Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells

Carolina D’Alesio, Grazia Bellese, Maria Cristina Gagliani, Cinzia Aiello, Elena Grasselli, Gianluca Marcocci, Angela Bisio, Sara Tavella, Tiziana Daniele, Katia Cortese, Patrizio Castagnola

ERBB2 is overexpressed in up to 20-30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2(+)) cells and whether CA might restore Tz sensitivity in Tz-resistant cells. We have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy. We report that, in ERBB2(+) BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21(WAF1) and CDKN1B/p27(KIP1). Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line. Our data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2(+) BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2(+) cancers.