Investigating the Importance of Transcription Factors on Proliferative Pathways in Cancer

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Date

2019-05

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The Ohio State University

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Abstract

Transcription factors (TFs) are the primary mechanism that cells use to respond to external stimuli. These TFs allow for inducible regulation of gene expression. For this reason, TFs can act as a proto-oncogenes, tumor suppressors, or secondary messengers and aid many hallmarks of cancer. Understanding how TFs interact in cancer-causing pathways could lead to a much better understanding of the disease. In both the Guttridge and Miles laboratories, the impact of transcription factors in cancer-causing pathways, NF-κB in the former and RB in the latter, was investigated. Elucidating the function of lesser-known molecules in these pathways could profoundly impact how we understand the development of several different types of cancer. The NF-κB pathway plays an important role in controlling the immune response. It is also constitutively activated in leukemia/lymphoma, AML, breast, colon, pancreas, liver, head and neck cancers and is activated by many oncogenes (Ras, Her2/neu, BCR-ABL, HTLV-1/TAX and et al). In transformed cells, NF-κB is thought to suppress the immune system and allow for tumor development. 35 possible candidate genes were tested for NF-κB-regulation with qRT-PCR. 12 of these genes showed differential gene expression between wild-type and mutant NF-κB cell lines, showing that they could be involved in immune suppression around cancer cells. The RB pathway, controlled by RB1, RBL1, and RBL2 genes, and the pocket proteins (pRB, p107, and p130) that these genes encode are critical for regulating E2Fs that promote cell proliferation and apoptosis. pRB is important tumor suppressor that is inactivated in almost all human cancers and E2F functions are also commonly mis-regulated in cancer. Retinoblastoma (RB), a rare tumor of the retina caused by the homozygous loss of RB1, is thought to originate in rod and cone retinal pigment epithelial (RPE) cells. Retinoblastoma susceptibility gene (RB1), which encodes pRB, was first tumor suppressor gene ever identified. However, the functions RBL1 and RBL2, which encode p107 and p130 respectively, are still unknown despite encoding similarly structured proteins and having similar importance to the RB pathway as RB1. CRISPR-Cas9 gene editing was used to create two knockout (KO) RPE cell-lines for each RBL1 and RBL2. Western Blotting confirmed that both RBL2 cell lines were homozygous CRISPR-KOs while the RBL1 cell lines were a homozygous CRISPR-KO and a heterozygous CRISPR-KO.

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Transcription Factors, Cancer, NF-κB, RB, Pathways

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