Published OnlineFirst September 15, 2017; DOI: 10.1158/1078-0432.CCR-17-1444 Biology of Human Tumors Clinical Cancer Research Monoclonal Antibodies Directed against Cadherin RGD Exhibit Therapeutic Activity against Melanoma and Colorectal Cancer Metastasis Ruben A. Bartolome1, Carmen Aizpurua2, Marta Jaen 1, Sofía Torres1, Eva Calvino~ 1, Juan I. Imbaud2, and J. Ignacio Casal1 Abstract Purpose: New targets are required for the control of advanced vascular-endothelial (VE)-cadherin–mediated b1 integrin activa- metastatic disease. We investigated the use of cadherin RGD tion in melanoma and breast, pancreatic, and colorectal cancer motifs, which activate the a2b1integrin pathway, as targets for cells. Antibodies provoked a significant reduction in cell adhesion the development of therapeutic monoclonal antibodies (mAb). and proliferation of metastatic cancer cells. Treatment with mAbs Experimental Design: Cadherin 17 (CDH17) fragments and impaired the integrin signaling pathway activation of FAK in peptides were prepared and used for immunization and antibody colorectal cancer, of JNK and ERK kinases in colorectal and development. Antibodies were tested for inhibition of b1integrin pancreatic cancers, and of JNK, ERK, Src, and AKT in melanoma and cell adhesion, proliferation, and invasion assays using cell and breast cancer. In vivo, RGD-specific mAbs increased mouse lines from different cancer types (colorectal, pancreatic, melanoma, survival after inoculation of melanoma and colorectal cancer cell and breast cancer). Effects of the mAbs on cell signaling were lines to cause lung and liver metastasis, respectively. determined by Western blot analysis. Nude mice were used for Conclusions: Blocking the interaction between RGD cadher- survival analysis after treatment with RGD-specificmAbsandmetas- ins and a2b1 integrin with highly selective mAbs constitutes a tasis development. promising therapy against advanced metastatic disease in colon Results: Antibodies against full-length CDH17 failed to block cancer, melanoma, and, potentially, other cancers. Clin Cancer the binding to a2b1 integrin. However, CDH17 RGD peptides Res; 24(2); 1–12. Ó2017 AACR. generated highly selective RGD mAbs that blocked CDH17 and See related commentary by Marshall, p. 253 Introduction In colon cancer, CDH17 is expressed at low levels in primary tumors or in regional lymph node metastases, as well as in There is a necessity to find new therapeutic targets to control poorly differentiated colon cancer tumors (7). However, metastatic spread in cancer. Therapeutic monoclonal antibo- CDH17 is overexpressed in advanced colorectal cancer liver dies (mAb) against EGFR or VEGF(R) are in clinical use for the metastasis (8), where it correlates with poor prognosis (9). It treatment of advanced metastatic colorectal cancer. However, is also highly expressed in gastric cancer, esophagus carcinoma, their impact on the final outcome of patients with metastasis is pancreatic cancer (10), and hepatocarcinoma (11). CDH17 still limited (1), probably due to the difficulty of assessing the facilitates liver colonization and metastasis in orthotopic mouse EGFR status of the patients (2, 3), adverse effects related to the colorectal cancer models after intrasplenic injection (9, 12). wide distribution of these molecules in healthy tissues, and the CDH17 binds a2b1 integrin through an RGD motif and induces lack of response in patients with KRAS mutations (4). b1 integrin activation, which leads to increased cell adhesion to Cadherin 17 (CDH17), also known as liver–intestine (LI)- Matrigel and type IV collagen, and increased proliferation (12). cadherin, is present in fetal liver and the gastrointestinal tract, The CDH17 RAD mutant does not induce integrin signaling but exhibiting elevated expression during embryogenesis (5). rather leads to a reduction in tumor growth and liver coloni- CDH17 localizes to the basolateral domain of hepatocytes and þ zation (12). In colorectal cancer metastatic cells, we have enterocytes, where it mediates intercellular adhesion in a Ca(2 )- described a nonconventional situation in which a2b1 integrin dependent manner to maintain tissue integrity in epithelia (6). binds to cadherins in a RGD-dependent manner, making it conformationally activatable and allowing it to bind to type IV collagen. This finding modifies the classical notion that a2b1 1 Department of Cellular and Molecular Medicine, Centro de Investigaciones integrin binds type I collagen using the GFOGER motifs in a 2 Biologicas, CSIC, Madrid, Spain. Protein Alternatives SL, Tres Cantos, Madrid, RGD- and conformation-independent way (13, 14). Previous Spain. data support a preferential trans activation model for cadherin/ Note: Supplementary data for this article are available at Clinical Cancer integrin interaction, although cis interaction cannot be ruled out Research Online (http://clincancerres.aacrjournals.org/). (12). No activation effects were observed for the RGD motif on Corresponding Author: J. Ignacio Casal, Centro de Investigaciones Biologicas avora6b4 integrins. (CIB-CSIC), Ramiro de Maeztu 9, Madrid 28040, Spain. Phone: 34 918373112; Fax: RGD motifs are also present in vascular-endothelial (VE)- 34 915360432; E-mail: [email protected] cadherin, CDH6 [fetal kidney (K)-cadherin], and CDH20. doi: 10.1158/1078-0432.CCR-17-1444 VE-cadherin is expressed in aggressive human melanoma cell Ó2017 American Association for Cancer Research. lines and cutaneous melanomas (15), but not in poorly www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 15, 2017; DOI: 10.1158/1078-0432.CCR-17-1444 Bartolome et al. from Dr. A Villalobos (IIB-CSIC Madrid, Spain). RKO, Colo320, Translational Relevance and HT29 human colon cancer cells; A375 human melanoma Many unsuccessful attempts have been made in the last cells; and the pancreatic cancer cell line PANC1 were purchased decades to target integrins with RGD peptides for cancer from the ATCC and passaged for all of the experiments fewer therapy. Here, we have demonstrated that cellular cadherin than 6 months after purchase. BLM, SKBR3, and MDA-MB-468 RGD motifs are equally efficient and more selective targets for were not authenticated in our laboratory. All cell lines were integrin inhibition in metastatic cells than RGD peptides from tested regularly for Mycoplasma contamination and cultured in the extracellular matrix proteins. We describe the development DMEM (Invitrogen) containing 10% FCS (Invitrogen) and anti- fi and functional characterization of cadherin RGD-speci c biotics at 37 C in a 5% CO2 humidified atmosphere. monoclonal antibodies that have demonstrated an extraordi- CDH17 polyclonal antibodies (H-167 and C-17), RhoGDIa nary efficiency in blocking b1 integrin activation and protect- (G-2), VE-cadherin (BV9), and FAK (A-17) were purchased ing mice against liver and lung metastasis from colorectal from Santa Cruz Biotechnology. CDH17 antibody (#141713) cancer and melanoma, respectively. Our data support that and Src (AF3389) were from R&D Systems. Blocking anti-b1 these highly promising results also can be extended to breast, (P5D2), a4(ALC1/1),anda5 (P1D6) integrin antibodies were pancreatic, and other tumors. In summary, these highly selec- from Abcam. b1 integrin antibody specificforhigh-affinity tive monoclonal antibodies open a new avenue for the treat- conformation (HUTS-21) and pY397-FAK (#611722) were ment of metastasis in different types of cancer. from BD Transduction Laboratories. Antibodies against phos- pho-Src (#2101), JNK (56G8), phospho-JNK (G9), ERK1/2 (L24F12), and phospho-ERK1/2 (#9101) were from Cell Sig- naling Technology. Anti-a3 integrin (AB1920) was from EMD a aggressive cell lines isolated from the same tumors (16). VE- Millipore. The antibody against 1 (TS 2/7) integrin was a kind cadherin has been involved in vasculogenic mimicry (the gift from Dr. C. Bernabeu (CIB-CSIC). ability to form novel blood vessel–like structures) in uveal CDH17-derived peptides (VSLRGDTRG, SLRGDTR, and melanomas (16). It is also expressed in Ewing sarcoma (17) LRGDT), VE-cadherin domain 2 (QGLRGDSGT), VE-cadherin and promotes breast cancer progression (18). Recently, we domain 3 (SILRGDYQD), CDH6 (DQDRGDGSL), CDH16 have demonstrated that knocking down VE-cadherin sup- (RAIRGDTEG), and CDH20 (DMDRGDGSI) peptides were syn- presses the lung colonization capacity of melanoma or breast thesized using solid phase chemistry with a Focus XC instrument cancer cells inoculated in mice, whereas preincubation with (AAPPTec). In the CDH17 9-mer VSLRGDTRG, Tyr at position VE-cadherin RGD peptides promotes lung metastasis for both 600 was replaced with a Val to facilitate synthesis and hydrophi- cancer types (19). Like CDH17 RGD peptides, VE-cadherin licity. The cyclic RGD peptide Cilengitide was from Selleck RGD peptides cause b1 integrin activation, suggesting that the Chemicals. a a mechanisms of action for both cadherins are similar (12). siRNAs against human 1 (SASI_Hs01_00067020), 3 a We hypothesized that blocking the cadherin RGD motifs (SASI_Hs01_00196571), and 2 integrin (19) subunits were would provoke an inhibition of liver and lung metastasis purchased form Sigma-Aldrich and transfected using jetPRIME through a2b1 integrin inhibition. Here, we investigated the reagent (Polyplus Transfection). use of 9-mer peptides containing the CDH17 RGD motif and Immunization and preparation of mouse mAbs their flanking sequences