Schedule-dependent activity of combined epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) targeting in PTEN negative breast cancer xenografts

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The PI3K/Akt/mTOR pathway is a key component in growth factor receptors (GFRs) signaling. However, its constitutive activation, as in cases of PTEN mutation, can lead to GFR independence and resistance to GFRs inhibitors. In previous studies on MDA-MB-468 human breast cancer cells which overexpress the EGFR and are PTEN negative, we observed a relative in vitro resistance to the anti-EGFR inhibitor gefitinib (G). This resistance was partially reversed, in a sequence dependent way, by the mTOR inhibitor everolimus (E). To dissect the effects of this combination and its sequence-dependency, we studied the antitumor activity of E, G and their combination in MDA-MB-468 xenografts. The study was designed to evaluate the effect of combinations with respect to each agent acting alone within the schedule and retaining the timings of exposure as in the combined treatments. Drug interaction was analyzed by calculating the ratio observed/expected effect (ratio < 1 super-additivity, >1 infra-additivity). When tumors reached 134 mm3 in size, mice were allocated into 4 groups: placebo (group 1), E 2.5 mg/kg/daily (group 2), G 100 mg/kg/daily (group 3), and E 2,5 mg/kg/daily + G 100 mg/kg/daily (group 4). After 10 days, mice of group 1 were divided into further 3 arms: continuation of placebo, E 5 mg/kg/daily (group 5) and G 200 mg/kg/daily (group 6); mice that started on day 1 with E (group 2) were subdivided in 2 arms: one half continued with E alone and the rest with E plus G 200 mg/kg/daily (group 7, i.e. E>> E + G); similarly, mice that started with G (group 3) continued with G alone or plus E 5 mg/kg/daily (group 8, i.e. G>> G + E). The total dose at 3 weeks of therapy was the same for the simultaneous and sequential groups. The anti-tumor activity of E and G alone was cytostatic rather than cytotoxic. E given simultaneously with G reduced tumor growth up to 90% as the result of a super-additive interaction (ratio= 0.8). When G was followed by E, the anti-tumor activity was additive (ratio=1). Conversely, with the opposite schedule (E>> E + G), the ratio ranged from 1.8 to 2.4, suggesting an infra-additive effect of the combination. Treatment effect on the upstream and downstream targets of mTOR was analyzed by immunohistochemistry. Compared to control, E significantly increased the intensity and number of cells with p-Akt, while G alone partially reduced p-Akt either in the nucleus or in the cytoplasm. Accordingly, no significant difference was found in p-Akt among combined treatments over control. Simultaneous exposure completely inhibited p-S6 and reduced the citoplasmatic expression of p-4E-BP-1 more than each drug given alone or in sequence. Dual targeting of EGFR and mTOR is a promising anti-tumor strategy to circumvent the low sensitivity of PTEN-negative cancer. The interaction between E and G is schedule dependent, the best combination being co-administration.