Although DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.

Purpose: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models. Experimental Design: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses. Results: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/ Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-wayANOVA, synergy by isobologram analysis).Increased activity of the combination correlated with more profound effects on G 1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations. Conclusion: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormonedependent breast cancers.

The orally bioavailable rapamycin derivative RAD001 (everolimus) targets the mammalian target of rapamycin pathway and possesses potent immunosuppressive and anticancer activities. Here, the antitumor activity of RAD001 was evaluated in the CA20948 syngeneic rat pancreatic tumor model. RAD001 demonstrated dose-dependent antitumor activity with daily and weekly administration schedules; statistically significant antitumor effects were observed with 2.5 and 0.5 mg/kg RAD001 administered daily [treated tumor versus control tumor size (T/C), 23% and 23-30%, respectively], with 3-5 mg/kg RAD001 administered once weekly (T/C, 14 -36%), or with 5 mg/kg RAD001 administered twice weekly (T/C, 36%). These schedules were well tolerated and exhibited antitumor potency similar to that of the cytotoxic agent 5-fluorouracil (T/C, 23%). Moreover, the efficacy of intermittent treatment schedules suggests a therapeutic window allowing differentiation of antitumor activity from the immunosuppressive properties of this agent. Detailed biochemical profiling of mammalian target of rapamycin signaling in tumors, skin, and peripheral blood mononuclear cells (PBMCs), after a single administration of 5 mg/kg RAD001, indicated that RAD001 treatment blocked phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 and inactivated the translational activator ribosomal protein S6 kinase 1 (S6K1). The efficacy of intermittent treatment schedules was associated with prolonged inactivation of S6K1 in tumors and surrogate tissues (>72 h). Furthermore, detailed analysis of the dose dependency of weekly treatment schedules demonstrated a correlation between antitumor efficacy and prolonged effects (>7 days) on PBMCderived S6K1 activity. Analysis of human PBMCs revealed that S6K1 also underwent a concentration-dependent inactivation after RAD001 treatment ex vivo (>95% inactivation with 20 nM RAD001). In contrast, human PBMC-derived eukaryotic initiation factor 4E-binding protein 1 was present predominantly in the hypophosphorylated form and was unaffected by RAD001 treatment. Taken together, these results demonstrate a correlation between the antitumor efficacy of intermittent RAD001 treatment schedules and prolonged S6K1 inactivation in PBMCs and suggest that long-term monitoring of PBMC-derived S6K1 activity levels could be used for assessing RAD001 treatment schedules in cancer patients.

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood–brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and β-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.