1 reagent (Nalgene, Rochester, NY) was used as previously . 2.4. Western blot analysis and ELISA Nuclear/cytosol Fractionation Kit (Bio Vision, Mountain View, CA, USA) was performed according to the manufacturer’s instruction using the tumor samples extracted from mice. Extracted proteins were separated by SDS–PAGE as previously described . The cytoplasmic fractions were probed with GAPDH, and nuclear fractions were probed with PARP (poly ADP-ribose polymerase). Human VEGF DuosetÒ ELISA Development System (R&D system, Minneapolis, USA) was performed according to the manufacturer’s instruction using the blood samples extracted from mice. 2.5. Generation of VEGF-speciﬁc shRNA stable cell lines For stable suppression of VEGF-A expression by short hairpin (sh)-activated gene silencing vector system, plasmids expressing shRNAs were constructed by synthesizing cDNA oligonucleotides bearing the target sequence, Xhol1 and HindIII linker, and then ligated into XhoI and HindIII sites of pSingle-tTs-shRNA vector (tTs-VEGF shRNA, Clontech). The target sequences for VEGF, corresponding to region at nucleotides 379–397 of human VEGF mRNA (GeneBank accession No. GI6631028), were 50 AAATGTGAATGCAGACCAA-30 . One day after transfection with the tTs-VEGF-A shRNA constructs, HT-1080 cells were grown in Dulbecco’s complete medium containing 1 g/LG418 for 3 days followed by an additional 6 days of culture in Dulbecco’s complete medium containing 400 mg/L G418 for the selection of stable transfectants. 2.6. Xenograft tumor model Balb/c nude mice were obtained from Orient (Seongnam, Korea) at 5–6 weeks of age. All mice were housed and handled in accordance with the Animal Research Committee’s Guidelines at KAIST (Daedeok Science Town, Daejeon, Republic of Korea). When tumors reached an average size approximately 3 cm3, water containing 200 mg/L doxycycline was fed to mice. Tumor volume was calculated using the following formula: V = 0.523 LW2 (L = length, W = width). Animals were anesthetized by ketamine and xylazine, perfused with PBS, and followed by ﬁxation with formalin (Sigma–Aldrich, St. Louis, MO, USA). Specimens were excised, immersed in formalin, and transferred to 30% sucrose (Sigma) solution. Immunohistochemistry was performed using the VECTASTAINÒ ABC-kit (Vector Laboratories, Inc., Burlingame, CA, USA) as manufacturer’s recommendation. 2.7. Statistical analysis Data are presented as the mean ± standard deviation (SD). Levels of signiﬁcance for comparisons between two independent samples were determined using the Student’s t-test. Groups were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test applied to signiﬁcant main effects (SPSS 12.0 K for Windows; SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Blockade of VEGF-A induced apoptotic cell death via inhibition of AKT and FAK Previously, we have demonstrated that VEGF blockade induced a signiﬁcant cell death in human glioblastoma and ﬁbrosarcoma cell lines . We further conﬁrmed the functional dependency of HT-1080 and U251-MG cells on VEGF-A by showing the dosedependent induction of cell death by inhibition of VEGF signaling using anti-VEGF-A monoclonal antibody, soluble VEGF receptor and receptor tyrosine kinase inhibitor (Fig. 1). To elucidate the anti-cancer mechanism of VEGF-A blockade, we examined the signal transduction pathways that are speciﬁcally activated by VEGF stimulation (Fig. 2A). Treatment with VEGF-A induced a time-dependent increase in FAK and AKT phosphorylation. Inhibition of VEGF-A-induced activation of AKT and FAK by pharmacological inhibitors induced a signiﬁcant cell death in a dose-dependent manner, which was signiﬁcantly suppressed by pretreatment of a pan-caspase inhibitor, z-VAD (Fig. 2B). The involvement of caspases was conﬁrmed by immunoblot analysis speciﬁc for active caspase-3 (Fig. 2C). Treatment with pharmacological inhibitors of AKT and FAK induced a signiﬁcant cell death even in the absence of exogenous VEGF-A (data not shown). The involvement of autocrine FAK and AKT activation in HT-1080 cells was conﬁrmed by immunoblot analysis (Fig. 2D). Similar results were obtained using U251-MG cells (data not shown). Interestingly, inhibition of FAK also suppressed phosphorylation of AKT; while speciﬁc inhibition of AKT had little effect on FAK phosphorylation, suggesting that FAK might be an upstream signaling event in this setting. To conﬁrm that, we further inhibited FAK expression using siRNAs to demonstrate that knockdown of FAK suppressed both FAK and AKT phosphorylation in HT-1080 cells (Fig. 2E). Similar results were also obtained in U251-MG cells (data not shown). 3.2. Inhibition of tumor-derived VEGF-A suppressed tumor growth independently from anti-angiogenic activities We constructed a doxycycline-inducible sh-VEGF HT1080 cell line (supplementary ﬁgure), then investigated the effect of speciﬁc targeting of tumor-derived VEGF-A in vitro and in vivo. First, we checked the knock-down efﬁcacy of VEGF-A in these cells in vitro (Fig. 3A). Treatment with tetracycline induced a signiﬁcantly reduction of VEGF-A accompanied by inhibition of AKT and FAK phosphorylation, conﬁrming that AKT and FAK are downstream signals responsible for VEGF-dependency. Secretion of VEGF-A was also signiﬁcantly suppressed by inducible expression of shRNA speciﬁc for VEGF-A (data not shown). Cell viability also decreased signiﬁcantly upon induction of VEGF knockdown in vitro (Fig. 3B). Such an anti-proliferative effect of VEGF knockdown was also observed in HT-1080 xenograft model transplanted with the doxycycline-inducible sh-VEGF HT1080 cells (Fig. 3C). The tumor-laden mice were fed with doxycycline when the tumors reached to an average size approximately 3 cm3. Control tumors grew to an average size of 6.24 ± 1.9 cm3 by 46 days after transplantation; while the shRNA-inducible tumors remained to an average size of 2.94 ± 0.9 cm3 by 46 days after 6 days of doxycycline treatment. The serum level of human-speciﬁc VEGF-A was signiﬁcantly lower in shRNA-inducible tumors compared to the control (Fig. 3D). We further investigated the possible therapeutic mechanisms of inhibition of tumor-derived VEGF-A by histochemical analysis. As expected, the vessel areas and number of CD31-positive endothelial cells were signiﬁcantly reduced in VEGF-suppressed tumors (Fig. 4A and B), which were consistent with previous results of ours and others [3,8]. To verify the effect of tumor vessel reduction, we next examined the level of HIF-1a protein, a hypoxia-sensitive protein, in the cytosol and nuclear fractions extracted from the tumors (Fig. 4C). In VEGF-suppressed tumors, the level of HIF-1a protein was substantially lower compared to the control tumors, indicating that the tumor vessel reduction was not associated with vessel disruption leading to tissue hypoxia but rather tumor vessel normalization. To conﬁrm the hypothesis, we analyzed the involvement of tissue hypoxia with tumor cell apoptosis in the core and peripheral regions of the tumors (Fig. 4D). In the central necrotic regions, both 223 J. Lee et al. / Cancer Letters 318 (2012) 221–225 A P <0.01 P <0.01 P <0.01 B P <0.01 * 95 90 * * * 85 * * * * 80 75 20 * * * HT1080 U251 ** ** * 10 0 0 2 5 10 0 2 5 10 - - - - - - - - VEGF-Trap - - - - - - - - 0 10 50 100 0 10 50 100 (mg/L) α-VEGF * P =0.046 Cell Death (%) Viability (%) P <0.01 HT1080 U251 100 (mg/L) SU1498 0 5 10 20 50 0 5 10 20 50 (μmol/L) Fig. 1. Dose-dependent anti-cancer effect of VEGF blockade. (A) HT-1080 and U251-MG cells were treated with varying doses of an anti-VEGF-A neutralizing antibody and VEGF-Trap for 72 h. Viability was measured by WST-1 assay. (B) Cells were incubated with varying doses of SU1498 for 20 h, and the cell death was measured by LDH assay. (n = 3; Tukey’s post hoc test was applied to signiﬁcant group effects in ANOVA, ÃP < 0.05, ÃÃP < 0.01). A B VEGF-A 0 ( / L) 10 30 60 120 P <0.001 P <0.001 (min) * 70 Cell Death (%) p-FAK FAK p-AKT AKT P =0.02 60 ** 50 ** 40 30 * * P =0.01 * 20 10 GAPDH 0 SC203950 LY294002 VEGF Z-VAD C (kDa) Caspase-3 32 Cleaved Caspase-3 19 GAPDH SC203950 LY294002 VEGF Z-VAD + - + + - + + - + + + + + + 0 5 10 20 50 50 - - - - - + + + + + + - - - - - + D - - - - - - (μmol/L) 0 5 10 20 50 50 (μmol/L) + + + + + + - - - - - + E p-FAK (Y397) p-FAK (Y576/577) p-FAK (Y397) p-FAK (Y576/577) FAK FAK p-AKT p-AKT AKT AKT GAPDH SC203950 LY294002 Wortmannin GAPDH - - - + - + scrambled si-RNA si-FAK + - - + Fig. 2. FAK and AKT are downstream signals of VEGF. (A) HT-1080 cells were incubated with VEGF-A (50 lg/L) for varying time periods, and the cell lysates were subjected to western blot analysis using antibodies speciﬁc for phosphor-FAK (Y397), total FAK, phosphor-AKT (S473), total AKT and GAPDH. (B) Cells were treated with varying doses of SC203950 (FAK inhibitor) and LY29400 (AKT inhibitor) in the presence of exogenous VEGF-A (50 lg/L) for 20 h. Cytotoxicity was measured by LDH assay. (n = 3; Tukey’s post hoc test was applied to signiﬁcant group effects in ANOVA, ÃP < 0.05, ÃÃP < 0.01). (C) Cells were incubated in the absence or presence of VEGF-A (50 lg/L) or Z-VAD (10 lmol/L) for 1 h, and further treated with SC203950 or LY294002 for 20 h. Cell lysates were subjected to immunoblot analysis using antibodies against total caspase-3, cleaved caspase-3 and GAPDH. (D) Cells were incubated in the absence or presence of varying doses of pharmacological inhibitors of AKT and FAK for 20 h, and the cell lysates were examined for expression of phosphor-FAK (Y397 and Y576/577), FAK, phospho-AKT (S473), AKT and GAPDH. (E) Cells were transfected with siRNA speciﬁc for FAK, and then examined for expression of various proteins by immunoblot analysis. VEGF-suppressed and control tumors exhibited high levels of HIF1a and active caspase-3. The number of HIF-1a positive cells was lower in VEGF-suppressed tumors even though the difference is not statistically signiﬁcant. On the contrary, in the peripheral regions where the blood perfusion is higher than the core regions , the level of HIF-1a protein was quite lower in both VEGF-suppressed and control tumors; however, the level of active caspase-3 was signiﬁcantly higher in VEGF-suppressed tumors. These results suggest that the reduction of tumor size in VEGF-suppressed tumors was not associated with tissue hypoxia. The in vivo involvement of downstream kinases for VEGF dependency was also conﬁrmed by immunoblot analysis using tumor extracts (Fig. 4E). 4. Discussion In the present study, we demonstrated that selective inhibition of tumor-derived VEGF-A can suppress tumor growth in vivo by inhibition of autocrine tumor growth signals. We further showed that FAK and AKT are the downstream signal transduction pathways responsible for VEGF-A dependency in human glioblastoma and ﬁbrosarcoma cells; while the FAK signaling seems to be upstream to the AKT pathway. This is the ﬁrst report to our knowledge in that anti-VEGF therapy might exert its anti-cancer effects in some VEGF-dependent malignancies independently from its anti-angiogenic effect whether it is disruptive or normalizing, 224 J. Lee et al. / Cancer Letters 318 (2012) 221–225 A B sh-Vector sh-VEGF-A VEGF-A sh-Vector p-FAK sh-VEGF-A Viability (%) 100 FAK p-AKT 90 * 80 70 AKT - Dox - + + GAPDH C - + - D + Dox - (n=3) 9 8 7 6 5 4 3 2 1 0 Dox + (n=4) Serum VEGF-A (rel. secretion) Tumor volume (cm3) Dox * * Dox (-/+) 1.75 Dox - 1.50 Dox + * * 1.25 1.00 * 0.75 0.50 40 41 42 43 44 45 46 0 Days after cancer cells implantation 3 6 Days after doxycyclin treatment Fig. 3. In vivo effect of VEGF silencing in tumor xenograft model. (A) and (B) HT-1080 cells stably expressing control or shVEGF vectors were incubated in the absence or presence of 2 mg/L doxycycline, and the cell lysates were examined for expression of VEGF-A, FAK and AKT. Cell viability was measured using WST-1 assay (a representative of more than three independent experiments. n = 5, ÃP < 0.05). (C) The effect of VEGF-A silencing on tumor growth in vivo (ÃP < 0.05). (D) Human-speciﬁc VEGF expression was determined by ELISA in the plasma of the mice. VEGF level is expressed as the quantity of relatively secreted human VEGF (n = 3; Tukey’s post hoc test was applied to signiﬁcant group effects in ANOVA, ÃP < 0.05; y scale 1.00 indicates 13500 ng/L of VEGF-A in the plasma). 60 50 ** 40 C # of CD31+ vessel/ microscopic fields B Vessel areas/ microscopic fields A 30 20 10 0 Dox C 90 + N C + N GAPDH 70 PARP 60 Dox - HIF-1α * 80 50 - sh-VEGF-A Dox E - Dox + sh-VEGF-A + VEGF-A p-FLK-1 D central region 500 # of positive stained cells peripheral region P =0.19 n.s P =0.01 400 FLK-1 HIF-1α Cleaved Caspase-3 p-FAK FAK 300 p-AKT 200 100 AKT 0 PCNA Dox - + - + GAPDH Fig. 4. Anti-cancer effect of VEGF silencing is not associated with anti-angiogenic action. (A) Blood vessel areas (pixels) and the number of CD31 positive vessels (B) were assessed by immunohistochemical staining in shVEGF expressing HT-1080 tumors. Magniﬁcation, Â200. (C) Fractionated lysates (C, cytosol fraction; N, nuclear fraction) from tumor samples was subjected to the immunoblot analysis for HIF-1a expression. GAPDH and PARP were measured to test the efﬁcacy of cell fractionation. (D) The number of HIF-1a and cleaved caspase-3 positive cells was analyzed in tumors. Peripheral region means the outside of three quarters in the section of tumor samples. Magniﬁcation, Â200. (E) The lysates from tumor samples were tested for VEGF-A, p-FLK-1, FLK-1, p-FAK, FAK, p-AKT, AKT, PCNA and GAPDH by immunoblot analysis. J. Lee et al. / Cancer Letters 318 (2012) 221–225 and that AKT and FAK signaling pathways can be potential therapeutic targets in VEGF-sensitive tumors. Multiple pro-angiogenic functions are attributed to VEGFs, including the activation of endothelial cell invasion, migration, and proliferation . Tight balance between pro-angiogenic and anti-angiogenic signals in the tumor microenvironment that governs the response of endothelial cells is critical. Accordingly, VEGF, one of the most effective pro-angiogenic factor, can critically shift this balance to angiogenesis process . In agreement with this, vessel areas and CD31-positive vessels were signiﬁcantly decreased when tumor-derived VEGF-A was speciﬁcally targeted. The reduction of tumor vessels seems to be the result of tumor vessel normalization rather than disruptive changes of tumorassociated vessels since there were less hypoxic markers in VEGF-suppressed tumors. Given that inhibition of VEGF decreases tumor growth in vivo, our results are consistent with previous studies [12,13]. However, the reduction of tumor sizes seems to be independent from both tumor vessel normalization and disruption, which are usually regarded the leading mechanisms responsible for anti-cancer effect of VEGF blockade [2,3,14]. In concordance with our initial hypothesis, we could conclude that the reduction of tumor growth was closely related to the autocrine or paracrine VEGF signaling in the cancer cells based on several ﬁndings. Firstly, the immunereactivity to active caspase-3, a marker for apoptotic cell death, was even higher in properly perfused tumor periphery of VEGFsuppressed tumors, indicating that increased cell death was not related to vessel disruption. Secondly, the tissue level of HIF-1a, a speciﬁc marker of hypoxia, was signiﬁcantly lower in VEGF-suppressed tumors, where the tumor growth was markedly inhibited. Lastly, the downstream signals responsible for tumor growth in vitro were also remarkably reduced in vivo in VEGF-suppressed tumors. 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