Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells

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.

Figure 1. Protein expression of the receptor tyrosine kinase ROR1 in melanoma cell lines.

Representative experiment (IF) showing the expression of ROR1 on the ESTDAB112 cell line using the anti-ROR1 (clone 3H9) mAb (40×). Nuclei were counterstained with DAPI (blue). A non-relevant isotype control mAb (mouse IgG1 isotype) was used as a negative control (A). Western blot analysis of ROR1 protein expression and phosphorylation in melanoma cells detected by a goat anti-ROR1 antibody, anti-p-tyrosine (PY99) and anti-p-serine (clone 4A4) mAbs (B). ROR1 protein was shown to be phosphorylated in all cell lines using immunoprecipitation of ROR1. A 130 kDa band corresponding to the fully glycosylated/phosphorylated ROR1 was observed. The T47D cell line was used as a ROR1 negative control .

Figure 2. Induction of apoptosis in melanoma cells using anti-ROR1 mAbs.

Frequency (%) of apoptotic/necrotic cells in Annexin-V⁺/PI⁺ (A) and XTT cytotoxicity assay (B) induced by anti-ROR1 mAbs in the absence of complement or immune effector cells [anti-ROR1 mAb clones 1A8 (□), 1E9 () and 5F1 ()3H9 (▪)]. Dot plot diagrams of apoptosis induced by anti-ROR1 mAbs (clones 1A8 and 3H9) in melanoma cells and ROR1 negative cell line T47D (Annexin-V/PI) (C). Western blot for cleaved PARP, caspase 8, 9 and MCL-1 expression in apoptotic ESTDAB049 and ESTDAB112 cells induced by the anti-ROR1 mAb clone 5F1 (D). (−) cells treated with a non-relevant isotype control mAb (mouse IgG1 isotype). (+) cells treated with the anti-ROR1 mAb clone 5F1. (S) cells treated with staurosporine.

Figure 3. Anti-ROR1 mAbs in complement dependent cytotoxicity (CDC).

Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V⁺/PI⁺) induced by 4 anti-ROR1 mAbs with (▪) or without (□) human complement using various ESTDAB (A, B), DFW and A375 melanoma cell lines (C). The T47D cell line did not express ROR1. ^*P = 0.01; ^**P = 0.001. P-values refer to comparison with and without complement for the respective mAbs. NR mAb: non-relevant isotype control mAb (mouse IgG1 isotype), C: Complement.

Figure 4. Cytotoxic effects of anti-ROR1 mAbs in the presence of NK cells (ADCC).

Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V⁺/PI⁺) induced by 4 anti-ROR1 mAbs and a non-relevant isotype control mAb (mouse IgG1 isotype) in the presence of NK cells at different target: effector ratios. Target cells: ESTDAB049 (□), 075 (), DFW (▪), A375 () (A) and ESTDAB081 (□), 094 (), 112 (▪) melanoma cells and T47D () as a ROR1 negative cell line (B). ADCC of the melanoma cells induced by the anti-ROR1 mAbs compared to the non-relevant isotype control mAb (mouse IgG1 isotype) as wells as to the T47D cell line was statistically significant (P = 0.05-0.0001).

Figure 5. Transfection of melanoma cells (n = 6) using ROR1 suppressing siRNA.

Downregulation of ROR1 mRNA (RT-PCR) (A). Downregulation of the ROR1 protein (130 kDa) expression (B). (−) untreated cells, (C) control siRNA treated cells, (+) ROR1 siRNA treated cells.

Figure 6. Apoptosis of melanoma cells treated with siROR1.

Dot plot (frequency) of apoptotic/necrotic melanoma cells (Annexin-V⁺/PI⁺) treated with siRNA, control siRNA and untreated. Within each quadrant the frequency of apoptotic cells is shown. Results are presented for the ESTDAB049, ESTDAB075, A375, ESTDAB112 (sensitive to apoptosis by anti-ROR1 mAbs) and ESTDAB081 (resistant to apoptosis by anti-ROR1 mAbs), The cell lines T74D cell line was used as a ROR1 negative control.