1.1 Background and objectives:
Malignant tumors of the breast are common among women, and they represent the number one threat to their health, especially triple-negative breast cancer. It is a relatively common cause of death for women. At present, immunotherapy has become an emerging method of cancer treatment. Cancer cells express increased levels of PVR, which is classified as a cell adhesion molecule. The molecule is expressed in a variety of tumor cells, including breast cancer, pancreatic cancer, gallbladder cancer, liver cancer, ovarian cancer, etc. In the development of tumors, it is involved in the adhesion, metastasis and proliferation of tumors. A PVR-mediated tumor growth mechanism does not just affect the growth of the tumor, but also influences the tumor immune environment, and takes part in immune escape to some extent. However, the expression of PVR is not limited to the area where immune cells exist and is uniformly expressed in tumor cells. Therefore, the expression and regulation of PVR should not be affected by external factors but caused by certain gene mutations in the tumor cells. In addition, PVR expression is related to the tumor's development stage. Early and late stages of tumor development are markedly different in their expression of PVR, and its role also differs. Additionally, PVR expression is closely related to that of PD-L1, a tumor immune target for which tumor immunotherapy is now more concerned, and this also affects the efficacy of tumor immunotherapy. As a result, the biomarker PVR was chosen for the purposes of this research.
1.2 Methods:
The aim of the study is to identify the targets that regulate PVR in breast cancer using CRISPR/Cas9 loss-of-function screening technology, as well as to strengthen the screening through fluorescence-activated cell sorting (FACS) to identify groups expressing high and low PVR expression levels. We transfected breast cancer cell line with the human protein kinase library. After three rounds of FACS cell sorting, the cell population with high and low expression of PVR was detected by flow cytometry and Western Blot, and the DNA with 500-fold coverage was collected for next-generation sequencing to identify the candidate gene.
1.3 Results:
In this study, we analyzed PIP4K2C as an important gene regulating PVR expression, and knocking it down resulted in increased expression of PVR in cells. Furthermore, we demonstrate that this regulation involves the AKT/mTOR signaling pathway and is found to promote cancer cell migration in RAS-mutated cells.
1.4 Conclusion:
Our results support the utility and effectiveness of using CRISPR/Cas9 combined FACS technology to screen immune targets of interest and provide new targets to regulate PVR expression. PIP4K2C also plays an important role in regulating the expression of PVR, and AKT/mTOR is its potential signaling pathway
