Targeting CDH17 in Cancer: When Blocking the Ligand Beats Blocking the Receptor?

Author Manuscript Published OnlineFirst on October 30, 2017; DOI: 10.1158/1078-0432.CCR-17-2823 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Targeting CDH17 in cancer: when blocking the ligand beats blocking the receptor?

John F. Marshall Centre for Tumour Biology Barts Cancer Institute Queen Mary University of London Charterhouse Square London EC1M 6BQ, UK

Running title: Antibody targeting CDH17 in cancer

Corresponding author: John F. Marshall Centre for Tumour Biology Barts Cancer Institute Queen Mary University of London Charterhouse Square London EC1M 6BQ, UK

E-mail: [email protected] Phone: +44(0)20 7882 3580

NB Author has no conflicts or disclosures

Funding Support: J. F. Marshall is supported by Cancer Research UK grant C355/A25137.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 30, 2017; DOI: 10.1158/1078-0432.CCR-17-2823 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Summary: Cadherin-17 (CDH17) has been implicated as pro-tumorigenic for many years but mechanisms have been unclear. A Spanish team have generated antibodies to an RGD-motif in CDH17 that inhibits integrin 21 binding to CDH17 and thereby inhibits integrin activation, tumorigenesis and metastasis. These reagents may have therapeutic potential.

Main text: In this issue of CLINICAL CANCER RESEARCH Bartolome and colleagues have taken a novel approach to prevent integrin-dependent tumorigenesis and metastasis by targeting the ligand instead of the receptor (1). Certainly the development of reagents to block the function of specific integrin adhesion molecules as a means of creating new strategies for the therapy of inflammation, fibrosis and cancer is a well established concept. In fact, at least with integrin targets on leukocytes and platelets, many pre-clinical studies have transferred successfully to the clinic (2). In contrast, targeting the binding site on the integrin ligand to achieve a similar biological goal is not a common strategy but has been successfully developed by the Casal group in Madrid, Spain (1).

The liver-intestine (LI)-cadherin CDH17 has been implicated in promotion of cancer for almost two decades (3). As noted by Bartolome and colleagues compared with normal tissue CDH17 expression is significantly increased in cancers of many organs but particularly of the gastrointestinal tract where it is often correlated with poor prognosis and the promotion of metastasis(1,4,5). However the mechanisms underlying these correlations were not wholly resolved. Over the last few years the Casal group has identified that integrin 21 is a receptor for CDH17. Thus when colon cancer cells expressing mutant CDH17 in colon cancer cells, where the RGD sequence of the membrane-proximal domain of CDH17 was mutated to an inactive RAD, were compared with cells expressing wild-type CDH17, it was found that only the wild-type CDH17 promoted activation of 1 integrins. To identify which integrin was involved in the binding siRNA experiments to knockdown each of the integrin subunits expressed in the test colon cancer cell line (4) revealed that 21 promoted interaction with CDH17 and that the RGD sequence in CDH17 was critical for this interaction. In fact 21 could be specifically isolated by affinity chromatography using a column coated with the cadherin domain 6 of CDH17 that contains the VSLRGDTRG binding site. CDH17 expression enhanced integrin activation and signalling and correlated with a moderately increased adhesion and proliferation of pancreatic cancer cells. Furthermore the authors generated RKO and KM12SM colon cancer cell lines expressing CDH17 wild-type protein or mutant RAD and showed that the RGD motif significantly enhanced tumour growth. Based on the sum of their data the authors presented a model whereby 21 on one cell bound to the membrane proximal RGD-site in CDH17 in trans on an adjacent cell (4 and Figure 1).

Several cadherin molecules including CDH16, CDH5 (VE-Cadherin), CDH6 and CDH20 possess an RGD motif (4). Bartolome and colleagues examined VE-cadherin which has two adjacent domains that have an RGD motif. Again the ability to activate 1

integrins was dependent on the presence 21. Incubation of MDA MB468 breast carcinoma and BLM melanoma cell lines with either of the two RGD peptide sequences from VE-cadherin was sufficient to enhance integrin activation, adhesion, proliferation, invasion and experimental metastasis to the lung (5). High expression of VE-cadherin enhanced experimental metastasis of lung and breast lines and correlated with poor survival in breast and melanoma patients. The authors posit that there is a general mechanism whereby 21 interacts with the RGD motif in CDH17 or VE-cadherin resulting in 1 integrin activation (it was not established if it was only 21 integrins that were activated) resulting in increased integrin signalling and adhesiveness which enhanced their tumorigenic and metastatic capacity.

In the current study published herein (1) the authors have developed upon this concept by generating monoclonal antibodies that bind to and block the RGD binding site in CDH17 in the hope of inhibiting this cadherin RGD/21 dependent pro-tumorigenic activity. Immunising mice with ovalbumin conjugated to the VSLRGDTRG 9-mer from CDH17 Bartolome and colleagues generated a panel of monoclonal antibodies. Several monoclonal antibodies (mAbs) had the ability to inhibit the activation of 1 integrins induced by RGD peptides derived from CDH17, but also VE-cadherin and CDH6, the foetal kidney cadherin. Inoculation of colon, melanoma or breast cancer cell lines in the spleen, together with anti CDH17-RGD peptide mAb 6.6.1 eliminated RT-PCR detection of metastatic cells in the lung or liver 4 days after inoculation. More excitingly, when mice injected intra-splenically with KM12SM colon cancer cells were given intravenous 6.6.1 mAb bi-weekly for two weeks there was a highly significant increased survival and reduction in the burden of liver metastasis. These data suggest that targeting the RGD sequence of CDH17 (and potentially therefore also VE-cadherin and CDH6) in tumour cells can block binding to 21 and potentially have a therapeutic effect.

The integrin 21 is perhaps more commonly associated with being a receptor for certain collagens (and laminin) through binding to the non-RGD site GFOGER (6) in the collagen sequence although it has also been reported to bind several other ligands including decorin, MMP-1, endorepellin, C1q and certain viruses (7). Thus by targeting the binding sites on the tumour-promoting ligands of 21, the therapeutic effect of the antbodies will be separated from the potential effects on other 21-ligand interactions that may be important for homeostasis or wound repair; for example fibroblast binding to collagen. It is possible that such an approach may reduce off-target effects seen for targeting other key molecules that have multiple ligands. Certainly some antibodies that inhibit integrins have been withdrawn from clinic due to toxicicty (2). With more biological information of their key ligand specificities perhaps targeting specific ligand motifs may have been therapeutically more successful. . The discovery that 21 binds to members of the cadherin family was first reported in 2002 when Whittard et al reported that human 21 bound to recombinant rodent E-cadherin in vitro, but only when the E-cadherin was presented as a pentameric construct, but not as monomeric E-cadherin (8). This binding was cation- dependent and appeared to be via interaction with the I-domain of the integrin. It

remains unclear whether these observations have any importance in binding to human E-cadherin. In contrast the data of Bartlome and colleagues now provide clear evidence that the integrin 21 can bind several other human cadherins via interaction with the RGD-motif (NB there is no RGD motif in E-cadherin). It is intriguing why in none of the studies from the Casal team was there any attempt to use 21-inhibitory antibodies. This would have provided integrin-specific proof in experiments where only siRNA was used alone to confirm 21 specificity.

The integrin 21 has itself been implicated in promoting metastasis of melanoma and ovarian cancer cells to the liver (9, 10) but in light of the data presented by Bartolome and colleagues, the mechanisms suggested (collagen-binding) might have to be revisited.

While the studies of the Casal group still seem far from human therapeutic studies they do offer a novel strategy that could potentially have clinical value. It will be welcome to see whether the therapeutic efficacy of these anti-RGD antibodies is enhanced by combination with conventional chemotherapies, anti-PD-L1/CTLA4 or anti tumour-microenvironment drugs. In fact these data could assist in generating the financial support for the journey to the clinic. Certainly this is an interesting area of biology that I hope we will see progressing positively.

References

1 Bartolomé RA, Aizpurua C, Jaén M, Torres S, Calviño E, Imbaud JI, Casal JI. Monoclonal antibodies directed against cadherin RGD exhibit therapeutic activity against melanoma and colorectal cancer metastasis. Clin Cancer Res. 2017; pii: clincanres.1444.2017. doi: 10.1158/1078-0432.CCR-17-1444.

2 Raab-Westphal S, Marshall JF, Goodman SL. Integrins as Therapeutic Targets: Successes and Cancers. Cancers (Basel). 2017;9(9). pii: E110. doi: 10.3390/cancers9090110. Review.

3 Grötzinger C, Kneifel J, Patschan D, Schnoy N, Anagnostopoulos I, Faiss S, Tauber R, Wiedenmann B, Gessner R. LI-cadherin: a marker of gastric metaplasia and neoplasia. Gut. 2001;49:73-81.

4 Bartolomé RA, Peláez-García A, Gomez I, Torres S, Fernandez-Aceñero MJ, Escudero-Paniagua B, Imbaud JI, Casal JI.. An RGD motif present in cadherin 17 induces integrin activation and tumor growth. J Biol Chem. 2014 289:34801-14. doi: 10.1074/jbc.M114.600502

5 Bartolomé RA, Torres S, Isern de Val S, Escudero-Paniagua B, Calviño E, Teixidó J, Casal JI. VE-cadherin RGD motifs promote metastasis and constitute a potential therapeutic target in melanoma and breast cancers. Oncotarget. 2017 8:215-227. doi: 10.18632/oncotarget.13832.

6 Knight CG, Morton LF, Peachey AR, Tuckwell DS, Farndale RW, Barnes MJ. The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens. J Biol Chem. 2000;275:35-40.

7 Zutter MM, Edelson BT. The alpha2beta1 integrin: a novel collectin/C1q receptor. Immunobiology. 2007;212:343-53. Review.

8 Whittard JD1, Craig SE, Mould AP, Koch A, Pertz O, Engel J, Humphries MJ. E- cadherin is a ligand for integrin alpha2beta1. Matrix Biol. 2002;21:525-32.

9 Yoshimura K, Meckel KF, Laird LS, Chia CY, Park JJ, Olino KL et al. Integrin alpha2 mediates selective metastasis to the liver. Cancer Res. 2009;69:7320-8.

10 Fishman DA, Kearns A, Chilukuri K, Bafetti LM, O'Toole EA, Georgacopoulos J et al. Metastatic dissemination of human ovarian epithelial carcinoma is promoted by alpha2beta1-integrin-mediated interaction with type I collagen. Invasion Metastasis. 1998;18:15-26.

Figure Legend CDH17/21 interactions promote cancer. The RGD containing domain (green) of CDH17 or CDH5 (aka VE-cadherin) is bound by integrin 21 on an adjacent tumour cell. This results in enhanced integrin activation, adhesion, proliferation, invasion, tumorigenesis and metastasis. All of these processes can be suppressed by antibodies raised against the VSLRGDTRG-motif of CDH17.

Figure 1: Tumor cell Tumor cell

A2 CDH17

B1 CDH5

Anti-VSLRGDTRG antibodies

B1 integrin activation Adhesion Proliferation Invasion Tumorigenesis Metastasis

Targeting CDH17 in cancer: when blocking the ligand beats blocking the receptor?

John F Marshall

Clin Cancer Res Published OnlineFirst October 30, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-2823

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2017/10/28/1078-0432.CCR-17-2823. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.