CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvß6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. METHODS: ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. RESULTS: In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. CONCLUSIONS: ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC.

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells.

CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.

A comparison of cell survival and heat shock protein expression after radiation in normal dermal fibroblasts, microvascular endothelial cells, and different head and neck squamous carcinoma cell lines.

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) shows increased radioresistance due to the manipulation of homeostatic mechanisms like the heat shock response. This study intended to comparatively analyze effects of ionizing radiation on different HNSCC cell lines (PCI) and normal human dermal fibroblasts (NHFs) and human dermal microvascular endothelial cells (HDMECs) to uncover differences in radiation coping strategies. MATERIALS AND METHODS: Proliferation (BrdU assay), apoptosis (caspase 3/7) and intracellular protein expression of heat shock protein (HSP)-70, and phosphorylated and total HSP27, determined by enzyme-linked immunosorbent assay (ELISA), were analyzed after exposure to increasing doses of ionizing radiation (2, 6, and 12 Gray, Gy). RESULTS: Cell count decreased dose-dependently, but PCI cell lines consistently showed higher numbers compared to NHF and HDMEC. Likewise, high doses reduced cell proliferation, but low-dose radiation (2 Gy) instead increased proliferation in PCI 9 and 52. Apoptosis was not detectable in PCI cell lines. Basic HSP70 expression was high in PCI cells with little additional increase by irradiation. PCI cells yielded high basic total HSP27 concentrations but irradiation dose-dependently increased HSP27 in HDMEC, NHF, and PCI cells. Phosphorylated HSP27 concentrations were highest in NHF. CONCLUSION: PCI cell lines showed higher resistance to dose-dependent reduction in cell number, proliferation, and protection from apoptosis compared to NHF and HDMEC. In parallel, we observed a high basic and radiation-induced expression of intracellular HSP70 leading to the assumption that the radioresistance of PCI cells is conferred by HSP70. CLINICAL RELEVANCE: HNSCC use HSP to escape radiation-induced apoptosis and certain subtypes might increase proliferation after low-dose irradiation.