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Browsing by Subject "intratumor heterogeneity"

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  • Lin, Linh (2023)
    Lung cancer, the current leading cause of death by cancer, can be categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), lung adenocarcinoma of NSCLC being the most common. Advances have been made in lung adenocarcinoma treatment based on the tumor genetic profile, especially with epidermal growth receptor (EGFR) gene mutated lung cancers. The initial responses of targeted therapy have promising outcomes, but the patients acquire drug resistance against the selective EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib. To find a way to prevent, revert, or bypass various EGFR TKI resistance mechanisms, they are studied in hopes of discovering new ways to inhibit or degrade EGFR, to target the bypass mechanisms, or tumor heterogeneity. This study aims to target intratumor heterogeneity of EGFR expression to prevent Osimertinib resistance/enhance Osimertinib efficacy in PC9 cells. It was detected that PC9 cells were a heterogeneous population, that could be separated into EGFR-low expressing and EGFR-high expressing cells. Based on clustered regularly interspaced short palindromic repeats (CRISPR) screening, EGFR-low cells had enriched expression of Ariadne RBR E3 Ubiquitin Protein Ligase 2 (ARIH2) and RING finger protein 7 (RNF7) genes compared to EGFR-high cells. These genes encode proteins that are part of a ubiquitylation complex E3-E3 ligase, possibly mediating proteasomal degradation of EGFR. To verify this, the genes are first knocked out (KO) in PC9 EGFR-low cells with CRISPR-Cas9 method. Second, parental cells are treated with pevonedistat, which is an E3-E3 ligase activator inhibitor. The effects of ARIH2/RNF7 KO cells, and pevonedistat-treated cells are analyzed with flow cytometry. Lastly, an in vitro drug experiment to see, if the combination of osimertinib and pevonedistat would have a synergistic effect in killing PC9 EGFR-low cells. RNF7 KO cells and pevonedistat-treated cells appeared to have elevated levels of EGFR, insinuating EGFR is proteasomally degraded by E3-E3 ligase. The results of in vitro drug experiment looked promising as the combination drug treatment seemed to be the most effective. The E3-E3 ligase is an appealing target for cancer therapy but it has not been researched much in lung cancer context. Also, pevonedistat is still on clinical trials and has cytotoxic effects, therefore, the proteasomal degradation pathway of EGFR requires to be more studied.