Fig 2: DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells(a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. ß-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against ß-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a). Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against ß-actin. (a-b) NS, p > 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; statistical significance was calculated using one-way ANOVA followed by Bonferroni test.

Fig 3: DDR1 affects IR – dependent transcription factors(a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against ß-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against ß-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human ß-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b), IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human ß-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Statistical significance was calculated using Student's t-test.

Fig 4: IR, DDR1 and IGF-1R expression in cultured cells(a) DDR1, IR and IGF-1R protein expression in various cell lines. A panel of human breast cancer cell lines (MCF-7, T47-D, ZR-75, BT-474, MDA-MB-157, MDA-MB-231, MDA-MB-468) were analyzed by western immunoblot for DDR1, IR and IGF-1R expression using specific polyclonal antibodies, as indicated. ß-actin antibody was used as control for protein loading. A representative blot of three independent experiments is shown. (b) qRT-PCR analysis of DDR1 and IR mRNA. Human DDR1 and IR mRNA levels were evaluated in all human cell lines shown in panel (a). Normalization was done using human ß-actin as housekeeping control gene. Data are presented as the mean±SEM (error bars) from three independent experiments.

Fig 5: DDR1 correlates with IR in human breast cancer datasetsStratification analysis of DDR1 and IR correlative expression was performed. Pearson tests were calculated by separating the patients in clinical classes. This analysis was computed for GEO datasets gse21653 (Panel A), gse42568, gse76124 (Panel B). Black dots showed nine different combinations of Pearson test for three DDR1 probes (1007_s_at, 210749_x_at, 207169_x_at) and three IR probes (213792_s_at, 226450_at, 226216_at). Values plotted in the histograms are shown as –log10 of Pearson p-values. ER: estrogen receptor status; PR: progesterone receptor status; L: pathological lymph node status; Ki67: Ki-67 protein status evaluated by immunohistochemistry; pT: pathological tumor size; NB: normal breast tissue; PB: pathological breast tissue; G: grading of gse42568 datasets. G* grading of gse76124 datasets.