Fig. 1. CDK2AP1 repression in OSCC is caused by non-mutational mechanisms and correlates with shorter disease-free survival.

A Stratification of tongue carcinoma patients based on CDK2AP1 staining intensities. The 45% threshold of negative tumor cells was established based on the analysis shown in Supplementary Fig. 1. For each patient, the staining intensity fraction (S0, S1, and S2) is expressed according to the grayscale. Patients (n = 100) are from the RONCDOC cohort (see Table 1 and “Materials and Methods”). B Kaplan–Meier analysis of the disease-free survival (DFS) probability established on the RONCDOC cohort of oral squamous cell carcinoma of the tongue. The red line refers to patients with more than 45% of tumor cells negative for CDK2AP1 (low/neg), while the blue line indicates those with less of 45% of tumor cell negative for CDK2AP1 (positive). DSF probability for CDK2AP1-low/neg patients was significantly decreased when compared with CDK2AP1-positive tumors (Log-rank P = 0.02). C Graphic representation of the results of the somatic mutation analysis relative to 438 head and neck squamous cell carcinoma tumors (HPV infections excluded) from the TCGA dataset (left panel), and to the panel of OSCC cell lines analyzed by exome sequencing (right panel). D CDK2AP1 western blot analysis of the panel of OSCC cell lines. The 12 KDa CDK2AP1 protein is observed exclusively in the HEK293T, OKF-6 (here employed as positive controls), and in the CA1, LM, and LUC4 cell lines. The tongue OSCC cell lines (SCC4, SCC9, SCC15, and SCC25) do not express the mature CDK2AP1 protein. β-actin (BACT) was employed as a loading control. The blot shown here is a representative example of 3 independent experiments. E CDK2AP1 RT-qPCR analysis of the protein-proficient (CA1, LM, and Luc4) and -deficient (SCC4, SCC9, SCC15, SCC25) oral cancer cell lines. The results are normalized based on GAPDH expression and are representative of three independent experiments.