Fig 2: p130Cas/BCAR1 and EREG expression are upregulated in distant metastases, and BCAR1 downregulation sensitizes CRC cells to FOLFIRI. (A) Cells were seeded in 6-well plates and treated after 1 d with vehicle control, 5-FU (20 µM) or FOLFIRI (20 µM 5-FU) for indicated times. BCAR1 mRNA expression was quantified with RT qPCR. Shown are expression levels in relation to vehicle control (set to 1) from one representative out of three performed experiments. (B–D) BCAR1 and EREG levels in patient samples of the GSE131418 data set were analyzed. Patient numbers for each investigated subgroup are presented in Table S3B. (C) BCAR1 and EREG expression in pre- and post-treatment patient samples. (D) BCAR1/EREG expression correlation calculated with Spearman correlation. Spearman coefficients (rs) are presented. (E) Cells were transfected with control or p130Cas siRNA and subsequently incubated with or without recombinant EREG (12.5 ng/mL) and increasing concentrations of FOLFIRI. Cell viability was measured with AlamarBlue assays. Shown are results from one representative out of three performed experiments. IC50 values are indicated. Lower panels, significant differences between response curves were estimated by one-way ANOVA and Bonferroni’s multiple comparison post-test approach. (A–E) ns, not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. IC50, half-maximal inhibitory concentration.

Fig 3: p130Cas/BCAR1 and EREG expression are negatively correlated in CRC cell lines and in right-sided and earlier stages of colorectal adenocarcinomas. (A,F–H) BCAR1 and EREG mRNA levels in patient samples of the TCGA CoAd data set were analyzed. Patient numbers for each investigated subgroup are presented in Table S3A. (A,F) BCAR1/EREG expression correlation calculated with Spearman correlation in the indicated subgroups. Spearman coefficients (rs) are shown. (B) Cells were transfected with control or p130Cas siRNA and subsequently incubated with or without recombinant EREG (12.5 ng/mL) or vehicle control. Proliferation assays were performed as described in Materials and Methods. Statistical differences between growth curves were calculated with the CGGC permutation test. (C) Cells were transfected with control, p130Cas, or EREG siRNA. BCAR1 and EREG mRNA expression was quantified with RT qPCR. Shown are expression levels in relation to control siRNA (set to 1) from one representative out of three performed experiments. (D,E) Cells were treated with recombinant EREG (12.5 ng/mL) or vehicle control for 24 h. (D) BCAR1 mRNA expression was quantified with RT qPCR. (E) Expression and phosphorylation (p-p130Cas, pooled phospho-antibodies) in WCE (20 µg) was investigated by IB. Band intensities were quantified by densitometric analysis. The ratio of p-p130Cas and p130Cas was calculated and related to vehicle control (set to 1). (A–H) ns, not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. si, short-interfering RNA.

Fig 4: BCAR1/p130Cas is strongly expressed in and associated with reduced survival of colorectal cancer patients (TCGA data set). (A) BCAR1 expression was compared in the indicated TCGA PanCancer Atlas data sets by Kruskal–Wallis test (*** p < 0.0001). (B) Overall survival (OS) of patients of the TCGA CoAd cohort expressing high (red curve) or low (blue curve) BCAR1 levels. HR, hazard ratio. CI, confidence interval. (C) Number of studies showing phosphorylation of certain motifs in the different p130Cas domains in CRC and breast cancer as curated at PhosphositePlus. P, proline-rich domain. SBM, bipartite Src binding motif.

Fig 5: BCAR1 expression is associated with poor outcomes in right-sided, stage I and II, MSS, or BRAF-mutated CRCs. Overall survival was analyzed in the indicated subgroups (TCGA CoAd data set) as described in Materials and Methods. CI, confidence interval.