Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

Non-small cell lung cancer (NSCLC) originates in the epithelia of the lung and persists as the leading cause of cancer-related death in the United States. Many studies have shown that hypoxia is prevalent in NSCLC tissue and negatively influences treatment outcome.

In previous studies, we investigated the efficacy of gamma-secretase inhibitor (GSI) treatment in an orthotopic mouse NSCLC model. Analysis of hypoxic areas of tumors from GSI treated animals showed reappearance of 4E-BP1 phosphorylation at threonine 37/46 residues (T37/46). 4E-BP1 phosphorylation is primarily mediated by mechanistic target of rapamycin complex 1 (mTORC-1), a major cell integrator of growth factors stimulation, nutrient availability, and environmental cues.

In this study, we sought to identify a putative target of gamma-secretase that could mediate this apparent reactivation of mTORC-1 activity in otherwise quiescent hypoxic NSCLC. We identified amyloid precursor protein (APP) as the substrate of gamma-secretase that mediates this anabolic reactivation upon GSI treatment. APP is a highly pleiotropic protein implicated in a plethora of cellular functions, including malignant growth. siRNA-mediated depletion of APP in NSCLC cell lines cultured in hypoxia enhanced 4E-BP1’s phosphorylation at T37/46.

Our results indicated that depletion of APP caused 4E-BP1 phosphorylation pattern rearrangement by affecting various proteins including ERKs and the pseudophosphatase STYX, a novel APP target. Additionally, APP altered the recruitment of eIF4A RNA helicase to the translation initiation complex. These changes coincided with a prominent increase in the global protein synthesis rate.

Since cell growth and division are inherently linked processes in both normal and cancerous cells, we investigated if depletion of APP had any effect on cell proliferation. Surprisingly, APP depletion resulted in a cyclin C-dependent G0 arrest of hypoxic NSCLC cells. This paradoxical uncoupling of cell growth and proliferation caused cellular abnormalities, including increased cell size and necrotic cell death. The observed changes were reversed by the overexpression of APP intracellular domain (AICD).

In conclusion, APP (via AICD) seems to mediate G0/G1 transitions and moderate cell growth rate. As inhibition of APP causes cell cycle arrest and necrosis, we propose that APP could be a novel therapeutic target for the eradication of hypoxic NSCLC.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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