Date of Award

2017

Embargo Period

8-1-2024

Document Type

Thesis

Degree Name

Master of Biomedical Science

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Bradley Shulte

Second Advisor

Victoria J. Findlay

Third Advisor

Robert M. Gemmill

Fourth Advisor

John Wrangle

Fifth Advisor

Hiu Wing

Abstract

Non-small cell lung cancers (NSCLC) with epidermal growth factor receptor (EGFR) gene mutations can exhibit a strong dependence on mutant EGFR signaling for growth and survival. They are also sensitive to EGFR tyrosine kinase inhibitors (TKIs), which provide superior clinical benefits to conventional chemotherapy. However, despite initial response, most patients experience relapse with resistant tumors within a year. This study aims to identify modifiers of dependence on mutant EGFR signaling and the mechanisms by which they do so in order to improve therapeutic strategies and outcomes. A genome-scale CRISPR-Cas9 genetic knockout screen was conducted to identify genes whose loss-of-function confer EGFR-TKI resistance. A pooled sgRNA library targeted more than 18,000 protein-coding human genes with multiple sgRNAs. The lung cancer cell line HCC827 was used as it is EGFR-mutant and sensitive to EGFR TKIs. Cells were transduced with the sgRNA library and cultured in the presence of erlotinib, an EGFR TKI, or DMSO control for 17 days. sgRNAs that were enriched in erlotinib-treated groups over control groups were identified, indicating genes whose loss- of-function confer TKI resistance. The RNAi gene enrichment ranking (RIGER) algorithm was applied to identify gene hits with enrichment of multiple sgRNAs. Top-ranked candidates include previously confirmed genes PTEN, NF1, NF2, TSC1, and TSC2; validating this system as a means to identify modifiers of EGFR dependence in HCC827 cells. A novel candidate gene is the E3 ubiquitin ligase HUWE1. I showed that suppression of HUWE1 by inducible short hairpin RNA (shRNA) in HCC827 cells re-activated AKT and ERK1/2 signaling pathways and increased cell survival in response to EGFR inhibition. These findings were confirmed in vivo by implanting mouse xenografts of HCC827 cells with suppressed HUWE1 expression and monitoring tumor development in response to erlotinib. Tumors with suppressed HUWE1 continued to grow into large tumors whereas control cells had durable tumor regression throughout the treatment period. We have shown that dependence on EGFR signaling can be decreased in EGFR-mutant lung cancer cells through mechanisms that involve the activation of AKT and ERK1/2 signaling pathways. Future studies involve identifying HUWE1 substrates/interactions that participate in tumor cell response to EGFR inhibitors, revealing a novel mechanism of resistance to EGFR-targeted therapy.

Rights

All rights reserved. Copyright is held by the author.

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