Figure 4
p65BTK is a novel oncogenic protein acting downstream of RAS/MAPK pathway and is overexpressed in colon cancers. (a) NIH3T3 cells transfected with empty vector or plasmids encoding p65BTK, p77 or mutated H-RAS (H-RASV12). p65BTK expression was assessed by p65BTK-specific polyclonal antibody BN49, whereas p77BTK was probed with a monoclonal antibody against the N-term of BTK (BD). (b) Phase contrast images of NIH3T3 transfected with empty vector or plasmids expressing p77BTK, p65BTK, H-RASV12; × 40 magnification. To note, p77BTK-transfected NIH3T3 maintain the same appearance of the empty vector-transfected untransformed fibroblasts, whereas p65BTK-transfected NIH3T3 are similar to H-RASV12-transformed fibroblasts. (c) In soft agar assay, p65BTK-transfected NIH3T3 fibroblasts showed a colony-forming activity higher than H-RASV12-transfected ones (× 10 magnification). Right: number of colonies (mean of three separate wells). (d) Focus assay of NIH3T3 cells transfected with empty vector, H-RASV12, p65BTK or p77BTK expression plasmids, grown in the absence or presence of BTK (Ibrutinib), RAS (FTI-277) or MEK1/2 (CI-1040) inhibitors; parallel samples of p65BTK-transfected cells were treated for 16 days with CI1040 or treated for 10 days with CI1040 followed by 6 days without drug; (× 10 magnification). (e) Immunohistochemical detection of p65BTK, hnRNPK and p-ERK-1/2 in formalin-fixed, paraffin-embedded specimens (× 40 magnification); tumour samples (T) showing predominant cytoplasmic hnRNPK expression and moderate to strong p-ERK-1/2 levels expressed the highest amounts of p65BTK, whereas low expression of p65BTK was detectable in peritumoural (PT) samples, in which hnRNPK was exclusively or predominantly nuclear and p-ERK-1/2 levels were very low. (f, g) Overexpression of p65BTK in patients with stage II colon cancer. Tissue microarray (TMA) analysis of p65BTK expression was performed in tumoural/peritumoural pairs of specimens from a cohort of 83 patients and results were grouped by comparing the expression in tumoural vs peritumoural tissues (f) and by the intensity of the staining in the tumour tissue (g).
