mTORC1 and mTORC2 regulate cell proliferation in Glioblastoma multiforme

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Human malignant glioblastoma multiforme (GBM) develops as the resultof stepwise accumulations of multiple genetic alterations. The PI3K/PTEN-AKT/mTOR signaling pathway plays a critical role in oncogenesis due to the loss of tumor suppressor PTEN. The downstream signaling kinase, mammalian target of Rapamycin (mTOR), controls cell survival, growth, and motility through two distinct multiprotein complexes, mTORC1 (a mammalian counterpart of TORC1) and mTORC2 (a mammalian counterpart of TORC2). Rapamycin (RAPA), an mTOR inhibitor, and its analogs are considered potential candidates for the treatment of GBM. We hypothesize that the contribution of the two complexes are interdependent, resulting in uncontrolled GBM growth and dissemination while providing a molecular basis for therapeutic intervention. The interplay of mTORC1 and mTORC2 was studied in cell lines LN18, U87, and U373. Immunohistochemical analysis demonstrated activated AKT (pAKT) expression in 55% of GBMs. The cells were treated with one of the following, RAPA, LY 294002 (LY), PDGF-BB, or fibronectin (FN), alone or in combination. Levels of phosphorylated AKT, p70S6K, S6K, STAT3, and RhoA, downstream components in the PI3K/PTEN pathway, were assessed by immunoblotting. Treatment with RAPA noticeably reduced activation of AKT at Ser473 by 61% and 72% at 1 and 3 h, respectively, and demonstrated enhanced activation from the baseline thereafter. Pre-treatment with RAPA or LY diminished PDGF-BB or FN stimulation of p70S6K at Thr389. RAPA treatment resulted in sustained suppression of phospho-S6K at Ser 235/236 through a 24h period. A combined treatment with RAPA and LY completely blocked the activation of S6K. Cell survival and growth by PDGF or FN was achieved via the modulation of STAT3; either RAPA or LY alone inhibit this induction, while combined treatment totally abolished PDGF-BB or FN induced phosphorylation of STAT3. Timed treatment with RAPA altered activation of RhoA, a downstream component of the mTORC 2 complex. Cellular motility was determined using radial monolayer cell migration and proliferation was assessed by MTT assay. Cell growth was suppressed 76% and 32% by treatment with LY and RAPA, respectively. Also, pre-treatment with LY plus RAPA reduced PDGF-BB induced cell proliferation. GBM cell migration was reduced by LY treatment but not by RAPA. The results of this study indicate that mTORC1 RAPA-dependent S6K phosphorylation regulates cell proliferation. Activation of mTORC2 is decreased initially but activated by prolonged RAPA treatment, which inhibits downstream S6K that acts as a negative feedback modulator of IRS-1, in turn promoting upstream pAKT signaling. Thus, combining PI3K/AKT and mTORC2 inhibitors in conjunction with RAPA treatment may provide novel targets for GBM therapy.