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Identification of genes that drive early-stage lung tumorigenesis and maintenance in the context of mutated KRAS and TP53
Lung cancer is by far the leading cause of cancer-associated deaths. Its high death rate is largely attributed to the difficulty in disease diagnosis at an early stage and lack of effective strategies for therapeutic intervention. Thus, understanding of cancer drivers that are defined as genes capable of causing lung cancer development is crucial for identification of accurate predictive methods and effective therapeutic strategies. Mutated KRAS and TP53 are well recognized drivers of multiple human cancers including lung cancer. Yet, directly targeting mutated KRAS or restoring wildtype p53 levels in KRAS/p53-mutated tumors still remains a therapeutic challenge. A rational alternative approach involves targeting additional genes that are critical for mediating KRAS;p53-driven lung tumorigenesis. This includes identification of genes that either (1) potentiate KRAS;p53-driven lung cancer initiation and progression, or (2) are necessary for maintaining the cancerous state of KRAS;p53-mutated lung tumors. Apart from protein-coding genes, a class of small non-coding RNA species called microRNAs (miRNAs) are also encoded in the human genome and known to regulate cancers. Importantly, global miRNA loss is prominent in various cancers including lung cancer. Loss of genes involved in miRNA biogenesis such as DROSHA and DICER have also been shown to promote lung tumorigenesis, suggesting the functional importance of miRNAs in lung cancer. Therefore, we hypothesize that certain protein-coding and miRNA genes encoded by the human genome are critical for mediating KRAS;p53-driven lung tumorigenesis. To identify these critical mediators of lung cancer, three distinct genome-wide CRISPR screens were proposed, two of which were conducted, resulting in discovery of several genes that potentially play critical roles in various cancer-associated processes. My major finding was that loss of ARPC3 or the actin-nucleating activity of the actin related protein 2/3 (Arp2/3) complex promoted anchorage-independent (AI) growth, a hallmark of cancer, in a non-cancerous KRAS;p53-mutated human bronchial epithelial cells (HBEC-KP). Importantly, the combination of mutated KRAS and loss of ARPC3 was sufficient for driving AI growth. Co-loss of p53 and ARPC3 was insufficient for the AI phenotype. Yet, both mutated KRAS and loss of p53 were positively contributing to AI growth in the HBEC-KP cells that lack ARPC3. In-depth mechanisms that led to ARPC3-driven AI and critical environmental factors that might influence this biological process were evaluated. Additionally, loss of several genes that mediate enhanced cell proliferation in HBEC-KP cells were discovered and individually evaluated for their functional impacts. In addition to critical genetic suppressors of cell growth and AI, genes essential for maintaining the cancerous state of a lung cancer cell line Calu6 were also identified through an in vivo screen. miR-92a and KDM2A, both known for their functions in promoting cancers, were validated for the roles that they played in promoting Calu6 cell growth and tumorigenesis. Additional top ranked miRNAs and relevant genes that are clinically relevant yet lacked functional understanding are also discussed in this work.