Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Subhra Mohapatra, Ph.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Srinivas Bharadwaj, Ph.D.

Committee Member

Validimir Uversky, Ph.D.


Cancer Biology, Cholesterol Metabolism, Drug Resistance, Mitochondrial Apoptosis


Lung cancer is the number one cause of cancer related death in both males and females. About 20% of all non-small cell lung cancer (NSCLC) patients are expected to harbor an epidermal growth factor receptor (EGFR) activating mutation. EGFR inhibitors have been shown to provide clinical benefits over chemotherapy for lung cancer patients with EGFR activating mutations. However, despite the initial clinical responses to these EGFR targeted therapies, long-term efficacy is not possible because acquired drug resistance hampers the effectiveness of these therapies. We found that a fiber inspired smart scaffold (FiSS) platform established in our laboratory allows growth of three-dimensional (3D) tumor-like aggregates (referred to as tumoroids), which resemble in vivo tumors. Tumoroids exhibit better drug resistance compared to two-dimensional (2D) cultures that lack ability to mimic the environment of the tumor microenvironment. As the FiSS acts as a better representation of the in vivo tumor environment, we have used the FiSS platform to help verify our most important monolayer findings before proceeding to in vivo studies. We have developed EGFR tyrosine kinase inhibitor (TKI) drug tolerant (DT) human lung cancer cell lines as model for de novo drug resistance. The drug sensitivity of the parental and DT cells on both the monolayer and the FiSS were determined by testing a panel of standard-of-care chemotherapeutics along with EGFR TKIs. A comparison of the drug sensitivity showed that parental cells were at least 2- fold more sensitive to the EGFR TKIs compared to the DT cells. Data mining the significantly differentially expressed proteins list generated by the mass spectroscopic analysis revealed that the protein expression is skewed in the EGFR TKI DT cell line as compared to the parental cell line. The cytochrome P450 protein CYP51A1, which is directly involved with cholesterol synthesis, was significantly up-regulated in the DT cells compared to the parental cells. Western blotting has confirmed the upregulation of CYP51A1 in DT cell lines. Differences in cholesterol synthesis between parental and DT cells were then studied using a variety of techniques including qPCR, western blotting, and cellular cholesterol assays. Total cellular cholesterol and more specifically, mitochondrial cholesterol, were found to be upregulated in DT cells, as well as parental cells treated with EGFR TKIs for as little as 48 hours. This upregulation of cholesterol synthesis was shown to be able to block the release of cytochrome C and stop the initial induction of apoptosis by EGFR TKIs. We then used the CYP51A1 inhibitor, ketoconazole, to downregulate cholesterol synthesis in the cells. The cellular effects of a combination therapy of ketoconazole and EGFR TKI was then studied in parental and DT cells. In both parental and DT cells, ketoconazole and EGFR TKIs acted synergistically to induce apoptosis and overcome the development of EGFR tolerance in these cells. Lastly, this combination therapy was shown to shrink the growth of flank tumors in an in vivo mouse model of EGFR TKI resistance. By studying the mechanisms of survival to initial EGFR TKI exposure, we were able to better understand how lung cancer cells withstand EGFR TKI treatment allowing time for the development of resistance and we used this knowledge to strategize potential ways of sensitizing the cells to EGFR TKIs.

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