Date on Master's Thesis/Doctoral Dissertation

12-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Beverly, Levi

Committee Member

Clem, Brian

Committee Member

Klinge, Carolyn

Committee Member

Siskind, Leah

Committee Member

Watson, Corey

Author's Keywords

Lung adenocarcinoma; bacteria; methionine; LAT1; Glycolysis; EMT

Abstract

Lung cancer is one of the most common forms of cancer diagnosed, and has the highest lethality rate across all other forms of cancer in the U.S. While current therapeutic options include chemo-, immuno-, and radio-therapy, the benefits of caloric or nutrient restriction on cancer cells has also been investigated. Due to the many biological functions associated with methionine, many have proposed a methionine restricted diet would lead to favorable outcomes when combating cancer. Although our cells are incapable of synthesizing methionine, the bacteria found in our microbiome can. Furthermore, recent discoveries suggest an independent microbiome found within tumors that portray increased methionine production. In this dissertation, I investigate: 1) the impact of nutrient restriction on cancer phenotypes, 2) the role of bacteria in this nutrient restricted environment, and 3) the direct impact of bacteria on cancer growth and survival. I show that low methionine availability lessens lung adenocarcinoma (LUAD) proliferation, which I rescued upon introduction of bacterial biomolecules. Additionally, our RNA-seq data revealed several altered pathways due to bacterially supplemented media, including glycolysis and Epithelial-to-Mesenchymal Transition (EMT). These results were verified using western blot, measuring lactate production, glucose uptake, and invasive potential. I hypothesized the increased glycolysis and EMT was attributed to activation of Toll-Like Receptor 4 (TLR4), a receptor well known to recognize bacterial lipopolysaccharides (LPS). To test my hypothesis, I treated LUAD cell lines with 1-Methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate (C34), a potent TLR4 inhibitor. My results indicated C34 inhibition of TLR4 did not reduce lactate production or invasive potential. To gain a better understanding of which biomolecule was responsible for this altered phenotype, I treated bacterially supplemented media with RNAse, charcoal stripping, or dialysis. From the results, I show bacterial RNA, lipids, and biomolecules greater than 3kDa influences HKII and Claudin in similar ways. However, bacteria RNA was shown to have opposite effects on cJUN expression compared to either bacterial lipids or biomolecules greater than 3kDa. Future work will be required to characterize these biomolecules and elucidate their role in tumor development. The characterization of the biomolecules will help in two-fold: 1) opening novel therapeutic options that targets and inhibits the bacteria’s ability to synthesize these biomolecules; 2) allowing for the characterization of bacteria into tumor promoting microbes and tumor suppressive microbes by using the biomolecules produced as markers.

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