Figure 5: ATM modulates HER2-positive cell sensitivity to therapeutic approaches.

(a) Viability assay represented in the histogram as mean±s.d. (n=6). SKBR3shR5 control cell lines and SKBR3shATM were incubated or not in the presence of Trastuzumab (Tzb, 20 μg ml⁻¹ for 72 h). Viability was assessed with the Cell Titer Glo Luminescent Assay and represented as the percentage of inhibition of viability measured in cells treated without Tzb. *P value for SKBR3shR5 versus SKBR3shR5+Tzb=0.001; P values were calculated using two-sided Student’s t-tests. (b) Viability assay represented in the histogram as mean±s.d. (n=3 for each condition). MCF7 and MCF-7HER2, interfered or not for ATM expression, were incubated (or not) in the presence of Tamoxifen (Tam 5 μM for 72 h). Viability was assessed as in a and represented as the percentage of inhibition of viability measured in cells treated without Tam (*P value=0.002). (c,d) Representative western blot showing phosphorylation of AKT on S473 (pS473-AKT) upon treatment with KU-55933 (10 μM for 48 h) or Trastuzumab (Tzb, 20 μg ml⁻¹ for 48 h) or Lapatinib (100 mM, for 48 h) (a) and upon infection with lentiviruses expressing shATM#1 or shATM#3 or shHER2 RNA interference or shGFP as control (b). (e) Proposed model depicting the interplay between HER2 and ATM kinase. ATM stabilizes HER2 protein enhancing HER2 interaction with HSP90, thus preventing HER2 ubiquitination and degradation. HER2 may promote the activation of several tumorigenic pathways (red arrow). ATM supports HER2’s ability to activate AKT signaling. Future experiments will clarify whether ATM directly modulates other HER2 signaling (black labels) dependently on or independently of its effect on HER2 (dashed arrow).