Fig 2: IRS4 in human breast cancer patient material and breast cancer cells.(a) Expression of IRS4-mRNA in 27 random human primary breast carcinomas (by qRT–PCR). Expression levels greater than median+s.d. (grey dashed line) were defined as positive (IRS4+) and greater than median+2 × s.d. (black dashed line) as highly positive (IRS4+++). (b) IRS4 Western blot of a subset of tumours from a. +++ are IRS4-high samples, +/- are samples at the borderline of IRS4-positive and IRS4-negative in a. (c) IRS4 mRNA-expression levels (log2 transformed) in 157 metastasized human primary breast carcinomas (microarray probe ILMN_1712774 of Illumina HumanHT-12 v4 Expression BeadChip). Log2-ratios greater than 1.5 × s.d. (grey dashed line) were defined as positive (IRS4+) and greater than 2 × s.d. (black dashed line) as highly positive (IRS4+++). (d) Overall survival of patients with IRS4-negative and IRS4-positive (combining IRS4+ and IRS4+++) tumours using the threshold depicted in c. **P<0.01 (log-rank test). (e) Distribution of the IRS4-negative and IRS4-positive (combining IRS4+ and IRS4+++) tumours using the threshold depicted in c over the indicated PAM50-based clinical subtypes. **P<0.01 (Pearson's ?2-test). (f,g) IRS4-mRNA expression in 30 HER2-positive (f) and 31 triple-negative primary breast carcinomas (g), determined by quantitative RT–PCR showing. Expression level cut-offs as in a. The tumour percentage (estimated by a pathologist) of each sample is indicated below. N/A indicates that tumour percentage was not available. (h) Western blot showing IRS4 protein, and phosphorylated (p-) and total AKT from MDA-MB-453 cells transduced with the indicated shRNAs. (i) Quantified IRS4-levels plotted against ratios of phosphorylated AKT over total AKT, derived from the blot in h. Linear regression and associated R2-values show a positive correlation. (j) Quantified IRS4 levels, derived from the blot in h, plotted against the corresponding cell proliferation rates, determined in triplicate by the Crystal Violet staining as in Supplementary Fig. 7f. Linear regression and associated R2-values show a strong positive correlation. (k) Representative soft agar growth scans of MDA-MB-453 cells, transduced with the indicated shRNAs, cultured for two weeks in absence of insulin, or vehicle. (l) Anchorage-independent growth (mean+s.d.), relative to Parental cells, of four independent experiments as in k. ***P<0.001, ****P<1 × 10-4 (Welch's t-test, compared with shGFP).

Fig 3: MMTV proviral insertions in the Irs4 locus and expression analysis.(a) Insertion map of a 300 Kb section of the X-chromosome comprising the Irs4 locus. The map shows the targeted Irs4 gene (blue) and non-targeted Col4a5 gene (grey). Rectangles indicate exons, where solid rectangles depict translated exons and open rectangles the UTRs. The lines interspacing the exons indicate introns. Arrowheads indicate the position and orientation (based on Ensembl build 67, NCBIm37) of MMTV proviral insertions in independent tumours, black arrowheads indicate insertions in which the MMTV-LTR putatively will act as a promoter, green or orange arrowheads (upstream or downstream, respectively) indicate insertions potentially acting as enhancer of the endogenous Irs4-promoter. One integration in this locus (white triangle) is more likely a spurious insertion improbable to activate Irs4 (but might activate Col4a5). (b,c) Expression of Irs4 (b) and Col4a5 (c) mRNA in a random series of independent MMTV-induced mammary tumours with (n=13) and a random series without (n=16) an insertion in the Irs4 locus, showing a strong correlation between MMTV-insertion in the Irs4 locus and Irs4 expression, but no correlation with Col4a5-expression. **P<0.01; NS, not significant (Welch's t-test). (d) The percentage each genotype is contributing to the tumours with (n=35) and tumours without (n=1,097) insertions in the Irs4-locus. No preference for Irs4 insertions in mammary tumours from any of the tested genotypes: NS, not significant (Pearson's ?2-test). (e) mRNA-expression of Irs gene family members at various stages of adult mammary gland development and embryogenesis (d, days) of wildtype BALB/c mice.

Fig 4: Proposed model of IRS4-induced signalling and therapy resistance.(a-c) Simplified PI3K/AKT signalling cascade in normal mammary cells expressing IRS1 and IRS2, or mammary cells expressing IRS4, in presence (a, b) or absence (c) of insulin/IGF1. (d,e) Model of signalling in HER2+ (ERBB2) breast cancer cells where ERBB3 (HER3) (d) or IRS4 (e) activates the PI3K/AKT-pathway. (f,g) Model of the effect of Trastuzumab or Lapatinib treatment in HER2+ breast cancer cells expressing ERBB3 (f) or IRS4 (g). Thickness of the arrows indicates strength of signalling. Red 'X' indicates no interaction/signalling. Dashed arrow indicates possible cross-talk.

Fig 5: Growth factor-independent cell proliferation in mammary cells expressing Irs4.(a) Growth curves of MCF10A-cells, stably transfected with Irs4 or empty vector, growing in the presence of the indicated serum (FBS) concentrations, supplemented with insulin (ins) or IGF1, or without supplement (-). Cell growth is represented as the local regression over quadruplicates. Results shown are representative for four independent experiments, wherein independently transduced cell cultures were used. (b,c) Quantification of maximum growth rates (b) and times until maximum confluency was reached (c) derived from growth curves in a and determined as outlined in Supplementary Fig. 3a. Data represented as mean+s.d. of quadruplicates. *P<0.05, **P<0.01, ****P<1 × 10-4 (Welch's t-test, compared with vector in each condition). (d) Growth curves of NMuMG-cells, stably transfected with Irs1, Irs2, Irs4 or empty vector. Conditions and data represented as outlined in a. Results shown are representative for two independent experiments. (e,f) Quantification of maximum growth rates (e) and times until maximum confluency was reached (f) derived from growth curves in d. Data represented as mean+s.d. of quadruplicates. *P<0.05 (Welch's t-test, compared with vector in each condition).