Fig 1.
ROR1 expression in lung adenocarcinoma.
(A) Flow cytometry analysis of ROR1 protein expression on the surface of NSCLC cell lines. The histograms on the right represent staining for ROR1 with polyclonal goat anti-ROR1 antibody. The background signal with normal goat IgG is shown in gray shadow. (B) Representative images of lung adenocarcinoma tissues (LA) detected by immunohistochemical analysis. Formalin-fixed, paraffin-embedded tissues were stained with polyclonal rabbit anti-ROR1 antibody or normal rabbit IgG. Tissue-bound ROR1 is shown in brown and the nucleus counterstained with hematoxylin is in blue (scale bar in the bottom right picture represents 50μm). A score of 0 indicates that none of the cells within the sample bound to the anti-ROR1 pAb; a score of 1 indicates low-level binding of the pAb on more than 25% of tumor cells; a score of 2 indicates low-level binding of the pAb on more than 50% of tumor cells or moderate-level staining on more than 25% of tumor cells; a score of 3 indicates moderate-level staining on more than 75% of tumor cells or high level staining on more than 50% of tumor cells. Controls include: Chronic lymphocytic leukemia lymph node (CLL LN) stained with normal rabbit IgG as negative control and with polyclonal rabbit anti-ROR1 antibody as positive control; Reactive hyperplasia of lymph node (RH LN) and tissues judged to be adjacent to cancer (TAC) stained with polyclonal rabbit anti-ROR1 antibody as negative controls. (C) A summary of immunohistochemical analysis for ROR1 staining in lung adenocarcinoma specimens. The proportion of lung tumor tissues found negative (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in the pie chart. (D-E) The proportion of lung adenocarcinoma tissues of different stages or sex found lacking staining (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in each bar. The number of different cases examined for each group is indicated in the parentheses. Statistical analyses was performed using Mann–Whitney U test. p = 0.048.
Table 1.
Drugs used and their IC50 values (μM) in NSCLC cells.
Fig 2.
Blocking ROR1 via siRNA inhibited tumor cell growth and induced apoptosis.
(A-C) PC9, XLA-07, and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for ROR1 protein expression with chimeric rabbit/human anti-ROR1 monoclonal antibody R12 by flow cytometry (A), growth-inhibition by MTS assay (B) and apoptosis induction by Annexin-V/PI staining (C). The height of each bar in the graph B provides the mean number of viable cells that are representative of more than three independent experiments. *** p<0.001, ** p<0.01 by Student’s t test.
Fig 3.
ROR1 activates the PI3K/AKT/mTOR signaling pathway.
PC9 and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for phosphorylated AKT at Ser-473 (p-AKT), phosphorylated mTOR at Ser-2448, and phosphorylated PTEN at Ser-380/Thr-382/383 (p-PTEN) by immunoblot analysis.
Fig 4.
Proposed model of ROR1-mediated tumor cell survival and proliferation via PI3K/AKT/mTOR signaling pathway.
Activation of ROR1 significantly enhanced phosphorylation of both AKT and mTOR, and deactivated PTEN, a negative regulator of PI3K/AKT.