EFFECT OF P53 GAIN-OF-FUNCTION KNOCK-IN MUTATION ON CD320 EXPRESSION

Abstract

Lung Cancer (LC) is a global public health challenge and the most prevalent cancer type. Tobacco smoking is the major risk factor and leading cause of death attributed to LC with an increased death rate in low and middle-income countries. Tp53 often known as the ‘master guardian of the genome’ is the most mutated gene in most cancers including LC—associated with poor prognosis. The association between p53 mutations and various tumors suggests its significant role, including its ability to drive tumor progression in a gain-of-function manner. CD320, a transmembrane receptor, facilitates vitamin B12 endocytosis; a cofactor crucial for DNA synthesis and integrity. Although ubiquitously expressed in many cell types, CD320 is highly expressed in actively proliferating cells, including cancers. Mutant p53 can protect tumor cells during oxidative stress, reprogramming both tumor cells and their microenvironment which is often associated with poor prognosis. Relatively, CD320 is highly expressed during the cell cycle and cellular reprogramming, highlighting the significance of vitamin B12 in the one-carbon pathway. Currently, there is no report highlighting a direct relation between p53 mutations and CD320 expression. We hypothesize that the p53 GOF mutation upregulates CD320 expression, making it a potential therapeutic target. To test our hypothesis, we designed and constructed p53 single guide RNA (sgRNA) and homologous donor repair (HDR) plasmid and knock-in R175 hotspot mutation at p53 R175 into the wild-type p53 gene of LC cell line (H460) using the CRISPR cas9 technology.