Establishment of a p53 Null Murine Oral Carcinoma Cell Line and the Identification of Genetic Alterations Associated with This Carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), ranks sixth in cancer incidence worldwide. To generate OSCC cells lines from human or murine tumors, greatly facilitates investigations into OSCC. This study describes the establishing of a mouse palatal carcinoma cell line (designated MPC-1) from a spontaneous tumor present in a heterozygous p53 gene loss C57BL/6 mouse. A MPC-1-GFP cell subclone was then generated by lentivirus infection resulting in stable expression of green fluorescent protein. Assays indicated that MPC-1 was a p53 null polygonal cell that was positive for keratinocyte markers; it also expressed vimentin and showed a loss of E-cadherin expression. Despite that MPC-1 having strong proliferation and colony formation capabilities, the potential for anchorage independent growth and tumorigenesis was almost absent. Like other murine MOC-L and MTCQ cell line series we have previously established, MPC-1 also expresses a range of stemness markers, various oncogenic proteins, and a number of immune checkpoint proteins at high levels. However, the synergistic effects of the CDK4/6 inhibitor palbociclib on other therapeutic drugs were not observed with MPC-1. Whole exon sequencing revealed that there were high rates of non-synonymous mutations in MPC-1 affecting various genes, including Akap9, Arap2, Cdh11, Hjurp, Mroh2a, Muc4, Muc6, Sp110, and Sp140, which are similar to that the mutations present in a panel of chemical carcinogenesis-related murine tongue carcinoma cell lines. Analysis has highlighted the dis-regulation of Akap9, Cdh11, Muc4, Sp110, and Sp140 in human HNSCC as indicated by the TCGA and GEO OSCC databases. Sp140 expression has also been associated with patient survival. This study describes the establishment and characterization of the MPC-1 cell line and this new cell model should help to advance genetic research into oral cancer.

Keywords: cancer; mouth; mutation; p53; palate.

Figure 1

Establishment of the MPC-1 cell line. (A) Left, an exophytic tumor growing on the palate of a C57BL/7 mouse. Right, the morphology of the established MPC-1 cell line. Magnification: ×100. (B) Genotyping of MPC-1, SAS, and OECM1 cells using PCR to amplify the PTGER2 gene. The differential amplification and sizes of the PCR products generated by different inputs and primers confirms the mouse origin of the MPC-1 cell line. H & M, both human and mouse; H, human; M, mouse. (C) Left, the morphology of the MPC-1-GFP cell subclone. Right, the fluorescence image of MPC-1 cells. Magnification: ×250. (D) The growth curves of the MPC-1, MPC-1-GFP, MTCQ1-GFP, and SAS cell lines. Human SAS and OECM1 cell lines, and murine MTCQ1-GFP OSCC cell subclone are served as controls to assay the origin or the grow potential of MPC-1 and MPC-1-GFP.

Figure 2

Confirmation of MPC-1 as a p53 null OSCC cell line. (A,B,D) Western blot analysis. (A) Analysis of the keratinocyte markers. The MPC-1 cell line expresses keratinocyte markers much more strongly than the MTCQ1 cell line. (B) MPC-1, MOC-L1, MTCQ1, and MTCQ2 are positive for expression of vimentin, but negative for expression of E-cadherin. The other murine OSCC cells express E-cadherin. (C) RT-PCR analysis. This shows a ~700 bps p53 transcript in MPC-1 and a ~1200 bps p53 transcript in MTCQ1 cell. Note that the bands with a mobility below 600 bps position are not shown in this picture. (D) The analysis of p53 protein in OSCC cells. High p53 protein expression levels can be seen in the cell lines harboring p53 mutations. MTCQ1 has a truncated p53 protein. No p53 expression can be seen in the MPC-1 cells. (E) Sequencing of bacteria colonies carrying RT-PCR products. This reveals the presence of the truncated transcript originally engineered into the knockout mouse (deletion of exon 2–6) in 50% of the white colonies, while four shorter truncated variants make up the remaining 50% colonies.

Figure 3

Phenotypic analysis of MPC-1 and MTCQ1. (A) Migration. Magnification: ×100. (B) Invasion. Magnification: ×100. (C) Colony formation. Magnification: ×2. *** p < 0.001; **** p < 0.0001. (D) Anchorage-independent growth. Magnification: ×100. (A–D), Left panels, quantification. It is performed 48 h (in A,B) and 10 day (in C,D) after cell seeding from at least duplicate analysis. Right panels, the representative fields or study sets. The SAS cells in (D) act as a side-by-side control. (E,F) Subcutaneous tumorigenesis in C57BL/6 mice and nude mice, respectively. MPC-1, n = 10; MTCQ1 (for side-by-side control), n = 1.

Figure 4

Gene expression profiling and the testing of the effects of various drugs on OSCC cells. (A) The heatmap of the normalized protein expression values (log₂ transformed) for the various cell lines. Right lower, gradient bar. (B) The heatmap of correlation values (γ) according to the IC₅₀ of the drugs used and protein expression values for MPC-1, MTCQ1, and SAS cells. Right lower, gradient bar. pEGFR*, normalize pEGFR to EGFR. * p < 0.05. (C) Representative correlation analysis between the IC₅₀ of drugs used and protein expression in various cells. (D) Analysis of the synergism between palbociclib and AG1478, and between cisplatin and taxol by means of CI for MPC-1 and MTCQ1 cells.

Figure 5

Gene mutations in murine OSCC cells. (A,B) Heatmap of the mutations present in the various murine OSCC cell lines. (A) Analysis according to the order of mutation frequency using the TCGA dataset. (B) Analysis according to the presence of mutations in nearly all murine OSCC cells. (A,B) Left, the mutation frequency based on the TCGA dataset. Right, the mutation frequency in murine OSCC cell lines. A red block indicates the mutated gene. (C) Heatmap of gene expression levels in the TCGA and GEO datasets. Right, gradient bar. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (D) Whiskers and a box diagram illustrating the expression levels of genes based on the TCGA human HNSCC database. N, normal tissue, T, HNSCC. (E) Kaplan–Meyer survival analysis of the five-year overall survival based on the TCGA human HNSCC database in relation to Sp140 expression.