The expression and methylation of PITX genes is associated with the prognosis of head and neck squamous cell carcinoma

Abstract

Background: The PITX gene family, comprising PITX1, PITX2, and PITX3, is critical in organogenesis and has been evolutionary conserved in animals. PITX genes are associated with the advanced progression and poor prognosis of multiple cancers. However, the relationship between the PITX genes and head and neck squamous cell carcinoma (HNSC) has not been reported. Methods: We used data from The Cancer Genome Atlas (TCGA) to analyze the association between PITX mRNA expression and clinicopathological parameters of patients with HNSC. The prognostic value of PITX genes was evaluated using the Kaplan-Meier plotter. Multivariate Cox analysis was used to screen out prognosis-associated genes to identify better prognostic indicators. The potential roles of PITX1 and PITX2 in HNSC prognosis were investigated using the protein-protein interaction (PPI) network, Gene Ontology (GO) analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The correlation between PITX1 and PITX2 expression or methylation and immune cell infiltration was evaluated using the tumor-immune system interaction database (TISIDB). MethSurv was used to identify DNA methylation and its effect on HNSC prognosis. Results: PITX genes expression was correlated with different cancers. PITX1 and PITX2 expression was lower in the patients with HNSC. In HNSC, PITX1 expression was significantly related to the clinical stage, histologic grade, and N stage, while PITX2 expression was only significantly related to the histologic grade. The high expression of PITX3 was significantly related to the histologic grade, T stage, and N stage. Survival analysis revealed that PITX genes had prognostic value in HNSC, which was supported by multivariate Cox analysis. PPI network and enrichment analysis showed that the genes interacting with PITX1 and PITX2 belonged predominantly to signaling pathways associated with DNA binding and transcription. Of the CpG DNA methylation sites in PITX1 and PITX2, 28 and 22 were related to the prognosis of HNSC, respectively. Additionally, PITX1 and PITX2 expression and methylation was associated with tumor-infiltrating lymphocytes (TILs). Conclusion: The PITX genes were differentially expressed in patients with HNSC, highlighting their essential role in DNA methylation and tumor-infiltrating immune cell regulation, as well as overall prognostic value in HNSC.

Keywords: Bioinformatic analysis; DNA Methylation; HNSC; PITX gene family; prognosis.

FIGURE 1

Expression of PITX genes in HNSC and normal tissue. (A) PITX1, (B) PITX2, and (C) PITX3 expression levels in different tumor types from TCGA. (D) PITX1, (E) PITX2, and (F) PITX3 expression difference in unpaired HNSC samples. p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

FIGURE 2

Relationship between PITX genes and clinicopathological parameters in patients with HNSC. The expression of PITX1, PITX2, and PITX3 markedly correlated with (A–C) clinical stage, (D–F) histologic grade, (G–I) T stage, (J–L) N stage, and (M–O) M stage. p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3

Kaplan-Meier survival curves for HNSC prognostic PITX genes. Kaplan-Meier survival curves for PITX1 showing (A) OS and (B) RFS; Kaplan-Meier survival curves for PITX2 showing (C) OS and (D) RFS; Kaplan-Meier survival curves for PITX3 showing (E) OS and (F) RFS.

FIGURE 4

The network and functional enrichment analysis of PITX1 and PITX2. (A) The protein-protein interaction (PPI) network for PITX1 and PITX2 (generated using GeneMANIA). The functions of PITX1, PITX2, and co-expressed genes were predicted by performing (B) GO and (C) KEGG analysis.

FIGURE 5

DNA methylation of PITX1 and HNSC prognosis. (A) The DNA methylation of PITX1 was determined using MethSurv; (B) The prognostic values of CpGs in PITX1, based on Kaplan-Meier survival analysis. Red to blue: high expression to low expression.

FIGURE 6

DNA methylation of PITX2. (A) The DNA methylation of PITX2 was determined using MethSurv; (B) The prognostic values of CpGs in PITX2, based on Kaplan-Meier survival analysis. Red to blue: high expression to low expression.

FIGURE 7

TIMER analysis of correlations between PITX1 and PITX2 expression and immune cell infiltration in HNSC. (A) Correlations between PITX1 expression and immune cell infiltration were evaluated using TIMER. (B) Correlation between PITX2 expression and immune cell infiltration levels using TIMER.

FIGURE 8

TISIDB analysis of correlations between PITX1 and PITX2 expression and immune cell infiltration across human cancers. (A) The relationship between PITX1 expression and 28 types of TILs across various human cancers. (B) The top six TILs displaying the strongest Spearman’s correlation with PITX1 expression in HNSC. (C) The relationship between PITX2 expression and 28 types of TILs across various human cancers. (D) The top six TILs displaying the strongest Spearman’s correlation with PITX2 expression in HNSC. Act_CD8, activated CD8⁺ T cells; Tcm_CD8, central memory CD8⁺ T cells; Tem_CD8, effector memory CD8⁺ T cells; Act_CD4, activated CD4⁺ T cells; Tcm_CD4, central memory CD4⁺ T cells; Tem_CD4, effector memory CD4⁺ T cells; Tfh, T follicular helper cells; Tgd, gamma delta T cells; Th1, type 1 T helper cells; Th17, type 17 T helper cells; Th2, type 2 T helper cells; Treg, regulatory T cells; Act B, activated B cells; Imm_B, immature B cells; Mem_B, memory B cells; NK, natural killer cells; CD56^bright, CD56^bright NK cells; CD56^dim, CD56^dim NK cells; MDSC, myeloid derived suppressor cells; NKT, natural killer T cells; Act DC, activated dendritic cells; pDC, plasmacytoid DCs; iDC, immature DCs; Mast, mast cell.

FIGURE 9

TISIDB analysis of correlations between PITX1 and PITX2 methylation and immune cells infiltration across human cancers. (A) The relationship between PITX1 methylation and 28 types of TILs across various human cancers. (B) The top six TILs displaying the strongest Spearman’s correlation with PITX1 methylation in HNSC. (C) The relationship between PITX2 methylation and 28 types of TILs across various human cancers. (D) The top six TILs displaying the strongest Spearman’s correlation with PITX2 methylation in HNSC. Act_CD8, activated CD8⁺ T cells; Tcm_CD8, central memory CD8⁺ T cells; Tem_CD8, effector memory CD8⁺ T cells; Act_CD4, activated CD4⁺ T cells; Tcm_CD4, central memory CD4⁺ T cells; Tem_CD4, effector memory CD4⁺ T cells; Tfh, T follicular helper cells; Tgd, gamma delta T cells; Th1, type 1 T helper cells; Th17, type 17 T helper cells; Th2, type 2 T helper cells; Treg, regulatory T cells; Act B, activated B cells; Imm_B, immature B cells; Mem_B, memory B cells; NK, natural killer cells; CD56^bright, CD56^bright NK cells; CD56^dim, CD56^dim NK cells; MDSC, myeloid derived suppressor cells; NKT, natural killer T cells; Act DC, activated dendritic cells; pDC, plasmacytoid DCs; iDC, immature DCs; Mast, mast cells.