Date of Award

8-1-2017

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Nie, Daotai

Abstract

The pseudokinase Tribbles pseudokinase 3 (TRIB3) is known as a regulator in cellular responses to a variety of stresses such as glucose insufficiency and (ER) stress. Recent studies reported an increased expression of TRIB3 in cancers and its correlation with the poor prognosis in cancer patients. However, other studies suggest TRIB3 as a tumor suppressor due to its inhibitory activities toward PI3K/Akt signaling. In the present study, we aim to investigate the role of TRIB3 in lung cancer cells and determine its functions in lung cancer progression and tumor cell responses toward chemotherapy. TRIB3 expression was determined by RT-PCR and Western blot. A lentiviral vector was used to overexpress TRIB3 in lung cancer cells. A CRISPR-CAS9 construct with guiding sequence matching to TRIB3 gene was used to knock out its expression. Cell proliferation was evaluated using MTS and trypan blue assays. Boyden chamber assay was used to assess the cell migration while cell cycle phases were determined using flow cytometry. Heat-map analysis was performed to assess the changes in the expressions of genes in cell cycle progression and apoptosis. In non-small cell lung cancer (NCI-H358) and (A549), TRIB3 expression was markedly stimulated during metabolic stresses. Overexpression of TRIB3 in cells led to decreased cell proliferation, migration, and the reduction of Akt phosphorylation at threonine 308, revealing an inverse relationship between TRIB3 and AKT activation. TRIB3 overexpression increased the cell cycle arrest at G0/G1 phase. QPCR and Western blot analyses of cell cycle related genes showed an upregulation of CDK inhibitors in (NCI-H358) TRIB3 overexpression cells, while depletion of TRIB3 led to the down-regulation of CDK inhibitors. The upregulation of the CKI p27 was consistent in all cell lines used in this study with overexpressed TRIB3 level. This overexpression also led to downregulation of LC3B and other autophagy markers while increasing the expression of apoptotic genes. Our study reveals complex roles of TRIB3 in lung cancer progression. Induced by metabolic stresses, TRIB3 blocked cell cycle progression through the upregulation of p27 as adaptive responses mechanism forcing quiescent states for a short period to help the cells in overcoming the stressful conditions. Eventually, TRIB3 induced cell death via apoptosis to aid in maintaining homeostasis microenvironment. TRIB3 deletion induced the cell death by necrosis manner, an indicator of the protective role of TRIB3 in lung cancer. Furthermore, TRIB3 aids in increasing the cell sensitivity toward cisplatin-based chemotherapy. The relationship between TRIB3/P27 is the cause of chemoresistance property toward proteasome inhibitor based chemotherapy in NCSLC. Our study reveals a significant role of TRIB3 in regulating cell cycle progression and apoptosis in NSCLC. Further studies are needed to determine whether and how TRIB3 functions as a metabolic gatekeeper during lung cancer progression.

Available for download on Friday, October 25, 2019

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