Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

Tumor genome analysis includes germline genome: are we ready for surprises?

We sought to describe the spectrum of potential and confirmed germline genomic events incidentally identified during routine medium-throughput somatic tumor DNA sequencing, and to provide a framework for pre- and post-test consent and counseling for patients and families. Targeted tumor-only next-generation sequencing (NGS) had been used to evaluate for possible druggable genomic events obtained from consecutive new patients with metastatic gastroesophageal, hepatobiliary or colorectal cancer seen at the University of Chicago. A panel of medical oncologists, cancer geneticists and genetic counselors retrospectively grouped these patients (N = 111) based on probability of possessing a potentially inherited mutation in a cancer susceptibility gene, both prior to and after incorporating tumor-only NGS results. High-risk patients (determined from NGS results) were contacted and counseled in person by a genetic counselor (N = 21). When possible and indicated, germline genetic testing was offered. Of 8 evaluable high-risk patients, 7 underwent germline testing. Three (37.5%) had confirmed actionable germline mutations (all in the BRCA2 gene). NGS offers promise, but poses significant challenges for oncologists who are ill prepared to handle incidental findings that have clinical implications for at risk family members. In this relatively small cohort of patients undergoing tumor genomic testing for gastrointestinal malignancies, we incidentally identified 3 BRCA2 mutations carriers. This report underscores the need for oncologists to develop a framework for pre- and post-test communication of risks to patients undergoing routine tumor-only sequencing.

Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

Multimodality Approaches to Treat Hypoxic Non-Small Cell Lung Cancer (NSCLC) Microenvironment.

We found both in vitro and in vivo that survival of NSCLC cells in a hypoxic microenvironment requires Notch-1 signaling. A hypoxic tumor environment represents a problem for NSCLC treatment because it plays a critical role in cancer resistance to chemotherapy, tumor recurrence, and metastasis. Here we targeted hypoxic tumor tissue in an orthotopic NSCLC model. We inhibited the Notch-1/IGF-1R/Akt-1 axis using 3 agents: a γ-secretase inhibitor or GSI (MRK-003), a fully humanized antibody against the human IGF-1R (MK-0646), and a pan-Akt inhibitor (MK-2206), alone or in various combinations including therapeutics currently in clinical use. All treatments but Akt inhibition significantly prolonged the median survival of mice compared with controls. GSI treatment caused specific cell death of hypoxic tumors. Tumors excised from mice displayed a significant reduction of markers of hypoxia. Moreover, GSI treatment caused reduced metastasis to the liver and brain. MK-0646 was not specific to a hypoxic tumor environment but substantially increased the median survival of treated mice compared with controls. NSCLC cells evaded MK-0646 treatment by specifically overactivating EGF-R both in vivo and in 5 cell lines in vitro. This phenomenon is achieved at the level of protein stability. MK-0646 treatment caused increased erlotinib sensitivity in NSCLC cells poorly responsive to it. Sequential treatment with MK-0646 followed by erlotinib prolonged median survival of mice significantly. When the 2 drugs were administered simultaneously, no survival benefit was observed, and this combination therapy proved less effective than MK-0646 used as single agent. Our data offer novel information that may provide insights for the planning of clinical trials in humans, likely for maintenance therapy of NSCLC patients.