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In previous studies we targeted hypoxic Non-Small Cell Lung Cancer (NSCLC) tumor tissue using gamma-secretase inhibitors (GSI) in a preclinical model of advanced NSCLC. GSI treatment of mice suggested reactivation of mTORC-1 in otherwise quiescent hypoxic NSCLC tissue. Methods included immunohistochemistry and cytochemistry, immunoblotting, immunoprecipitation, siRNA-mediated gene knockdown, nucleotide and amino acid analogs incorporation, FACS analysis, various imaging techniques, Q-PCR, gene transfection, cap binding assays, RNA and antibodies arrays. GSI treatment of mice showed a re-induced phosphorylation of 4E-BP1 at T37/46 in hypoxic tumor masses. In vitro studies indicated a role for APP in this phenomenon. APP downregulation caused a rearrangement in 4E-BP1's phosphorylation pattern at residues T37/46, S65 and T70, reversed by overexpression of the APP C-terminal domain. Increased recruitment of eIF4A on the mRNA cap was observed. These events coincided with a drastic increase in the rate of global protein synthesis, an event that appeared to be mTOR-independent. Meanwhile, NSCLC cells with depleted APP were arrested in G0/G1 through a cyclin C-mediated mechanism. Cells arrested in G0/G1 with augmented rate of global protein synthesis (both cap- and IRES-dependent) increased their size and underwent a substantial necrotic cell death due to cell membrane permeabilization. These results indicate a novel role for APP in synchronizing the rate of protein synthesis and cell cycle progression, raising questions on how these processes interact in malignant transformation. The data presented here suggest that cells of different origin, including NSCLC cells, have developed some mechanisms to couple cell growth with cell cycle progression independent of overlapping signals which derive, among others, from the PI3-K pathway. APP seems to play a central role in this synchronization, and its loss of function causes cell size abnormalities and death. Citation Format: Anna Sobol, Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan Weber, Maurizio Bocchetta. Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C40.

In a previous study we found both in vitro and in an orthotropic NSCLC model in SCID mice that survival of NSCLC cells in hypoxic microenvironment is dependent upon Notch-1 signaling. Active Notch-1 was expressed in hypoxic tumor microenvironment only. Notch-1 provides survival stimuli through exacerbated Akt-1 activation due to suppression of PTEN transcription and positive regulation of the transcription of IGF-1 and its receptor. Hypoxic tumor environment is a problem for NSCLC treatment because it is responsible for cancer resistance to chemotherapy, tumor recurrence and metastasization. Also, hypoxic tumor microenvironment is quiescent and represses mTORC-1 activity. We show a number of experiments aimed at targeting hypoxic tumor tissue in the orthotropic NSCLC model. We attempted to target the Notch-1/IGF-1R/Akt-1 axis using three agents (a γ-secretase inhibitor, MRK-003; a fully humanized antibody against the human IGF-1R that promotes the receptor degradation, MK-0646; a pan-Akt inhibitor, MK-2206, all drugs provided by Merck Inc.) alone or in various combinations. We also used in combination studies drugs that are used for the treatment of advanced NSCLC, namely cisplatin and Erlotinib. The orthotropic model was established using two cell lines non-dependent on Notch-3 signaling. siRNA to Notch-3 in A549 and H1437 affects neither the growth rate nor the survival of these cells. All treatments (besides MK-2206) significantly prolonged the median survival of mice compared to controls (16 mice/experimental arm). MRK-003 treatment yielded specific death of hypoxic tumor areas. The tumors excised from the mice reaching an end-point displayed a significant reduction of markers of hypoxia as assessed by RT-qPCR using human specific primers. Moreover, qPCR measurements of the ratio between human/mouse GAPDH in DNA extracted from the whole brains and livers showed a significant reduction in metastasization in MRK-003 treated mice. MK-0646 was not specific to hypoxic tumor environment, but substantially increased the median survival of treated mice compared to controls. NSCLC cells evaded MK-0646 treatment by activating EGF-R and ErbB3 exclusively both in vivo and in five cell lines in vitro. This phenomenon is achieved at the level of protein stability. One outcome of MK-0646 treatment is that cells poorly or not responsive to Erlotinib become more responsive to it. Sequential treatment with MK-0646 (three weeks), then combination of MK-0646 plus Erlotinib prolonged median survival of mice dramatically. If the two drugs where administered in combination from day one no survival advantage was observed in median survival compared to mice treated with Erlotinib alone and was actually less effective than MK-0646 used as single agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5227. doi:1538-7445.AM2012-5227