ErbB receptors modulation in ER+/HER2+ cell lines and study of pathways involved in ER/HER2/Rb cross-talk. A, Quantification of Western blot bands (see Supplementary material for details) for the ErbB receptors (left panel pEGFR Tyr845, middle panel pHER3 Tyr1289, and right panel pHER2 Tyr1248) in BT474 (red bars), MDA-MB-361 (light blue bars), and ZR-75–30 (yellow bars) after 72 hours of treatment with P, F, PF, and PFHPert. B, Quantification of the Western blot bands of phosphorylated Akt (Ser473; top panel) and 4EBP1 (Ser65; bottom panel). A and B, Dotted lines indicate the reference ratio in DMSO. Data were mean ± SEM of two or three experiments and in Supplementary Fig. S8 only one representative blot was showed. P value by Mann–Whitney test, two-tailed (*, P ≤ 0.05). All values were calculated after correction for the loading marker (β-actin or GADPH) and normalization by the control sample in DMSO. C, Summary of the effects of treatments with palbociclib (left panel), palbociclib/fulvestrant (middle panel), and PFHPert (right panel) on their molecular targets and on the different observed outcomes (blue arrow) in HER2+ and in HER2low breast cancer cells. Exposure to palbociclib and palbociclib/fulvestrant promotes a quiescence status in G0 or G0–G1 that shares the features with a senescence status that is transient and reversible after drug removal. In this condition, a survival pathway is induced through reactivation of the RTKs/Akt/mTOR axis. Addition of anti-HER2 drugs blocks this escape and promotes sustained senescence (characterized by p53 activation, MDM2 degradation, p21 upmodulation, Cyclin D1 reduction, and mTOR inhibition). This scheme applies to breast cancer cells carrying either a constitutive (HER2 amplification/overexpression) or a functionally inducible (HER2low cells) activation of the ErbB family of receptors. Abs, antibodies.