Neoplasia (New York, N.Y.), 2014, Vol.16 (1), p.43-W19
Abstract Phosphoinositide 3-kinase (PI3K) pathway, in addition to its pro-proliferative and antiapoptotic effects on tumor cells, contributes to DNA damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mammalian target of rapamycin (mTOR) inhibitor, would induce an efficient antitumor effect in BRCA-competent triple negative breast cancer (TNBC) model when combined with ABT888 and carboplatin. Mechanism-based in vitro studies demonstrated that GDC-0980 treatment alone or in combination led to DNA damage (increased pγH2AXS139 ; Western blot, immunofluorescence), gain in poly ADP-ribose (PAR), and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin with a time-dependent 1) decrease in proliferation signals (pAKTT308/S473 , pP70S6KT421/S424 , pS6RPS235/236 ), PAR/poly(ADP-ribose) polymerase (PARP) ratios, PAR/pγH2AX ratios, live/dead cell ratios, cell cycle progression, and three-dimensional clonogenic growths and 2) increase in apoptosis markers (cleaved caspases 3 and 9, a pro-apoptotic BH3-only of Bcl-2 family (BIM), cleaved PARP, annexin V). The combination was effective in vitro in BRCA-wild-type PIK3CA -H1047R-mutated BT20 and PTEN-null HCC70 cells. The combination blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, CD31, phosphorylated vascular endothelial growth factor receptor, pS6RPS235/236 , and p4EBP1T37/46 as well as an increase in cleaved caspase 3 immunohistochemistry (IHC) levels. Interestingly, a combination with GDC-0941, a pan-PI3K inhibitor, failed to block the tumor growth in MDA-MB231. Results demonstrate that the dual inhibition of PI3K and mTOR regulates DDR. In a BRCA-competent model, GDC-0980 enhanced the antitumor activity of ABT888 plus carboplatin by inhibiting both tumor cell proliferation and tumor-induced angiogenesis along with an increase in the tumor cell apoptosis. This is the first mechanism-based study to demonstrate the integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor action of PARP inhibitor in TNBC. Movie W1 Three-dimensional projection movie showing nuclear pyH2AXS139 foci in vehicle-treated MDA-MB468 cells at 24 hours. Movie W2 Three-dimensional projection movie showing the effect of GDC-0980 alone on nuclear pyH2AXS139 foci in MDA-MB468 cells at 24 hours Movie W3 Three-dimensional projection movie showing the absence of cytoplasmic cleaved caspase 3 in vehicle-treated MDA-MB468 cells at 72 hours. Movie W4 Three-dimensional projection movie showing abundance of cytoplasmic cleaved caspase 3 in GDC-0980 + ABT888 + carboplatin-treated MDA-MB468 cells at 72 hours.
Adaptor Proteins, Signal Transducing - metabolism ; Animals ; Antineoplastic Agents - pharmacology ; Apoptosis - drug effects ; Benzimidazoles - pharmacology ; Carboplatin - pharmacology ; Caspase 3 - metabolism ; Cell Cycle - drug effects ; Cell Line, Tumor ; Cell Proliferation ; Drug Screening Assays, Antitumor ; Female ; Gene Expression Regulation, Neoplastic ; Genes, BRCA1 ; Genes, BRCA2 ; Humans ; Ki-67 Antigen - metabolism ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Oncology ; Phosphatidylinositol 3-Kinases - metabolism ; Phosphoproteins - metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 - metabolism ; Poly(ADP-ribose) Polymerase Inhibitors ; Proto-Oncogene Proteins c-akt - metabolism ; Ribosomal Protein S6 Kinases, 70-kDa - metabolism ; TOR Serine-Threonine Kinases - metabolism ; Triple Negative Breast Neoplasms - metabolism ; Vascular Endothelial Growth Factor A - metabolism
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