Inhibition of vascular endothelial growth factor (VEGF)-165 and ...

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Inhibition of vascular endothelial growth factor (VEGF)-165 and semaphorin 3A–mediated cellular invasion and tumor growth by the VEGF signaling inhibitor ZD4190 in human colon cancer cells and xenografts Quang-De´ Nguyen,1 Sylvie Rodrigues,1 Christelle M. Rodrigue,1 Christine Rivat,1 Clara Grijelmo,1 Erik Bruyneel,2 Shahin Emami,1 Samir Attoub,1 and Christian Gespach1 1 Institut National de la Sante et de la Recherche Medicale U673, Universite ´ Pierre et Marie Curie-Paris 6, Molecular and Clinical Oncology of Solid Tumors, Ho ˆpital Saint-Antoine, Paris, France and 2Laboratory of Experimental Cancerology, Ghent University Hospital, Ghent, Belgium

Abstract We recently showed by DNA microarray analysis that vascular endothelial growth factor (VEGF) receptor (VEGFR) is expressed in HCT8/S11 human colon cancer cells, suggesting that several angiogenic factors may target colon cancer cells themselves. In this study, transcripts encoding the VEGF-165 and semaphorin 3A (Sema3A) receptors and coreceptors Flt-1, KDR/Flk-1, plexin A1, and neuropilins NP-1 and NP-2 were identified by reverse transcription-PCR in the human colon cancer cell lines HCT8/S11, HT29, HCT116, and PCmsrc. Collagen invasion induced by VEGF-165 and Sema3A in HCT8/S11 cells (EC50, 0.4 – 1 nmol/L) required p42/44 mitogen-activated protein kinase and signaling through RhoA/Rho-kinase – dependent and – independent pathways, respectively. As expected, the VEGFR signaling inhibitor ZD4190 selectively abrogated the proinvasive activity of VEGF in collagen gels (IC50, 10 nmol/L) and chick heart fragments. We identify a novel function for

Received 1/25/06; revised 5/29/06; accepted 6/15/06. Grant support: Institut National de la Sante et de la Recherche Medicale, AstraZeneca, and Fund for Scientific Research Flanders (Brussels, Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Q-D. Nguyen, S. Rodrigues, S. Attoub, and C. Gespach contributed equally to this work. The current address for S. Attoub is Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. Requests for reprints: Christian Gespach, Institut National de la Sante et de la Recherche Medicale U673, Universite ´ Pierre et Marie Curie-Paris 6, Molecular and Clinical Oncology of Solid Tumors, Ho ˆpital Saint-Antoine, 75571 Paris Cedex 12, France. Phone: 33-1-43453477; Fax: 33-1-49284694. E-mail: [email protected] Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-06-0044

VEGF-165 and Sema3A as proinvasive factors for human colorectal cancer cells. Interestingly, oral administration of the single drug ZD4190 to athymic mice (50 mg/kg/d, once daily) inhibited by 70% the growth of HCT8/S11 tumor cell xenografts. Combinations between the src tyrosine kinase inhibitor M475271 and ZD4190 or cisplatin resulted in additive therapeutic activity against LNM35 human lung tumor xenografts. Our data have significant implications for new therapeutic approaches and individualized treatment targeting VEGFR and src signaling pathways in combination with established clinical drugs at primary tumors and distant metastases in colon and lung cancer patients. [Mol Cancer Ther 2006;5(8):2070 – 7]

Introduction Vascular endothelial growth factor (VEGF)-165 belongs to homodimeric glycoprotein family of angiogenic factors consisting of 121, 145, 165, 189, and 206 amino acids generated by alternative splicing. Such molecular diversity suggests distinct roles for these VEGF variants. Five additional members of this family, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor, have been characterized (1). The biological effects of VEGF-165 are mediated by tyrosine kinase receptors VEGF receptor (VEGFR) 1 (Flt-1), VEGFR2 (KDR/Flk-1), and VEGFR3 (Flt-4) and modulated by the neuropilin VEGF coreceptors NP-1 and NP-2 (2). Flt-1 has a 10-fold higher affinity for VEGF but weaker tyrosine kinase activity than KDR. Both Flt-1 and KDR are primarily expressed by vascular endothelial cells (3). Activation of Flt-1 and KDR mediates cell migration and mitogenic responses in monocytes and endothelial cells (4, 5). Cellular effects of VEGFR are mediated at least in part through their association with neuropilins because NP-1 binds both Flt-1 and KDR, whereas NP-2 binds Flt-1, to transduce VEGF signals (6, 7). Neuropilins are 130 to 140 kDa cell surface glycoproteins regulating neuronal guidance and angiogenesis and are expressed by neurons, endothelial cells, mesotheliomas, and prostate and breast carcinomas (2, 8, 9). Neuropilins also function as coreceptors for the semaphorin family of axon guidance molecules. Semaphorin 3A (Sema3A) binds NP-1 only, whereas Sema3B, Sema3C, and Sema3F bind both NP-1 and NP-2 (10). The intricacy of the VEGF and semaphorin signaling pathways is further increased by intermolecular and intercellular cross talk between VEGFR through the formation of functional heterodimers and transphosphorylations. The situation is probably much more complex, especially Mol Cancer Ther 2006;5(8). August 2006

Molecular Cancer Therapeutics

in cancer cells, because signaling cross talk between plexin and Met receptors, which contain a semaphorin domain, triggers intracellular activation of the Met tyrosine kinase (11). Plexins are direct signaling receptors for membranebound semaphorins and are acting as neuropilin coreceptors for the secreted and soluble forms of the class III semaphorins Sema3A/Sema3F (12). Sema3A and Sema3F are respective agonists of the ligand-binding subunits NP-1 and NP-2 acting in association with A-type plexin as signal-transducing subunit (13, 14). Four different families of plexins A (1 – 4) have been identified in the mammalian system. Moreover, NP-1 and plexin A1 are sufficient to reconstitute a functional Sema3A receptor in Cos-7 cells (12). In this case, the NP-1/plexin A1 complex exhibits an enhanced affinity for Sema3A compared with NP-1 alone. NP-1 can also serve as a heterophilic cell-cell adhesion molecule (15, 16). Secreted semaphorins have been initially described as inhibitory signals to prevent axonal growth pathfinding through binding to transmembrane plexin receptors during the development of the nervous system. Opposing signals were subsequently described, including promotion of chemotaxis, attraction, and axon/dendrite outgrowth, according to cellular targets and receptor combinations activated by semaphorins (17). We have reported the early and persistent increase in VEGF transcript levels at early stages of the neoplastic progression in human colorectal mucosa (18). In addition, VEGF-B and VEGF-C as well as KDR, Flt-1, and Flt-4 are constitutively expressed during the adenomacarcinoma transition and later stages associated with distant metastases. VEGF-165, a major regulator of normal and pathologic angiogenesis, is induced by hypoxia and secreted by tumor cells and stromal fibroblasts. Subsequently, VEGF-165 targets endothelial cells at the vicinity of growing tumors. This multifunctional cytokine is well characterized as a potent paracrine angiogenic agent. In agreement with this initial model, cancer cell – derived VEGF exerts pleiotropic effects on preexisting endothelial cells and tumor neovascularization. During this angiogenic switch, VEGF regulates endothelial cell proliferation, migration, invasion, survival, and branching morphogenesis. During the identification of genes differentially expressed in HCT8/S11 human colon cancer cells stably transfected by the signal transducer and activator of transcription 3h (STAT-3h), we have observed that the Flt-1, NP-1, and NP-2 genes are down-regulated by this physiologic inhibitor of the STAT-3 oncogene (19). Thus, we have proposed that VEGF and VEGFR function as proinvasive factors in human colon cancer cells themselves. To address this hypothesis, we have undertaken a complementary study on the expression and functional roles of VEGF/Sema3A and their receptors in human colorectal cancer cell lines with special reference to their invasive properties and tumorigenic potential following the administration of the new VEGFR tyrosine kinase inhibitor ZD4190 (20). Mol Cancer Ther 2006;5(8). August 2006

Materials and Methods Cell Culture and Reagents Human colon cancer cells HCT8/S11, lung cancer cells LNM35 (21), and pleural mesothelioma cells MSTO-211H were maintained in RPMI 1640 (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal bovine serum (Roche Molecular Biochemicals, Meylan, France). HT29, HCT116, PCmsrc colon cancer cells (22), and MDCKts.src kidney cancer cells were cultured in DMEM (Life Technologies, Cergy Pontoise, France) supplemented with 10% to 20% fetal bovine serum and antibiotics. Primary cultures of human umbilical vascular endothelial cells were obtained from Clonetics-BioWhittaker and maintained between passages 5 and 15 in the EBM-2 bullet medium (BioWhittaker, Emerainville, France). Recombinant human Sema3A, VEGF-165, leptin, and transforming growth factor-a were from R&D Systems Europe Ltd. (Oxon, United Kingdom). PD98059 and SB203508 were from Calbiochem (Meudon, France). The Rho GTPase inhibitor Clostridium botulinum exotoxin C3 transferase was a gift from Dr. Gilles Flatau (Institut National de la Sante et de la Recherche Medicale U627, Nice, France). The Rho-kinase (ROK) inhibitor Y27632 was kindly provided by Yoshitomi Pharmaceutical Industries Ltd. (Osaka, Japan). Tyrosine kinase inhibitors targeting src (M475271) and VEGFR signaling (ZD4190 and M319390) were from AstraZeneca (Macclesfield, United Kingdom). RNA Isolation and Reverse Transcription-PCR Total cellular RNA was extracted using the Trizol reagent according to the manufacturer’s instructions (Invitrogen). Total RNA (500 ng) was reverse transcribed using the SuperScript II reverse transcriptase in the presence of hexarandom primers (Invitrogen). Fragments of the human Flt-1, KDR, neuropilins, plexin A1, and Sema3A cDNAs were amplified by PCR in the presence or absence of [a-33P]dCTP (GE Healthcare, Orsay, France) using the following primers (35 cycles, 56jC): Flt-1, 5¶-GCACCTTGGTTGTGGGTGAC-3¶ (forward) and 5¶-CGTGCTGCTTCCTGGTCC-3¶ (reverse); NP-1, 5¶-ACGATGAATGTGGCGATACT-3¶ (forward) and 5¶-AGTGCATTCAAGGCTGTTGG-3¶ (reverse); NP-2, 5¶-GTGACTGGACAGACTCCAAG-3¶ (forward) and 5¶-CGAACACAATCTGGTACTCC-3¶ (reverse); plexin A1, 5¶-ATCAACTCGCCCCTGCAGAT-3¶ (forward) and 5¶-CGCGTCACCTGCTTCTCGGT-3¶ (reverse); Sema3A, 5¶-AACTTGGATCATTGAGCCAC-3¶ (forward) and 5¶-GGATTTTCAGATTGTGTGGC-3¶ (reverse); and KDR/Flk-1 (35 cycles, 62jC), 5¶-CATGTGGTCTCTCTGGTTGTG-3¶ (forward) and 5¶-TCCCTGGAAGTCCTCCACACT-3¶ (reverse). Control glyceraldehyde-3-phosphate dehydrogenase amplification was done using the following primers: 5¶-ATCACCATCTTCCAGGAGCG-3¶ and 5¶-CCTGCTTCACCACCTTCTTG-3¶ (28 cycles, 58jC). PCR products were resolved on a 2% to 6% agarose gels and detected by ethidium bromide or autoradiography. Mitogen-Activated Protein KinaseAssay and Western Blot For the p42/p44 mitogen-activated protein kinase (MAPK) assay, cells were plated and cultured for 24 hours

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at 37jC on 100-mm Petri dishes in RPMI 1640 containing 10% fetal bovine serum and shifted to RPMI 1640 containing 1% fetal bovine serum for another 24-hour period. HCT8/S11 cells were treated at 37jC for the indicated times with transforming growth factor-a (20 nmol/L) or VEGF (2.4 nmol/L). Cells were rapidly washed with ice-cold PBS and lysed with ice-cold buffer containing 20 mmol/L HEPES (pH 7.5), 10 mmol/L EGTA (pH 8.0), 2.5 mmol/L MgCl 2 , 25 mmol/L, 1% Triton X-100, 40 mmol/L h-glycerophosphate, 2 mmol/L sodium vanadate, 20 Ag/mL leupeptin, 20 Ag/mL aprotinin, 1 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride. The lysates were centrifuged at 14,000 g for 10 minutes at 4jC to yield the whole-cell extract. Samples (100 Ag protein) were denatured and subjected to SDS-PAGE in 10% acrylamide. Activated forms of MAPK were analyzed by Western blot analysis using antiphosphorylated p42/p44 MAPK or anti – extracellular signal-regulated kinase 1/2 antibodies (1:1,000 dilution in PBS-Tween 20; Promega). Membranes were washed with PBS-Tween 20 and incubated for 1 hour with anti-rabbit horseradish peroxidase – linked secondary antibody (1:2,000). The immunoreactive bands were detected by the enhanced chemiluminescence detection kit (Promega, Charbonnie`res, France) and quantified with the ImageQuant software (GE Healthcare, Orsay, France). Invasion Assays For invasion of collagen type I gels, HCT8/S11 cells were harvested using Moscona buffer and trypsin /EDTA and seeded on top of the collagen type I gels (Upstate Biotechnology, Lake Placid, NY). Cultures were incubated for 24 hours at 37jC in the presence or absence of the indicated agents. The depth of cell migration inside the gels was measured using an inverted microscope. Invasive and superficial cells were counted in 12 fields of 0.157 mm2. The invasion index corresponds to the ratio of the number of cells invading the gel over the total number of cells counted in each field. For chick heart invasion assays, HCT8/S11 spheroids (diameter, 0.2 mm) were confronted on top of semisolid medium with precultured heart fragments (diameter, 0.4 mm) isolated from 9-day-old chick embryos (23). Confronting cultures were incubated for 4 to 7 days in 1.5 mL culture medium and a gyratory shaker at 120 rpm under a controlled atmosphere containing 10% CO2 in air. Cells were fixed in Bouin-Hollande for histologic determination of HCT8/S11 cell invasiveness. For each experimental condition, at least two confrontations were examined. Tumor Growth HCT8/S11 cells (4 106) and LNM35 cancer cells (1 106) were injected s.c. into the flank of 6-week-old athymic NMRI female mice (nu/nu; Elevage Janvier, Le Genest St Isle, France). One week after inoculation, when tumors had reached 70 mm3, animals (10 for each group) were treated by the anticancer drugs as specified. Tumor dimensions were measured with calipers every 3 days. Tumor volume (V) was calculated using the following formula: V = 0.4 a b 2, with ‘‘a’’ being the length and ‘‘b’’ the width of the tumor. Animals were sacrificed 21 days after treatment initiation, and tumors were excised

and weighed. There was no weight loss or any other manifest sign of toxicity. The statistical significance between experimental values was assessed by the unpaired Student’s t test, and P < 0.05 was considered to be statistically significant.

Results Human Colorectal Cancer Cells Harbor Functional VEGF and Sema3A Receptors Linked to MAPK Signaling As shown in Fig. 1A, HCT8/S11 cells expressed the VEGFR Flt-1, NP-1, and NP-2 transcripts, whereas PCmsrc colon cancer cells were only positive for NP-1 and NP-2. The transcripts encoding KDR, NP-1, and NP-2 were also

Figure 1.

Expression of VEGF and Sema3A receptors and coreceptors and their functional coupling with p42/p44 MAPK activation in human colon and lung cancer cells. A, expression of the transcripts encoding Flt-1, KDR, neuropilins NP-1 and NP-2, and plexin A1 by reverse transcriptionPCR in human umbilical vascular endothelial cells (HUVEC ), colon cancer cells HCT8/S11, HT29, PCmsrc, and HCT116, and LNM35 lung cancer cells. Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) cDNA served as internal control of the reverse transcription-PCR and loading. PCR products were resolved on 2% to 6% agarose gels and detected by ethidium bromide staining (left ) or autoradiography (right ). B, top, phosphorylation of the p42/p44 MAPK in HCT8/S11 cells treated for 2 to 60 min with either VEGF-165 (100 ng/mL), Sema3A (10 ng/mL), or the epidermal growth factor receptor agonist transforming growth factor-a (TGFa ; 20 nmol/L). Columns, mean of three to four experiments; bars, SE. Bottom, effect of the VEGF signaling inhibitor ZD4190 (1 mmol/L) preincubated for 2 h with HCT8/S11 cells (right ) on the subsequent extracellular signal-regulated kinase activation by VEGF-165.

Mol Cancer Ther 2006;5(8). August 2006


clearly identified in HCT116 human colon cancer cells. In contrast, only NP-1 was detected in HT29 colon cancer cells. Because NP-1 and NP-2 are also coreceptors for the plexin receptors binding the class III semaphorins, we next investigated for the presence of plexin A1 in these models. Interestingly, these four colon cancer cell lines and the LNM35 lung cancer cells were positive for the plexin A1 semaphorin receptors (Fig. 1A). The Flt-1 and KDR amplimers were abundant in human umbilical vascular endothelial cells, the essential VEGF target. These primary endothelial cells were also positive for NP-1/NP-2 and plexin A1 expression. To determine the functionality and biological significance of the VEGF/Sema3A receptors in colon cancer cells, we first determined whether VEGF stimulated p42/p44 MAPK activation in HCT8/S11 cells. Treatment of HCT8/S11 cells with VEGF (100 ng/mL) caused a rapid and strong phosphorylation of p42/p44 MAPK, a downstream marker of VEGF signaling (Fig. 1B, top). Activation was observed 2 minutes after the addition of VEGF was maximal at 5 to 10 minutes (4-fold increase) and declined to basal levels within 30 to 60 minutes. The extent of p42/p44 stimulation by VEGF was comparable with that produced by the mitogenic and angiogenic peptide transforming growth factor-a acting through the epidermal growth factor receptor in HCT8/S11 cells (24). Similar kinetics of p42/ p44 phosphorylation was observed after the addition of Sema3A. As expected, preincubation of HCT8/S11 cells with ZD4190 precluded the ability of VEGF to induce MAPK activation because this VEGFR signaling inhibitor inhibited by 82% and 73% VEGF-induced extracellular signal-regulated kinase activation observed at 5 and 10 minutes in control cells, respectively (Fig. 1B, bottom). Effect of VEGF and Sema3A on Colon Cancer Cell Invasion We next examined the invasive potential of HCT8/S11 cells in response to VEGF-165 and Sema3A. Both peptides elicited collagen type I invasion according to the potencies EC50 = 0.3 nmol/L VEGF and 2 nmol/L Sema3A (Fig. 2A). Because p42/p44 signaling pathways can regulate tumor cell growth, survival, and invasion, we asked whether VEGF- and Sema3A-induced invasion requires MAPK activation. Invasive responses induced by the two peptides were abolished by pharmacologic inhibitors targeting p42/ p44 and p38 MAPK/stress-activated protein kinase and phosphatidylinositol 3-kinase (Fig. 2B). Blockade of Rho GTPases, ROK, and VEGFR tyrosine kinase by ZD4190 selectively abrogated invasion induced by VEGF-165 but had no effect on the invasive activity of Sema3A, suggesting that these cellular responses are mediated through distinct receptors and signaling networks. ZD4190 dose dependently reversed collagen invasion induced by VEGF165 (IC50, 10 nmol/L ZD4190; data not shown). Our data are coherent with the inhibitory potency IC50 of ZD4190 = 50 nmol/L for human umbilical vascular endothelial cell growth, a selective pharmacologic response at KDR/Flt-1 – mediated signal transduction (20). The proinvasive activity of VEGF-165 was also inhibited in a dose-dependent Mol Cancer Ther 2006;5(8). August 2006

Figure 2.

Effect of VEGF and Sema3A on cellular invasion and their connection with several proinvasive signaling pathways in HCT8/S11 human colon cancer cells. A, dose effect of VEGF-165 and Sema3A on collagen type I invasion. HCT8/S11 cells were incubated for 24 h in the presence or absence of the indicated effectors. Invading cells were scored as described in Materials and Methods. Points, mean of three independent experiments; bars, SE. B, effects of pharmacologic inhibitors targeting phosphatidylinositol 3-kinase [wortmannin (WORT )], p42/p44 MAPK (PD098059), and p38 MAPK/stress-activated protein kinase (SB203508), elements of the Rho-ROK cascade [C. botulinum exotoxin C3 transferase (C3T ) and Y27632, respectively], and KDR/Flt-1 signaling (ZD4190) on cellular invasion induced by VEGF-165 (100 ng/mL) and Sema3A (10 ng/mL). Columns, mean of three to four experiments; bars, SE. C, invasion of collagen type I gels by HCT8/S11 cancer cells stably transfected by expression vectors encoding dominant-negative forms of the small GTPases (DN-RhoA, DN-Rac1, and DN-Cdc42) and ROK (DNROK) and the constitutively activated form of the RhoA antagonist DCRhoD. Cells were incubated for 24 h at 37jC in the presence or absence of VEGF-165 (100 ng/mL) and Sema3A (10 ng/mL). Columns, mean of three to four experiments; bars, SE.

manner by the KDR/Flt-1 tyrosine kinase inhibitor M319390 after simultaneous addition with 2.4 nmol/L VEGF (IC50, 100 nmol/L) or 2 hours of preincubation (IC50, 60 nmol/L). M319390 also abrogated VEGF-induced angiogenesis in the chick chorioallantoic membrane assay (data not shown; ref. 25). The selectivity of ZD4190 on VEGF-165 signaling was further sustained by its inability to reverse the invasive responses induced by leptin (26) and the src oncogene in HCT8/S11 and MDCKts.src cancer cells (data not shown). In agreement with our pharmacologic data, the VEGF proinvasive signals are interrupted by introduction of dominant-negative forms of RhoA, ROK, and the RhoA antagonist DC-RhoD into HCT8/S11 cells

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(27) as shown in Fig. 2C. As expected, blockade of the RhoROK axis by these interfering vectors is not associated with a decreased invasive potential of HCT8/S11 cells treated with Sema3A. Conversely, the dominant interfering mutant of Rac (DN-Rac) abolished both VEGF and Sema3Ainduced invasion, whereas the DN-Cdc42 did not halt collagen invasion promoted by VEGF and Sema3A. Interestingly, DN-EGFR interfering with the epidermal growth factor receptor signaling pathways in HCT8/S11 cells abolished the VEGF and Sema3A proinvasive signals but was ineffective on the invasive response induced by the intestinal trefoil factor (data not shown) as expected (24). We next confronted the HCT8/S11 cells with cultured embryonic chick heart fragments as an invasion signature linked to disruption of several biological barriers, including matricellular proteins and cell-cell adhesion complexes, in the host tissue. Histologic sections of the coculture system were examined under control conditions and in the presence of ZD4190 either alone or combined with VEGF165. Invasion index was scored on serial histologic sections according to grade 2 with confronting cancer cells found at the periphery or occupying the outer fibroblastic cell layers of the heart fragment (absence of invasion) and grade 3 when confronting cancer cells replaced the cardiac muscle cells to more than half. As shown in Fig. 3, HCT8/S11 cancer cell penetration into the healthy tissue was limited to the periphery of the explants (grade 2, n = 4). The same invasion score was observed after treatment with 1 and 10 Amol/L ZD4190 (n =4). In contrast, addition of VEGF resulted in a predominant invasion of the heart tissue by HCT8/S11 cells (grade 3, n = 3), a malignant response reversed by simultaneous addition of the VEGFR signaling inhibitor ZD4190 at 1 mmol/L (grade 2, n = 2; grade 3, n = 2) or 10 mmol/L (grade 2, n = 4). Our data reveal that these two ZD4190 concentrations respectively halved and abrogated the proinvasive activity of VEGF in this assay. Anticancer Activity of Therapeutic Associations Using the Tyrosine Kinase Inhibitors ZD4190, M475271, and Cisplatin in Colon and LungTumor Xenografts As shown previously and in the present study, ZD4190 is a selective VEGF signaling inhibitor targeting mainly the

Figure 3. Effect of VEGF-165 on the penetration of cultured embryonic heart fragments by HCT8/S11 colon cancer cells. Typical experiment showing the invasion of the host tissue by HCT8/S11 cells cultured for 4 to 7 d in the presence or absence (control) of VEGF (100 ng/mL). Light micrographs of paraffin sections stained with H&E.

KDR/Flt-1 tyrosine kinases (20). The ability of ZD4190 administered by oral gavage to reduce the growth of HCT8/S11 human colon cancer xenografts was therefore investigated and compared with the response of tumor xenografts induced by Flt-1/KDR-negative LNM35 lung cancer cells. Daily oral administration of ZD4190 (50 mg/kg) for 3 weeks resulted in a remarkable and persistent reduction (70%) of HCT8/S11 tumor growth (Fig. 4A). To extend our data to another preclinical model, the anticancer activity of ZD4190 was investigated in athymic mice receiving the highly tumorigenic LNM35 human lung cells as xenografts. The VEGF signaling inhibitor ZD4190 was tested alone and compared with the src tyrosine kinase inhibitor M475271 (28). Because cisplatin is a standard clinical drug in the treatment of lung cancer patients, other comparisons were made using cisplatin combined with M475271 (Fig. 4B). In coherence with data in Fig. 4A, daily administration of ZD4190 by oral gavage reduced the growth of LNM35 human tumor xenografts by 45% at day 21. Inhibition of LNM35 tumor xenograft growth by ZD4190 was comparable with that produced by the anticancer agent cisplatin. Combined treatment by ZD4190 and subeffective doses of the src signaling inhibitor M475271 resulted in a remarkable potentiation of the ZD4190 therapeutic effect. This association of src and VEGFR tyrosine kinase inhibitors produced a sustained inhibition of tumor growth by 80% at day 21. These additive effects are the result of the molecular selectivity of these two drugs on tyrosine kinases because ZD4190 was ineffective against src-induced invasion in MDCKts.src cells. Interestingly, subeffective doses of the src inhibitor M475271 showed additive responses with cisplatin.

Discussion Much focus has been placed on the role of VEGF acting as a key regulator of cancer progression through several mechanisms targeting vascular and lymphatic endothelial cell growth, survival, invasive migration, and morphogenesis of new vessels in growing tumors (1). The neoplastic progression of human colorectal tumors is associated with increased VEGF levels and sustained expression of its receptors VEGFR at early stages of the adenoma and carcinoma transitions (18). Because the initial proposal that hypoxia is a major signal for cancer cell – derived VEGF secretion and action on endothelial cells at the immediate proximity of the growing tumors, recent advances increased the complexity of tumor angiogenesis and cancer progression to metastasis. For example, both cancer cells and tumor stromal cells are involved in the secretion of VEGF in the tumor microenvironment (29). Moreover, bone marrow – derived endothelial cells and VEGFR1/Flt-1 – positive hematopoietic progenitors participate in tumor angiogenesis and metastasis by enhancing primary tumor neoangiogenesis and preparing a premetastatic niche with resident tumor fibroblasts (30). In the present study, we bring Mol Cancer Ther 2006;5(8). August 2006


Figure 4.

Therapeutic effect of the VEGF and src signaling inhibitors ZD4190 and M475271 on the growth of established human colon and lung cancer xenografts. A, nude mice were xenografted s.c. with HCT8/ S11 colon cancer cells (4 106 cells per animal) and treated by daily administration p.o. of the VEGF signaling inhibitor ZD4190 (50 mg/kg for 21 d). Drug administration started when the tumor became palpable 7 d after inoculation. B, animals were xenografted with human lung LNM35 cancer cells (106 cells per animal) and treated by ZD4190 and the src signaling inhibitor M475271 (each at 50 mg/kg, daily administration p.o. for 21 d) either alone or combined together. Data were compared with cisplatin alone (i.p. injections of 1 mg/kg/d the first 5 d) or combined with M475271 (50 mg/kg, daily administration p.o. for 21 d). Points, mean (n = 10 animals per group); bars, SE.

additional evidence for this complexity by showing that several human colon cancer cell lines express functional VEGF-165 and semaphorin receptors and coreceptors. The VEGFR KDR/Flk-1, Flt-1, and Flt-4 are not detected in normal human colon epithelial crypts (18). Interestingly, Flt-1, NP-1, and NP-2 are induced in a STAT-3– dependent manner in HCT8/S11 colon cancer cells (19). In the present study, we found that several colon cancer cell lines express functional VEGF and semaphorin receptors and coreceptors at various levels. As oncogenic STAT-3 activation is a frequent event in human solid tumors and leukemia, our data suggest that human colon cancer cells are controlled by autocrine and paracrine loops using VEGF and aberrant expression of VEGFR and coreceptors as the consequence of the neoplastic progression. In agreement with this notion, we show that VEGF-165 Mol Cancer Ther 2006;5(8). August 2006

receptors are coupled to oncogenic signals inducing MAPK p42/p44 activation in the present study. Moreover, collagen invasion by HCT8/S11 cells exposed to VEGF165 and Sema3A is abrogated by pharmacologic inhibitors targeting the MAPK p42/p44, p38, and phosphatidylinositol 3-kinase. As expected, the invasive potential of VEGF-165 is reversed by the VEGFR signaling inhibitor ZD4190 and blockade of the Rho-ROK axis by C. botulinum exotoxin C3 transferase and Y27632. In contrast, the invasion-stimulating activity of Sema3A was not reversed by ZD4190 and inhibitors of the Rho-ROK axis. Based on these observations, it seems that VEGF-165 and Sema3A function as proinvasive agents in human colon cancer cells by inducing common and divergent signaling pathways. Additional support for our interpretation is provided by reports that VEGF and Sema3 members exert divergent biological effects on cell survival and chemotaxis by virtue of their differential interactions either with VEGF-165 receptors Flt-1/KDR-Flk-1 in complexes with NP-1/NP-2 or with Sema3A receptors plexin A1 in association with the nonreceptor tyrosine kinase NP-1 (31, 32). Thus, we propose that the relative abundance and mobilization of the ligand-binding subunits NP-1/NP-2 and concomitant expression of their signal-transducing subunits Flt-1/Flk-1 and plexin A1 trigger simultaneously common and divergent signaling networks for cancer cell migration and invasiveness. In agreement with this hypothesis, Rac small GTPase activation is involved in VEGF-165/Flt-1/Flk-1 and Sema3A/plexin A1 signaling cascades (33) and cellular invasion in the present study. The guanine nucleotide exchange factor FARP2, an immediate downstream signaling molecule of the Sema3A receptor plexin A1, associates with plexin A1 in the presence of NP-1 and exerts a guanine nucleotide exchange factor activity for Rac but not for Rho or Cdc42 (33). This is consistent with our data showing that Sema3A-induced collagen invasion was Rac dependent and unaffected by RhoA and Cdc42 blockade. Similarly, down-regulation of RhoA-GTP is observed during plexin/ semaphorin signaling and on transient interaction of p190 Rho-GAP with plexin (34). Thus, RhoA inhibition is permissive for the semaphorin signals. Recent studies indicate that vascular endothelial cells express Sema3A, a repulsive axon guidance cue, suggesting that Sema3A may control the invasive growth and guidance of cancer cells toward blood vessels. In coherence with this hypothesis and our data, the p42/p44 MAPK pathways are activated during chemotactic responses induced by Sema3A (35). VEGF autocrine loops may also target cancer cells and signal transducers and activators of transcription 3 signaling in human leukemia and solid tumors of the digestive and urogenital systems (36 – 40). The detection of Sema3A transcripts in HCT8/S11, HT29, and LNM35 cancer cells (data not shown) also argues in favor of the implication of plexin A1/NP1-mediated autocrine regulation loops. Thus, the ‘‘atypical’’ expression of VEGF/semaphorin receptors and ligands in human colon and lung cancer cells in the present study

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and other solid tumors may have a major effect on cancer cell growth, survival, and invasion as well as tumor vascularization, metastasis, and selective homing of cancer cells in target organs. Taken together with recent research in VEGF signaling, our data highlight the implication of VEGF and semaphorin receptors and coreceptors in several detrimental functions associated with the neoplastic progression about their effect on vascular endothelial cells, tumor stromal cells, and immune cells and cancer cell themselves. Our data suggest that colon cancer cells may display multiple dependencies on VEGF and semaphorin signaling for tumor growth, cell invasion and survival, and chemoresistance. As ZD4190 exerts anti-invasive activity against VEGF-165 in the collagen and chick heart fragment assays, we explored its potential therapeutic action as anticancer agent. ZD4190 given as a single drug reduced by 50% to 80% tumor growth in animals bearing human colon and lung xenografts. The therapeutic effect of ZD4190 was more pronounced in tumor xenografts established from HCT8/ S11 colon cancer cells versus LNM35 lung cancer cells, suggesting that this VEGFR drug is acting exclusively through inhibition of tumor angiogenesis in this KDR/Flt1 – negative cancer cell line. Determination of the VEGFR status in cancer cells by laser capture microdissection in colon and lung tumors may identify a subset of patients that will most specifically take an advantage of therapeutic interventions based on VEGFR signaling inhibitors. Interestingly, we found that the src inhibitor M475271 potentiated the antitumor activity of the oral VEGF signaling inhibitor ZD4190 and cisplatin. Similarly, M47521 (10 nmol/L 1 Amol/L) abrogated the spontaneous invasive phenotype of MSTO-211H cancer cells in collagen type I (invasion index: 10 F 1.2%). However, this constitutive invasive phenotype observed in this malignant pleural mesothelioma cell line was insensitive to ZD4190. Combinatorial strategies are therefore required to halt several receptor and nonreceptor kinases in association with classic clinical drugs disrupting the integrity of the DNA. New drugs targeting the semaphorin pathways in human solid tumors will help to improve the therapeutic effect of interventions directed against cancer cells, vascular, and other stromal cells in human solid tumors. In support to this approach, a recent report identified a new compound disrupting VEGF-165 signaling through NP-1 and KDR (41).

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