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. 2009 Dec 29;106(52):22299-304.
doi: 10.1073/pnas.0905152106. Epub 2009 Dec 10.

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

Saskia M Brachmann  1 , Irmgard Hofmann, Christian Schnell, Christine Fritsch, Susan Wee, Heidi Lane, Shaowen Wang, Carlos Garcia-Echeverria, Sauveur-Michel Maira
Affiliations

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

Saskia M Brachmann et al. Proc Natl Acad Sci U S A. .

Abstract

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials VSports手机版. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235. .

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Conflict of interest statement

The authors declare a conflict of interest. S. M. B. , C. S. , C. F V体育安卓版. , S. Wang, C. G. -E. and S. -M. M. are employees of Novartis Pharma. S. Wee is an employee of Bristol-Myers Squibb. H. L. is an employee of Basilea Pharmaceuticals.

Figures

Fig. 1.
Fig. 1.
NVP-BEZ235 induces cell death through induction of apoptosis in a subset of breast cancer cell lines. (A) The mentioned cell lines were incubated with increasing concentrations of NVP-BEZ235 for a period of 72 h. Then, the cells were fixed and the effect on viability was recorded and plotted as a percentage of the number of cells present at the treatment start (100% straight line). All points beyond this line are indicative of cell death. (B) MDA-MB361, MCF-7 and MDA-MB231 cells were incubated either for 48 h with the indicated amount (in nM) of NVP-BEZ235 or for 3 h with 1 μM of staurosporine (st). The corresponding cell extracts were then analyzed by Western blot for the indicated markers. Legend: *, uncleaved form; arrow, cleaved form; (1), PIK3CA mut.; (2), HER2 amp./PIK3CA-H1047R; (3), HER2 amp./PIK3CA-E545K; (4), HER2 amp; (5), PTEN alterations; (6), KRAS mut. This legend applies to all of the Figures.
Fig. 2.
Fig. 2.
Antiproliferative and cell death induction activities of PI3K, Akt, and mTORC1 inhibitors in breast cancer cell lines. (A) MDA-MB231 (Left), MCF-7 (Center), and MDA-MB361 (Right) cells were incubated with increasing concentrations of either RAD001, PI-103, ZSTK474, NVP-BEZ235, or C124017 for a period of 72 h. Cells were then fixed and effect on viability plotted as described. The dashed line represents the concentration for which the compound is able to kill 50% of the cells (LD50 values). (B) Raptor protein knock-down was induced in the HCC1954(3) cell line by the addition of doxycycline (200 ng/mL) to the growth media. Seventy-two hours later, the cells were analyzed for the effects on proliferation (Right) and signaling events (Left), either in absence or presence of the indicated amounts of the PI3K inhibitor ZSTK474.
Fig. 3.
Fig. 3.
Apoptosis induction by the PI3K inhibitors involves the executioner caspase. Caspase-2. (A–C). MDA-MB361, MCF-7, and MDA-MB231 cells were incubated either for 48 h with the indicated amount (in nM) of NVP-BEZ235 (A), ZSTK474 (B), or RAD001 (C) for 48 or 3 h with 1 μM of staurosporine (st). The corresponding cell extracts were then analyzed by Western blot for the indicated markers.
Fig. 4.
Fig. 4.
NVP-BEZ235 activities in HER2 amplified and PTEN deficient breast cancer lines. (A) Extracts from the indicated cell lines were pretreated for 1 h (HCC1419 only) or not (all others) with either NVP-BEZ235 or lapatinib (1 μM) and analyzed either directly (lysates) or indirectly after immune-precipitation with anti-p85 immobilized beads (IP: p85) for their HER2, pHER2, and pHER3 levels. (B) Same as in A, but all of the cell lines were treated for 1 h with either NVP-BEZ235 or lapatinib (1 μM). (C) The indicated cell lines were pretreated with 1 μM of NVP-BEZ235 for 1 h, and the corresponding cell extracts analyzed for the indicated proteins. (D) PTEN knock-down was induced (+) or not (−) in the HCC1954 cell line stably expressing inducible shRNA hairpins against PTEN by addition of doxycycline (200 ng/mL) to the culture media. Seventy-two hours later, NVP-BEZ235 (1 μM) was added to the media for either 1 or 24 h. Then, the corresponding cell extracts were analyzed for PTEN, Akt and activated Akt (1-h time points), and cleaved PARP levels (72-h time points).
Fig. 5.
Fig. 5.
NVP-BEZ235 has antitumor activity against MDA-MB361, tumors, and induces apoptosis in vivo. Female Harlan nude mice bearing orthotopic MDA-MB361 tumors were treated p.o., once per day, either with 45 mg/kg of NVP-BEZ235 or with the vehicle control. Tumor volumes were recorded during the treatment period and T/C values were calculated. (B) One hour after the last dose, MDA-MB361 tumors from the efficacy study (A) were excised and tumor extracts analyzed by Western blotting for the level of expression of the indicated proteins.
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