CYR61 and AVB5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Research Article

Yan Monnier,1,2 Pierre Farmer,2,3 Gregory Bieler,1,2 Natsuko Imaizumi,1,2 Thierry Sengstag,2,3 Gian Carlo Alghisi,1 Jean-Christophe Stehle,4 Laura Ciarloni,1 Snezana Andrejevic-Blant,4 Raphael Moeckli,5 Rene´-Olivier Mirimanoff,6 Simon L. Goodman,7 Mauro Delorenzi,2,3 and Curzio Ru¨egg1,2

1Division of Experimental Oncology, Centre Pluridisciplinaire d’Oncologie, Faculty of Biology and Medicine, University of Lausanne, 2Swiss Institute for Experimental Cancer Research, National Centre for Competence in Research Molecular Oncology, and 3Swiss Institute of Bioinformatics, Epalinges Office, Epalinges, Switzerland; 4Institute of Pathology, Faculty of Biology and Medicine, 5Institute of Applied Radiophysics, and 6Department of Radio-Oncology, Centre Hospitalier Universitaire Vaudois and Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland; and 7Merck KGaA, Department of Preclinical Oncology Research, Darmstadt, Germany

Abstract irreversible growth arrest (2). In addition, recent findings indicate Radiotherapy is widely used to treat human cancer. Patients that radiotherapy rapidly and persistently alters the tissue locally recurring after radiotherapy, however, have increased microenvironment by modulating the cell phenotype, tissue risk of metastatic progression and poor prognosis. The clinical metabolism, and the intercellular cross-talk (3). Increasing management of postradiation recurrences remains an unre- evidence indicates that these stromal changes may contribute to solved issue. Tumors growing in preirradiated tissues have the antitumor effects of radiotherapy. However, clinical and an increased fraction of hypoxic cells and are more metastatic, experimental observations also indicate that irradiated stroma a condition known as tumor bed effect. The transcription may have potential deleterious effects on tumor behavior (3). factor hypoxia inducible factor (HIF)-1 promotes invasion and Experimentally, tumors growing within a previously irradiated bed metastasis of hypoxic tumors, but its role in the tumor bed tend to be more invasive and to form more metastases, an effect effect has not been reported. Here, we show that tumor cells known as the tumor bed effect (4, 5). Clinically, adjuvant radio- derived from SCCVII and HCT116 tumors growing in a therapy significantly improves local tumor control in patients preirradiated bed, or selected in vitro through repeated cycles undergoing conservative surgery (6). However, tumor recurrences of severe hypoxia, retain invasive and metastatic capacities within a preirradiated field are associated with higher risk of when returned to normoxia. HIF activity, although facilitating metastasis and poor prognosis compared with recurrences outside metastatic spreading of tumors growing in a preirradiated the irradiated area (7–10). The clinical management of this bed, is not essential. Through gene expression profiling and condition is a challenge, and the underlying cellular and molecular gain- and loss-of-function experiments, we identified the mechanisms are still matter of investigation (11). matricellular protein CYR61 and AVB5 integrin as proteins Although a limiting factor for tumor growth, intratumoral cooperating to mediate these effects. The anti-AV integrin hypoxia has been associated with local tumor invasion, metastasis monoclonal antibody 17E6 and the small molecular AVB3/ formation, and shorter disease-free survival in a number of human AVB5 integrin inhibitor EMD121974 suppressed invasion and tumors, including head and neck cancer (12), cervical cancer metastasis induced by CYR61 and attenuated metastasis of (13, 14) and soft tissue sarcoma (15). Experimental tumors with a tumors growing within a preirradiated field. These results high hypoxic fraction do more frequently metastasize compared represent a conceptual advance to the understanding of the with tumors with low hypoxic fractions (16, 17). Experimental tumor bed effect and identify CYR61 and AVB5 integrin as tumors growing in preirradiated tissues and human tumors proteins that cooperate to mediate metastasis. They also locally recurring after radiotherapy have an increased fraction of identify AV integrin inhibition as a potential therapeutic hypoxic cells (18–20). Rofstad and colleagues (21) have recently approach for preventing metastasis in patients at risk for shown that enhanced invasion and metastasis observed in tumors postradiation recurrences. [Cancer Res 2008;68(18):7323–31] growing within a preirradiated bed involves hypoxia-mediated up-regulation of metastasis-promoting gene products, in parti- Introduction cular, the receptor for the urokinase-type plasminogen activator receptor. These results suggest that primary tumors recurring Radiotherapy has proven therapeutic efficacy in human cancer after radiotherapy show aggressive behavior because of increased management. Around 50% to 60% of cancer patients are treated environmental hypoxia leading to transcriptional activation of by radiotherapy in the course of their disease (1). The antitumor metastasis-promoting genes. The transcription factor hypoxia effect of radiotherapy involves tumor cell DNA damage leading to inducible factor (HIF)-1 is likely to play a key role in this process. p53-mediated apoptosis, mitotic cell death, and senescence-like HIF-1 mediates adaptive responses to hypoxia and activates the transcription of many genes encoding for proteins regulating angiogenesis, glucose metabolism, intracellular pH, cell prolifera- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). tion/survival, tumor invasion, and metastasis (22, 23). Therefore, Requests for reprints: Curzio Ru¨egg, Division of Experimental Oncology, Centre activation of a metastatic program by HIF-1 seems as an attractive Pluridisciplinaire d’Oncologe, 155 Chemin des Boveresses, CH1066 Epalinges, mechanism by which tumor cells growing into a preirradiated Switzerland. Phone: 41-21-692-5853; Fax: 41-21-692-5872; E-mail: [email protected]. I2008 American Association for Cancer Research. hypoxic microenvironment invade and metastasize. However, doi:10.1158/0008-5472.CAN-08-0841 hypoxia-induced up-regulation of prometastatic molecules alone www.aacrjournals.org 7323 Cancer Res 2008; 68: (18). September 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research may not entirely explain the sustained aggressive behavior of respectively. HS, lines derived from parental cells through in vitro hypoxia these tumors because once hypoxic tumor cells reach normoxic selection. In vitro conditions, they may revert back to their initial phenotype. We invasion assay. Matrigel (3.5 mg/mLfor SCCVII and 0.5 mg/mL j therefore hypothesized that, in addition to hypoxia-mediated up- for HCT116) was polymerised at 37 C in the upper Transwell chambers (pore size, 8.0 Am; Corning Life Sciences). Serum-starved (24 h) SCCVII regulation of prometastatic genes, hypoxia forming within tumors (8 Â 104) or HCT116 (5 Â 104) cells were seeded in the upper chamber and growing in preirradiated tissue could generate a selective pressure incubated in full medium for 24 h. EMD inhibitors were used at 5 Amol/L, favoring the outgrowth of highly metastatic tumor cell variants 17E6 at 10 Ag/mL. Filters were fixed with 4% PFA, stained with 0.5% CV, retaining an aggressive phenotype upon escape from hypoxia. and migrated cell counted in 4 random fields per membrane under a Here, we provide experimental evidence supporting this microscope (n = 4). Values represent means F SD. hypothesis by demonstrating that SCCVII and HCT116 tumor cells Microarray hybridization. For each type of cell line (PA, NIR, IR, and derived from tumors growing in a preirradiated bed, or selected HS), three lines (#1, #2, and #3) were derived from three independent in vitro through repeated cycles of sever hypoxia, retain an invasive tumors or by in vitro hypoxia selections and passaged at least twice in vitro and metastatic phenotype even when returned to normoxia. We under standard conditions. RNA was obtained using RNeasy kit (QIAGEN). identified the matricellular protein CYR61 and integrin aVh5 Probe synthesis and Murine Genome 430 2.0 Genechip Array (Affymetrix Ltd) hybridization were performed at DNA Array Facility (DAFL). Micro- as proteins that cooperate to mediate these effects. Furthermore, array data are available at Gene Expression Omnibus8 under the access a we found V integrin inhibitors to suppress lung metastasis number GSE 11357. formation of tumors growing within a preirradiated field. These Mice irradiation, tumor growth, and metastasis. Six- to 10-wk-old À À observations extend our knowledge on the mechanisms involved RAGg2 / mice (Charles River Laboratories) were anesthetized (ketamine, in the tumor bed effect and open novel therapeutic perspective for 100 mg/kg, i.p.), placed in a lead jig allowing exposure to a 25 Â 25-mm field, the management of local tumor recurrences after radiotherapy. and irradiated (10–20 Gy single dose) using an X-ray unit (PHILIPS; RT 250) at 220 kV and 20 mA, with a 0.5-mm Cu filter. Two weeks later, tumors were initiated by injecting 104 (SCCVII) or 106 (HCT116 or HEPA1) Materials and Methods cells per mouse in 100 ALserum-free medium s.c. in the lower-right back Antibodies, chemicals, proteins, and peptides. Bovine serum albumin (at the level of the hip) of the mice (6 or more mice per group). Mice were (BSA), paraformaldehyde (PFA), fibronectin, vitronectin, and 4¶,6-diamidino- inspected daily and tumors measured with a caliper every 4 d. Tumor volume 2-phenylindole were purchased from Sigma Chemie. Antibodies were (V) was calculated as V = k/6 Â a Â b2, where a is the longer and b is the purchased as follows: monoclonal antibody (mAb) P1F6 (anti-aVh5) from shorter of two orthogonal diameters. For EMD121974, or 17E6 treatment, Becton Dickinson; mAb LM609 (anti-aVh3) from Chemicon International; mice were injected i.p. with 0.75 mg/mouse/d or 1 mg/mouse/wk, Lia 1/2 (anti-h1) from Beckman Coulter; and rabbit anti-CYR61 from respectively, starting when tumors reached a size of f10 mm3. For lung Abcam, goat anti-CYR61 (sc-13100; Santa Cruz Biotechnology), and mAb metastasis quantification, mice were sacrificed 32 d after tumor cell injection 356108 (anti-human CYR61) from R&D Systems, biotinylated and purified in all conditions (control versus preirradiated mice). Lungs were removed rat anti-CD31 mAb from PharMingen, and anti–HIF-1a mAb R&D Systems. and fixed in 4% PFA, embedded in paraffin, and a total of 4 coronal Antibody 17E6 was produced in house (Merck Farma). Antibodies to mouse sections separated by 500 Am per lungs were produced and H&E stained. aV (RMV-7), h3 (HMh3), and h1 (9EG7) integrins were kindly provided by The total number of nodules per 4 sections (= metastasis per animal) was Dr. B.A. Imhof (Department of Pathology, CMU, University of Geneva, determined under a binocular microscope (Leica; MZ16). Values stated for Geneva, Switzerland). The EMD121974 (the inner salt form of cyclic-(Arg- each group of animals are means of these numbers F SD. All experiments Gly-Asp-[D-Phe]-[N-Me-Val])) and EMD135981 (the inner salt form of cyclic- with mice were approved by the cantonal veterinary service. (Arg-[h-Ala]-Asp-[D-Phe]-Val)) cyclopeptides were synthesized by Dr A. Histopathologic and immunohistochemistry analyses. For tissue Jonczyk (Merck KGaA, Darmstadt, Germany; ref. 24). morphology evaluation, standard H&E staining procedures were performed Cell lines and cell culture. The mouse oral squamous cell carcinoma on paraffin-embedded tissues. For immunohistochemistry of mouse tumors, line SCCVII was kindly provided by Dr. R. Hill (Division of Applied detection of microvessels and tumor hypoxia was done on frozen sections Molecular Oncology, Ontario Cancer Institute, Princess Margaret Hospital, using a modified double staining protocol combining a biotinylated rat Toronto, Canada). The murine hepatoma lines HEPA1-C1C7 (ARNT- anti-CD31 for detection of mouse endothelial cells and a monoclonal functional) and HEPA1-C4 (ARNT deficient) were obtained from Dr I. hydroxyprobe-1 for detection of pimonidazole adducts (Chemicon Interna- Desbaillet-Hakimi (Centre Hospitalier Universitaire Vaudois, Lausanne, tional). Pimonidazole hydrochloride was injected into tumor-bearing animals Switzerland). The human colorectal carcinoma line HCT116 was purchased 15 to 90 min before sacrifice. Briefly, a total of 4 sections separated by 2 to from American Type Culture Collection (#CCL-247). For all experiments, 5 mm were taken for each tumor, fixed for 10 min in acetone, and blocked for cells were grown in DMEM high-glucose supplemented with 10% FCS and 5 min in 10% normal goat serum. Slides were incubated with primary 1% Penicillin/Streptomycin and maintained in a humidified incubator at antibodies (1:50 dilutions in 0.5% BSA) for 2 h at room temperature, and

37jC with 5% CO2. All cell culture reagents were purchased from Invitrogen. washed and incubated with anti-mouse EnVision horseradish peroxidase and To derive cell lines from tumor grafts, tumor-bearing mice were sacrificed Streptavidin-AP conjugate (Dako) at 1:200 dilution for 30 min. Antibody by cervical dislocation, tumors were excised under sterile conditions, complexes were revealed by 3,3¶-diaminobenzidine (DAB) treatment (Roche) mechanically disrupted with a scalpel, and digested for 2 h at 37jC with for 9 min followed by Fuchsin+ (Dako) for 20 min. Slides were mounted using collagenase A (Roche Applied Bioscience). To obtain a single-cell aqueous solutions. Quantification of microvascular density was done by suspension, digested tissues were filtered through an 80-Am mesh to calculating the Chalkley score (25). To this purpose, the three most vascular remove tissue debris. Recovered cells were seeded on tissue culture Petri areas were chosen subjectively from each tumor section. A 25-point Chalkley dishes (TPP) and maintained for several passages under standard culture eyepiece graticule was applied to each chosen area, and the number of conditions before use in experiments. microvessels hit by the graticule points was recorded. The Chalkley count for For in vitro hypoxia selection, exponentially growing cells were cultured an individual tumor was taken as the mean value of the three graticule counts. F in a hypoxic chamber (Billups-Rothenberg) at 0.1% O2 for 5 d. Cells were Statistical analysis. Results are expressed as mean SD. Data were t in vitro in vivo allowed to recover for 3 d at 21% O2. A total of three cycles were performed analyzed by Student’s test for both and experiments. for each cell line. Cell lines nomenclature were as follows: PA, parental lines; NIR and IR, lines derived from tumors grown on a nonirradiated or irradiated mice, respectively; mNIR and mIR, lines derived from lung metastases of tumors grown on a nonirradiated or irradiated mice, 8 http://www.ncbi.nlm.nih.gov/geo

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P values of <0.05 were considered significant. Error bars depict SD. For statistical analysis of gene expression data, see Supplementary Data. Microarray data analysis, lentiviral constructs, Western blotting, real- time reverse transcription-PCR, and fluorescence-activated cell sorting analyses are described in Supplementary Data.

Results Tumors growing in previously irradiated stroma are more hypoxic and show increased invasion and metastasis. Tumors locally recurring after radiotherapy (e.g., head and neck, rectal, or breast cancers) often regrow within preirradiated stroma. To experimentally mimic this condition, we locally irradiated the lower back of healthy mice (10 and 20 Gy single dose) and, 2 weeks later, implanted tumor cells s.c. within this area. The biological effect on healthy tissue of such single doses corresponds to cumulative does of up to 60 Gy delivered during fractionated radiotherapy, depending on the chosen a/h values, and are therefore of clinical relevance (26). Stroma preirradiation inhibited local growth of SCCVII (murine squamous cell carcinoma) and HCT116 (human colon carcinoma) tumor cells in a dose-dependent manner (Supplementary Fig. S1A). CD31 staining and pimonidazole staining revealed a dose-dependent decrease of microvascular density and the appearance of larger hypoxic regions in tumors growing in preirradiated skin compared with control tumors (Fig. 1A and B). The appearance of hypoxia was paralleled with the appearance of local invasion (Supplementary Fig. S1B) and with a dose-dependent increase in tumor necrosis (Fig. 1C), and lung metastasis (Fig. 1D). Tumor growth in preirradiated stroma selects for invasive and metastatic variants independently of HIF. The increased metastasis formation of tumors growing in a preirradiated stroma raised the possibility that tumor aggressiveness was a conse- quence of sustained tumor hypoxia. Because hypoxia inducible factors, in particular, HIF-1, induce expression of proinvasive and metastatic molecules, we first investigated whether HIF activation was necessary and sufficient for the observed increased rate of metastasis. To address this question, we examined the metastatic capacity of two congenic tumor cell lines with either functional (HEPA1-C1C7) or nonfunctional (HEPA1-C4) HIF (27) by injecting them in control and in locally preirradiated mice. Preirradiation of the prospective microenvironment strongly decreased the growth of both HEPA1-C1C7 and HEPA1-C4 lines in a dose- dependent manner (Supplementary Fig. S1C). The absence of HIF activity in HEPA1-C4 cells inhibited tumor growth on both irradiated and nonirradiated stroma compared with HEPA1-C1C7 grafts (Supplementary Fig. S1C) and strongly reduced the metastatic ability of HEPA1-C4–derived tumors developing on both nonirradiated and preirradiated stroma (Fig. 2A). This result is consistent with the role of HIF as a key mediator of metastasis. However, despite the overall reduced ability of HIF-deficient tumor cells to metastasize, the relative increase (4- to 6-fold) in lung metastasis formation of tumors growing in preirradiated versus Figure 1. Tumors growing in irradiated stroma have reduced microvascular nonirradiated stroma, was not affected (Fig. 2A). This result density, are more hypoxic, necrotic, and metastatic. A, SCCVII tumors were suggested that additional, HIF-independent, mechanisms were stained for vessels (CD31, red) and hypoxic regions (pimonidazole, brown). Tumor growing in preirradiated stroma are less vascularized and are more involved in the increased aggressiveness of tumors growing in hypoxic relative to controls. B, quantification of microvascular density (Chalkley preirradiated microenvironments. One possible mechanism was score) for SCCVII and HCT116 tumors. P values are relative to 0 Gy controls. C, quantification of tumor necrosis of SCCVII and HCT116 tumors growing in the selection of highly metastatic tumor cell variants by the preirradiated versus nonirradiated stroma. D, quantification of lung metastasis of hypoxic microenvironment. SCCVII and HCT116 tumors growing in preirradiated versus nonirradiated To explore this hypothesis, we rederived cell lines from tumors stroma. Microvascular density, hypoxia and necrosis in primary tumors and metastasis in the lungs were monitored at day 32 after tumor cell implantation in that had developed in preirradiated and nonirradiated stroma both control and preirradiated mice. The tumor size of the corresponding (IR and NIR lines, respectively), and compared their invasive and primary tumors is shown in Supplementary Figure S1A. www.aacrjournals.org 7325 Cancer Res 2008; 68: (18). September 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research metastatic capacities with their respective PA lines. IR lines were We used a quantile-quantile plot to graphically identify those genes more invasive in vitro under normoxic conditions (Fig. 2B) and deviating more markedly from the bulk of the observations and more metastatic to the lungs when reinjected s.c. into nonirradi- representing best candidates for significant effects (data not ated mice (Fig. 2C). The in vitro growth rates of these lines and the shown). The transcript encoding for cysteine-rich protein 61 in vivo growth of derived tumors were indistinguishable (data not (CYR61), a member of the CCN (CYR61/CTGF/NOV) family of shown). Importantly, in all derived lines, HIF-1a protein levels were matricelluar proteins regulating cell growth, differentiation, low under normoxia and were effectively induced by hypoxia survival and migration in development, tissue remodeling, and (Fig. 2D), thereby excluding that invasive/metastatic cell subpopu- repair (28), was prominently up-regulated in IR cells. Western lations were selected on the basis of constitutive HIF-1a activation. blotting analysis validated increased CYR61 protein expression in These data show that preirradiated stroma selects for invasive the SCCVII-IR line (Fig. 3C). Consistent with this observation, we and metastatic tumor cell subpopulations retaining invasive and found that levels of CYR61 protein were also stably increased under metastatic capacities even upon return to normoxia and in the normoxic conditions in the HCT116-IR line compared with the absence of constitutive HIF-1 activation. HCT116-PA and HCT116-NIR lines (Fig. 3C). Elevated levels of Gene expression profiling of selected tumor cells. Although CYR61 mRNA and protein were also observed in SCCVII lines the role of HIF in promoting hypoxia-dependent invasion and derived from lung metastases of tumors growing in preirradiated metastasis is well-established, little is known about alternative stroma (mIR) relative to metastases of tumors growing in molecular pathways conferring a metastatic phenotype enduring nonirradiate stroma (mNIR) and parental cells (PA) consistent beyond this selection process. To identify genes linked to the with a role of CYR61 in selecting metastatic variant from tumors metastatic capacity of tumor cell escape variants, we performed growing in preirradiated stroma (Supplementary Fig. S2). gene expression studies of SCCVII-PA, SCCVII -NIR, and SCCVII -IR To see whether hypoxia alone was sufficient to drive the lines under normoxic conditions. Unsupervised gene expression selection of aggressive tumor cells expressing elevated levels of clustering analysis grouped the three conditions separately, CYR61, we selected SCCVII hypoxia-resistant lines in vitro through confirming distinct and stable gene expression profiles (Fig. 3A repeated cycles of cell culture under harsh hypoxic (0.1% pO2) and B). Ninety-two probe sets were differentially regulated between conditions (called HS line). Global gene expression analysis of the IR and NIR conditions at a cutoff corresponding to a false three hypoxia-selected versus three PA SCCVII lines confirmed discovery rate (FDR) of <0.01, and the top 17 probe sets that CYR61 was among the top up-regulated gene in HS cells representing 16 genes at a FDR of <0.001 (Supplementary Table S1). (FDR < 0.001; data not shown). Western blotting analysis validated

Figure 2. Hypoxia selects for invasive and metastatic tumor cell populations independently of HIF activation. A, HIF-deficient (HEPA1-C4) and HIF-competent (HEPA1-C1C7) cells were injected in nonirradiated (0Gy) and 10 or 20 Gy preirradiated stroma. HIF deficiency had a global effect on metastasis but did not abrogate the increased metastatic spreading of tumors growing in preirradiated stroma. P values are relative to 0 Gy controls. B, tumor lines derived from tumors growing in nonirradiated (NIR) or preirradiated (IR) stroma were tested for their invasive capacity in an in vitro Matrigel invasion assay. IR lines are highly invasive compared with PA and NIR lines. C, the same lines were injected s.c. to nonirradiated mice and lung metastasis formation quantified. IR lines are more metastatic to the lung. P values are relative to PA lines. D, Western blotting showing HIF-1a protein level in normoxia (N; 21% O2) and in hypoxia (H; 24 h; 0.1% O2) in all SCCVII and HCT116 variants tested.

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Figure 3. Gene expression analysis of SCCVII-PA, SCCVII-NIR, and SCCVII -IR cell lines. The experiment was done using biological triplicates (#1, #2, and #3): NIR and IR lines were derived from three independent tumors, cultured independently for three passages, and probed independently for gene expression. For the PA line, three sublines were derived, cultured separately for three passages, and probed independently. A, unsupervised clustering analysis of gene expression profiles of SCCVII-PA, SCCVII-NIR, and SCCVII -IR cell lines shows that the three biological conditions have distinct gene expression profiles forming distinct clusters. B, gene expression visualized with color-coded intensity values (Heatmaps: red, high expression; green,low expression). Shown are the 28 probesets of the SCCVII-IR versus SCCVII -NIR comparison that have a FDR not superior to 10% and an estimated fold change of at least 2. Left, hierarchical clustering of the probeset; right, gene symbol annotation and Affymetrix probeset-ID. C, validation of increased CYR61 protein expression in SCCVII and HCT116 IR cells compared with NIR and PA cells by Western blotting.

increased CYR61 protein expression in HS cells (Fig. 4A). As for IR regulation of CYR61 protein expression by shRNA in IR lines cells, HIF-1a protein levels in HS cells were low under normoxia and attenuated Matrigel invasion in vitro and reduced lung metastasis high under acute hypoxia, such as in PA cells (Fig. 4B). When tested in vivo (Fig. 5B). Identical results were obtained with SCCVII cells in vitro for their invasive capacity, or in vivo for their ability to form (data not shown). lung metastasis, HS cells behaved similarly as IR lines (Fig. 4C). These data showed that CYR61 is necessary and sufficient to CYR61 promotes invasion and metastasis. Experimental data promote invasion and tumor metastasis formation in the lung. indicate that CYR61 promotes tumor progression by stimulating Invasion and metastasis induced by CYR61 require AVB5 tumor cell growth, angiogenesis, anchorage independence, and integrin. CYR61 is reported to exert many of its biological invasion (29, 30). Thus far, however, evidence for a prometastatic activities through integrin adhesion receptors (33, 34), including role of CYR61 was only correlative (28, 30–33). To directly test those integrins (e.g., aVh3 and aVh5) promoting invasion and whether the up-regulation of CYR61 in the IR and HS lines played metastasis (35–37). We found SCCVII and HCT116 cells to express a causal role in their aggressive phenotype, we performed gain aVh5 but not aVh3 integrin by cell surface detection of the and loss of function experiments on CYR61. CYR61 over- receptor (Fig. 6A and data not shown). Modulation of CYR61 expression in PA lines increased Matrigel invasion in vitro and expression level (i.e., overexpression and knockdown) had no effect promoted lung metastasis formation in vivo (Fig. 5A). Down- on aVh5 cell surface level (data not shown). www.aacrjournals.org 7327 Cancer Res 2008; 68: (18). September 15, 2008

Taken together, these results show that tumor cell–derived CYR61 cooperates with tumor cell aVh5 integrin to mediate invasion and metastasis.

Discussion Tumors recurring within a previously irradiated field have increased probability of developing metastasis (4, 8–10). This is a clinically relevant situation because 10% to 20% of the patients treated by radiotherapy will relapse within the irradiated field and progress to form metastases. To date, there are no effective therapeutic options to prevent or treat these relapses. The presented data give new insights in the mechanisms involved and provide a rational for a therapeutic approach to prevent postradiation tumor escape.

Figure 4. In vitro HS SCCVII cells express elevated levels of CYR61 and are more invasive and metastatic. A, SCCVII cells selected in vitro through three rounds of severe hypoxia (5 d; 0.1% O2) alternated to normoxia (3 d; 21% O2) have increased levels of CYR61 protein. B, Western blotting showing HIF-1a protein level in normoxia (21% O2) and in hypoxia (24 h; 0.1% O2)inPAandHS SCCVII cells. C, PA and HS SCCVII cells were tested for their invasive capacity in an in vitro Matrigel invasion assay and lung metastatic capacity upon s.c. injection in nonirradiated mice. HS lines are more invasive and metastatic compared with PA cells.

To test for the functional role of aVh5 in mediating the proinvasive and prometastatic effects of CYR61, we performed invasion and lung metastasis experiments in the absence or presence of aV integrin inhibitors. Because of the availability of the 17E6, an anti-human–specific aV blocking antibody (38), functional experiments were mostly done using the human HCT116 lines. The aVh3/aVh5-specific antagonist EMD121974 (24), currently in clinical development in oncology as cilengitide (39), was also used in vitro and in vivo to obtain clinically relevant proof-of-concept experiments. Treatment with EMD121974, or with 17E6, inhibited in vitro Matrigel invasion by IR, HS, and CYR61-overexpressing HCT116 lines (Fig. 6B). EMD121974 and 17E6 treatment reduced invasion of PA, IR, and CYR61-overexpressing cells below basal (i.e., no treatment) levels, consistent with a role of aVh5 in both basal and CYR61-enhanced invasion. These results also show that the aVh5-dependent invasion-promoting effect of tumor cell–derived CYR61 was cell autonomous because no other cells were present in the assay. To test whether such a cell-autonomous effect also occurred in vivo, we injected HCT116 CYR61 or mock transfectants into nonirradiated mice and started 17E6 treatment when tumors were palpable (f10 mm3). MAb 17E6 specificity for human aV allowed us to rule out any putative effect on the host microenvironment. 17E6 treatment inhibited enhanced lung metastasis capacity of HCT116 cells overexpressing CYR61 (Fig. 6C). To obtain a clinically relevant proof-of-concept whether aVh5 integrin inhibition could be a therapeutic approach to prevent metastatic spreading of Figure 5. CYR61 promotes invasion and metastasis. A, CYR61 cDNA- transduced HCT116 cells have increased in vitro invasive and in vivo metastatic tumors growing within a preirradiated field, we injected HCT116 capacities compared with PA cells. Increased CYR61 protein expression in cells into preirradiated or control mice and treated them with HCT116 transduced with CYR61 cDNA is shown by Western blotting. B, CYR61 EMD121974. EMD121974 treatment effectively prevented metasta- protein knockdown in HCT116-IR lines by CYR61-specific shRNA reduces D in vitro invasive and in vivo metastatic capacities of HCT116-IR cells. Western sis formation (Fig. 6 ). Importantly, primary tumor growth was not blotting shows reduced CYR61 protein expression in HCT116 cells (lines#1 affected (data not shown). and #2) transduced with CYR61-specific shRNA. Ctrl, control.

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A first key finding of this study is the demonstration that tumors HIF-1 gene expression signature was seen in IR and HS lines. growing within a preirradiated stroma are selected for escape The fact that escape variants retain enhanced adhesive, invasive, variants that retain an invasive and metastatic phenotype even and metastatic capacities upon return to normoxia, suggest a upon return to normoxia. Hypoxia seems as the main driving force mechanism involving hypoxia-mediated selection rather than in this process because in vitro selection through repeated cycles adaptation, which by definition is transient. A selection mechanism of hypoxia generated highly metastatic tumor cells that behaved is consistent with an emerging paradigm of cancer metastasis, indistinguishably from the in vivo obtained escape variants (i.e., whereby cells with a basal predisposition to metastasize are already increased levels of CYR61 expression, enhanced aVh5-depedent present in the primary tumor, and the full development of their invasion, and metastasis). The role of hypoxia in promoting tumor metastatic capacity requires the expression of only a few specific progression and metastasis is well-documented by experimental complementary functions in addition to the bulk of features and clinical observations, including in the context of the tumor bed promoting growth and survival at the primary site (43, 44). Thus, effect (13–16, 21, 40–42). It has been largely considered that hypoxia the risk of metastasis is proportional to the fraction of tumor cells promotes invasion and metastasis by eliciting an adaptive response expressing these complementary functions. Indeed, we have found a through HIF-1–dependent transcriptional activation of metabolic limited number of differentially expressed genes between parental and invasive programs (22, 23). Our results, however, suggest that and in vivo escape variants or in vitro hypoxia-selected variants. HIF-1–dependent events, although important, are not essential for These differentially expressed genes (signature) include those (i.e., metastatic spreading of hypoxic tumors: HIF-deficient cells can CYR61 and ITGB5) causally involved in metastasis. form enhanced metastasis when growing in irradiated skin; HIF-1 The second key finding of this work is the identification of levels are not constitutively increased in escape variants; and no CYR61 and aVh5, as molecules cooperating in promoting

Figure 6. aVh5 integrin mediate invasion and metastasis induced by CYR61. A, flow cytometry analysis of HCT116 cells showing expression of aVh5. aVh3 is not expressed in HCT116 cells. B, in vitro invasion of HCT116-IR and HCT116-HS and of CYR61-overexpressing HCT116-PA cells is blocked by integrin the aVh5/aVh3 inhibitors EMD121974 and by mAb 17E6, a pan-anti–aV integrin blocking antibody. C, inhibition of tumor cell aV integrin (17E6) abolished enhanced lung metastasis formation of tumors formed by HCT116 CYR61 overexpressing cells in nonirradiated mice. D, EMD121974 treatment suppressed enhanced lung metastasis of tumors formed by HCT116 cells injected into preirradiated stroma.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research metastasis. CYR61 may belong to the class of prometastatic protein preirradiated tissue selects for stable, aggressive tumor cell expressed in the primary tumor, such as ID1, CXCL1, cyclo- variants. Our data identifies CYR61 and aVh5, as molecules that oxygenase 2, EREG, and matrix metalloproteinase 1, whose cooperate to promote metastasis, and suggest a therapeutic expression increases with metastatic ability (45). Importantly, approach for preventing escape, which can be promptly tested in CYR61 was not induced by hypoxia, consistent with a previous the clinic. The proposed model is summarized in Supplementary study demonstrating that, in advanced melanoma, CYR61 expres- Fig. S3. sion occurs independently of hypoxia (46). Intriguingly, HRG, fibroblast growth factor 2, platelet-derived growth factor, and Disclosure of Potential Conflicts of Interest transforming growth factor h, four cytokines promoting cancer progression were reported to induce CYR61 expression (47), raising No potential conflicts of interest were disclosed. the possibility that CYR61 expression may be driven by those genetic or epigenetic events promoting malignant progression. Acknowledgments Integrin contribution to cancer progression is well-recognized (48). Received 3/5/2008; revised 5/15/2008; accepted 6/15/2008. The role of aVh5 in promoting metastasis, however, has remained Grant support: Swiss National Science Foundation (SNF, 3100A0 118079), Oncosuisse (OCS 0181212-12-2005 and 02020.02.2007), Gebert Ru¨f Stiftung, the unclear because divergent results were reported using different National Center for Competence in Research Molecular Oncology, a research experimental models (35, 49). aVh5 was shown to mediate CYR61- instrument of the SNF, and the Medic Foundation. stimulated fibroblast migration (50) and to promote resistance to 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 apoptosis in MCF-7 cells (51). Here, we provide original evidence with 18 U.S.C. Section 1734 solely to indicate this fact. that aVh5 is essential for CYR61-induced metastasis. It remains We thank F. Lejeune for continuous support, G. Christofori, F. Bosman, Ph. Monnier, B.A. Imhof, and I. Stamenkovic for discussion; R. Hill, B.A. Imhof, I. Debaillet to be seen whether CYR61 induces these effects by acting as a Akimi, L. Lau, L. Naldini, R. Iggo, A. Follenzi, A. Jonczyk, and M. Frech for kindly canonical aVh5 ligand, or by modulating aVh5 function indirectly providing reagents; O. Hagenbuchle, K. Harshman (DAFL), L. Ponsonnet, and the through an alternative receptor and intracellular signaling events. Microscopy, Imaging Morphology Facility at Swiss Institute for Experimental Cancer Research for help with experiments, R. Driscoll for reading of the article and the In conclusion, this study represents a conceptual advance to University of Lausanne DAFL and Swiss Institute for Bioinformatics Vital-IT project the understanding of the tumor bed effect by demonstrating that for infrastructure support.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Yan Monnier, Pierre Farmer, Gregory Bieler, et al.

Cancer Res 2008;68:7323-7331.

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