Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

Mohammad Hojjat-Farsangi,Hodjatallah Rabbani,Hakan Mellstedt,Ali-Ahmad Bayat,Jafar Mahmoudian,Amir Hossein Daneshmanesh,Mahmood Jeddi-Tehrani,Fatemeh Ghaemimanesh,Benjamin Edward Rich

doi:10.1371/journal.pone.0061167

Abstract

The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

Highlights

Melanoma is a skin cancer arising from melanocytes located in the epidermis
receptor 1 (ROR1) was constitutively phosphorylated in chronic lymphocytic leukemia (CLL) and cell lines of different origins [16,18]
In the present study we could show that melanoma cell lines expressed a 130 kDa ROR1 protein, corresponding to the fully glycosylated isoform [33]

Summary

Introduction

Melanoma is a skin cancer arising from melanocytes located in the epidermis. The incidence of melanoma is rapidly increasing. The frequency of melanoma is only 4% of all dermatological cancers but responsible for 80% of the mortality in skin cancer. Detection and treatment may improve prognosis [1]. A series of melanoma-associated antigens (MAGE) has been identified on melanoma cells [2,3,4]. Large efforts have been done to use different MAGEs for immunotherapy of melanoma patients, but most clinical trials have failed [5]

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Conclusion