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. 2016 Mar 15;6(4):714–746.

Figure 2.

Figure 2

Effect of BEZ235 and comparison on the effects of BEZ235 and RAD001 in HER2+ breast cancer cells: Western blot showing BEZ235 time/dose-dependently blocked activation of AKT and its downstream effectors in total lysates from HER2+ [trastuzumab-sensitive BT474 (A) & SKBR3 (B)], trastuzumab-resistant [BT474HerR (C)] breast cancer cells. Moreover, time course experiments revealed that long-term exposure to low concentration of BEZ235 resulted in an increase in p-AKT levels. Such an effect on p-AKT was completely abolished in cells incubated at higher concentrations (100 and 200 nM) of the inhibitor (see lane 8, 9 and 11, 12 in all three figures (A-C), BEZ235. Differential effects of BEZ235 versus RAD001 on intracellular signaling: BT474 (D), BT474HerRR (E), HER2+/PIK3CA mutated HCC1954 (F) and UACC893 (G) cells were treated with BEZ235 (50 nM) or RAD001 (1 and 2 nM) for different time periods (1 hr, 6 hrs, 24 hrs, and 48 hrs) and then lysed and analyzed by Western blots. Data show that both drugs completely blocked p-P70S6K, p-S6RP but only BEZ235 consistently blocked phosphorylation of AKT (Ser 474 and Thr 308) and 4EBP1. As expected RAD001-induced AKT activation was observed. Data also showed that a combination of BEZ235 plus RAD001 at sub-optimal concentration might have an additive effect on the PI3K-AKT-mTOR pathway molecules (lane 5, 9, 13 & 17 in D, E). The effect of BEZ235 was independent of the mutation profile of the cell cultures used. (H) PI3K pathway inhibition and ERK phosphorylation in HER2+ or HER2+/PIK3CA mutated breast cancer cells: Immunoblots of BT474, BT474HR, HCC1954 and UACC893 for indicated time points with different PI3K pathway inhibitors (dual PI3K/mTOR inhibitor, BEZ235 or mTOR1 inhibitor, RAD001) at the indicated concentration (nM). Our data show that mTOR1 inhibition induces ERK phosphorylation in all 4 cell lines. Importantly, BEZ235 at 50 nM concentration blocked phosphorylation ERK except UACC 893 cell lines where phosphorylation of ERK is upregulated following the treatment of BEZ235 (for the indicated time points). It has been known that level of HER3 is high in UACC cell line and it is resistant against trastuzumab and lapatinib (Slamon DJ 2010 Mol Can Ther 9: 1489).