Date of Award

8-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Nie, Daotai

Abstract

Mammalian homolog Tribbles (Trbs) is a newly characterized protein family that includes three different isoforms: Trb1, Trb2, and Trb3. Tribbles are serine/threonine kinases lacking catalytic activity, thus their classification as pseudokinases. Despite their catalytic inactivity, Tribbles can interact with different proteins and regulate different biological functions. The most studied tribble family member, Trb3, was reported to play a major role in Drosophila's ventral furrow formation. Further studies revealed that Trb3 is also involved in diabetes, stress-response, and development. Previously, Trb3 upregulation was detected in certain types of cancer but its function remains unknown. The goal of our study is to gain a better understanding of the biological function of Trb3 in cancer, including the molecular mechanism of action. Using the cohort analysis, we identified higher levels of Trb3 in the lung tumor compared to the normal tissue. Furthermore, higher Trb3 expression in the lung tissue was associated with a poor survival in cancer patients. Silencing of Trb3 in A549 promoted cell growth. On the other hand, overexpression of Trb3 in NCI-H358 inhibited cell growth. The analysis of cell cycle gene profiling revealed a decrease in several genes that are essential for cell cycle progression in S phase in Trb3 overexpressed NCI-H358. The cell proliferation protein, Ki67, was also decreased in Trb3 overexpressed NCI-H358 cells. Moreover, Tb3 overexpressed cells formed higher colony number in soft agar assay and depicted higher migration ability in the Boyden chamber assay. Mesenchymal markers SNAIL, TWIST and N-cadherin were upregulated while epithelial E-cadherin was significantly reduced. Interestingly, prosurvival protein Akt was also reduced post Trb3 overexpression. Trb3 expression was associated with a poor survival. However, we discovered that Trb3 overexpression inhibited cell growth. Thus, we hypothesized that Trb3 expression might contribute to tumorigenesis during cellular metabolic stress. In order to understand the potential role of Trb3 in metabolic stress, NCI-H358 cells were treated with five different cellular stressors to mimic the tumor microenvironment. All stressors used were shown to induce endogenous Trb3 expression. Moreover, stress proteins ATF4, CHOP and ASNS were induced by all stressors. One of the stressors used was rotenone, an inhibitor of the complex I of the electron transport chain. Rotenone treatment induced Trb3 expression. This expression inversely correlated with cytochrome C expression. Furthermore, Trb3 expression positively correlated with the expression of mitophagic genes PINK1, Parkin and p62, which suggest that Trb3 is induced during ROS-mediated oxidative stress to participate in the clearing of damaged mitochondria. This targeted clearing of the mitochondria, a process known as mitophagy is essential for the cell survival of the lung cancer cells. Last, Trb3 overexpression rendered cancer cells resistant to docetaxel and cisplatin, two chemotherapeutic drugs used in lung cancer treatment. On the other hand, Trb3 depleted cells were more sensitive to the drugs. Our results suggest that Trb3 is activated in the primary tumor to promote metabolic adaptation through cell cycle arrest and the inhibition of aerobic glucose metabolism through Akt inhibition. Furthermore, Trb3 is essential during cell survival post ROS-mediated stress and participates in the clearing of damaged mitochondria during mitophagy. Last, stress-mediated activation of Trb3 confers lung cancer cells with chemoresistance and suggest that Trb3 could be a potential target in lung cancer therapy.

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