Abstract 3979: CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts

Abstract

Abstract Non-melanoma skin cancers (NMSCs) constitute the largest number of cancers diagnosed in the United States, with an estimated 1 in 5 people affected by the age of 70. The most notable NMSCs are squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), which are considered keratinocyte cancers due to their origin. Although the majority of NMSCs can be removed with surgery or curettage, these options are not always available if the tumor occurs in an inoperable location or becomes metastatic. While non-surgical treatment modalities are also currently available, they have not been fully effective in NMSC management. Therefore, novel mechanistic-based therapeutic strategies are needed. The polo-like kinases (PLKs) are a family of serine/threonine kinases that have been found to play multiple roles in regulating the cell cycle. PLK4 is a structurally unique member of this family, which has been shown to be essential for precise centriole duplication. However, PLK4 has been shown to be dysregulated in certain cancers. Though the role of PLK4 in NMSC is not fully understood, we previously showed that PLK4 was significantly overexpressed in NMSC cell lines at both mRNA and protein levels as compared to normal keratinocytes, and its small molecule inhibition resulted in anti-proliferative responses in NMSC cell lines (Cancer Res 2018; 78 [13 Suppl]: Abstract nr 547). Here, we further validated our previous observation by determining the effects of CRISPR/Cas9-mediated knockout (KO) of PLK4 in the A431 human SCC line, both in vitro and in vivo. Our data demonstrated significantly reduced cell growth of multiple A431 PLK4 KO clones compared to wild-type (WT) cells, as measured by RealTime-Glo MT cell viability and trypan blue exclusion assays. In addition, we found significant reduction in clonogenic survival of A431 PLK4 KO cells, as measured by colony formation assays. Using a Human Cancer Pathway Finder RT2 Profiler PCR array, we identified 18 of the 84 genes tested to be greater than 1.6-fold differentially regulated after PLK4 KO in these cells. Employing Ingenuity Pathway Analysis (IPA) software, we identified that the regulation of the epithelial-mesenchymal transition by growth factors was one of the top canonical pathways modulated by PLK4 KO. To determine the in vivo relevance of our in vitro data, we compared the tumorigenicity of the A431 WT and PLK4 KO cells in nu/nu mice. The mice (n=7 per group) were injected subcutaneously with 5 x 105 A431 WT or PLK4 KO cells and tumors were allowed to grow for further analyses. We found a significant reduction in tumor volume in A431 PLK4 KO xenografts as compared to A431 PLK4 WT xenografts. Taken together, these findings support our previous results that suggest PLK4 has pro-proliferative roles in NMSC and should be further studied as a potential novel target for skin cancer management. Citation Format: Mary A. Ndiaye, Debra R. Garvey, Gagan Chhabra, Chandra K. Singh, Nihal Ahmad. CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3979.