P1-12-14: Genetic Ablation or Pharmacological Inhibition of Autophagy Suppresses Intrinsic Resistance of Breast Cancer to HER2−Targeted Therapies.

Abstract

Abstract Autophagy, an evolutionary conserved catabolic process whereby cells generate energy and building blocks by promoting large-scale recycling of cytoplasmic macromolecules and organelles, represents a novel drug-targetable molecular mechanism underlying de novo (primary) refractoriness of HER2 gene-amplified breast cancer (BC) to HER2 inhibition. JIMT-1 cells, which were obtained from a HER2-positive BC patient that rapidly progressed on trastuzumab (Herceptin™) ab initio and that show cross-resistance to multiple HER1/2 inhibiting drugs including lapatinib (Tykerb™), were found to constitutively exhibit an enhanced autophagic vesicle content as assessed by immunoblotting of endogenous ATG8/LC3 lipidation and confocal imaging of the recruitment of ATG8/LC3 to autophagic vesicles. A significant decrease in the expression status of the specific autophagy receptor p62/SQSTM1 -a protein selectively degraded by autophagy- confirmed further a constitutive activation of the autophagic flux in trastuzumab-refractory JIMT-1 cells. When the Human Autophagy RT2 Profiler™ PCR Array was employed to profile the expression of 84 genes involved in autophagy, ATG12 was the most differentially up-regulated gene in JIMT-1 cells (>10-fold) as compared with trastuzumab-responsive SKBR3 cells. Upon collection of the transcriptional profile of the ATG12 gene across two sets of > 50 widely used BC cell lines, HER2+ BC cells with well-established de novo resistance to trastuzumab were characterized by expressing significantly higher levels of ATG12. When lentiviral-delivered small hairpin RNA was employed to stably & specifically knock-down ATG12 gene, JIMT-1 ATG12-shRNA cells were more significantly growth-inhibited by trastuzumab (up to 5-fold) than parental JIMT-1 cells. Moreover, the half-maximal inhibitory concentration (IC50) values for the small-molecule HER1/2 Tyrosine Kinase Inhibitors gefitinib, erlotinib and lapatinib were drastically reduced by up to 10 times in response to ATG12 gene silencing. Knockdown of autophagy-regulatory genes other than ATG12 (e.g. ATG5, ATG8/LC3B) similarly suppressed refractoriness to trastuzumab as well as co-existing resistance to other HER1/2-targeted agents. Knockdown of autophagy-specific genes, however, did not alter basal sensitivity of JIMT-1 cells to multiple cytotoxics including doxorubicin, cisplatin, paclitaxel and vinorelbine. When lysosomal degradation was pharmacologically inhibited by using the antimalarial lysosomotropic drug chloroquine we found a massive accumulation of abnormal autophagolysosomes that promoted synergistic re-sensitization of JIMT-1 cells to the growth inhibitory and apoptotic activity of trastuzumab. In summary, autophagy-specific genes appear to play a potent protective role against HER2 inhibiting drugs currently in use. Given that genetic and pharmacological targeting of autophagy was found to be detrimental to intrinsic BC refractoriness to HER2−targeted therapies, our current findings may provide rationale for novel, chloroquine-based therapeutic approaches aimed to circumvent primary-resistance and potentiate the efficacy of both trastuzumab and lapatinib in patients treated for HER2−positive BC disease. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-14.