Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Toxicology and Cancer Biology

First Advisor

Dr. Qiou Wei

Second Advisor

Dr. Daret St. Clair

Abstract

Sulfiredoxin (Srx) is an antioxidant enzyme that can be induced by oxidative stress. It promotes oncogenic phenotypes of cell proliferation, colony formation, migration, and metastasis in lung, skin and colon cancers. Srx reduces the overoxidation of 2-cysteine peroxiredoxins in cells, in addition to its role of removing glutathione modification from several proteins. In this study, I explored additional physiological functions of Srx in lung cancer through studying its interacting proteins. Protein disulfide isomerase (PDI) family members, thioredoxin domain containing protein 5 (TXNDC5) and protein disulfide isomerase family A member 6 (PDIA6), were detected to interact with Srx. Therefore, I proposed that TXNDC5 and PDIA6 are important for the oncogenic phenotypes of Srx in lung cancer.

In chapter one, I presented background information about the role of Srx as an antioxidant enzyme in cancer. I also explained the functional significance of PDIs as oxidoreductase and chaperones in cells. In chapter two, I verified the Srx-TXNDC5/PDIA6 interaction in HEK293T and A549 cells by co-immunoprecipitation and other assays. In TXNDC5 and PDIA6, the N-terminal thioredoxin-like domain (D1) is determined to be the main platform for interaction with Srx. The Srx-TXNDC5 interaction was enhanced by H2O2 treatment in A549 cells. Srx was determined to localize in the endoplasmic reticulum (ER) of A549 cells along with TXNDC5 and PDIA6. This localization was confirmed by both subcellular fractionation and immunofluorescence imaging experiments. In chapter three I focused on studying the physiological function of Srx interacting proteins in the ER. A549 subcellular fractionation results showed that TXNDC5 facilitates Srx retention in the ER. Moreover, TXNDC5 and Srx were found to participate in chaperone activities in lung cancer. Both proteins contributed in the refolding of heat-shock induced protein aggregates. In addition, TXNDC5 and PDIA6 were found to enhance the protein refolding in response to H2O2 treatment. Conversely, Srx appeared to have an inhibitory effect on protein folding under same treatment conditions. Downregulation of Srx, TXNDC5, or PDIA6 significantly reduced cell viability in response to tunicamycin treatment. TXNDC5 knockdown decreased the time required for the splicing of X-box binding protein-1 (XBP-1). In either knockdown Srx or TXNDC5 cells, there was an observable decrease in the expression of GRP78 and the splicing of spliced XBP-1. These results suggest a possible role of Srx in unfolded protein response signaling. TXNDC5 and PDIA6, similar to Srx, contribute to the proliferation, anchorage independent colony formation and migration of lung cancer cells.

In this dissertation I concluded that Srx TXNDC5, and PDIA6 proteins participate in oxidative protein folding in lung cancer. Srx and TXNDC5 can modulate unfolded protein response (UPR) sensor activation and growth inhibition. Furthermore, TXNDC5 and PDIA6 can promote tumorigenesis of lung cancer cells. Therefore, the molecular interaction of Srx with TXNDC5/PDIA6 has the potential to be used as novel therapeutic targets for lung cancer treatment.

Digital Object Identifier (DOI)

http://dx.doi.org/10.13023/ETD.2016.176

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