Upregulation of Translationally Controlled Tumor Protein Is Associated With Cervical Cancer Progression

Abstract

Outside a few affluent countries with adequate vaccination and screening coverage, cervical cancer remains the leading cause of cancer-related deaths in women in many countries. Currently, a major problem is that a substantial proportion of patients are already at an advanced cancer stage when diagnosed. There is increasing evidence that indicates the involvement of translationally controlled tumor protein 1 (TPT1) overexpression in cancer development, but little is known about its implication in cervical cancer. We assessed the levels of TPT1 in surgical tissue and sera of patients with cervicitis, cervical intraepithelial neoplasia III, and cervical cancer, as well as in normal and cancerous cervical cell lines. Gene sets, pathways, and functional protein interactions associated with TPT1 were identified using the TCGA data cohort of cervical cancer. We found that the TPT1 expression was significantly increased in cervical cancer tissue compared to all nonmalignant cervical tissues, including samples of cervicitis, cervical intraepithelial neoplasia III, and normal controls. Serum level of TPT1 was also increased in cervical cancer patients compared to healthy subjects. Furthermore, elevated TPT1 expression was significantly correlated with lymph node metastasis and a low differentiation degree of the cancer. In the cancerous tissues and cell lines, selective markers of PI3K/AKT/mTOR pathway over-activation, apoptosis repression, and EMT were detected, and their interaction with TPT1 was supported by biometrics analyses. Our results, for the first time, demonstrate a strong correlation of upregulated TPT1 expression with cervical cancer progression, suggesting that TPT1 might provide a potential biomarker for cervical cancer progression.

Keywords: PI3K/Akt/mTOR pathway; TPT1; biometrics; cervical cancer; diagnosis.

FIGURE 1

Elevated TPT1 expression in cervical cancer tissues and cells. (A) Protein expression of TPT1 in tissues of cervical cancer patients, assessed by WB and pairwise compared to paracancerous tissue (left) and quantified (right). P: paracancerous tissue; C: cancerous tissue. (B) Relative TPT1 gene transcription in the paired cervical cancer tissues, measured by qRT-PCR. (C) TPT1 protein expression in normal human cervical epithelial cell line HCerEpic and cervical carcinoma cell lines SiHa and HeLa, analyzed by WB (left) and quantified (right). (D) Relative TPT1 gene transcription in the cell lines, measured by qRT-PCR. WB images are representative of all samples tested. All experiments were repeated at least three times. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Increased abundance of TPT1 protein in the tissue and sera of cervical cancer compared to cervicitis, CINIII, and controls. (A) Tissue IHC staining of TPT1 protein in the control and different cervical lesions including cervicitis, CINIII, and cervical cancer. Scale bars: x200, 50 μm; x400: 10 μm. (B) Serum TPT1 concentration in CINIII and cervical cancer patients, compared to normal healthy controls, measured by ELISA. IHC images are representative of all samples tested. *p < 0.05; NS: statistically not significant.

FIGURE 3

GSEA enrichment analysis of TCGA data on gene sets and pathways associated with low (A) or high (B) TPT1 expression in cervical cancer. The horizontal bar, gradient-filled from red to blue, represents where genes in each gene set appear in the ranked list of genes. Genes on the left side (red) correlate most strongly with the phenotype. The vertical black lines represent the running enrichment scores as the projection of individual genes onto the horizontal ranked gene list. The bottom ranking matric in gray, moving from above zero (positively correlated) to below zero (negatively correlated), measures a gene’s correlation with the phenotype profile. Statistical variables of GSEA analysis are displayed in each image. ES, enrichment score for the gene set, reflecting the degree to which this gene set is over-represented at the peak (furthest from 0.0) of the entire ranked list of genes; p-value, statistical assessment of the significance against null distribution; NES, normalized enrichment score calculated by adjusting ES for gene set size or multiple hypothesis testing across analyzed gene sets; FDR (false discovery rate q-value), the estimated probability that a given NES represents a false positive finding. p < 0.05 and FDR < 0.25 were considered statistically significant.

FIGURE 4

STRING PPI network analyses of functional connections of TPT1 with key proteins of PI3K/AKT/mTOR signaling, apoptosis, and EMT pathways. Line thickness indicates the strength of data support, and circles in different colors represent individual proteins with the abbreviated name. The number above each line is the combined association score that indicates a probability of the existence of a real functional association between assigned proteins, calculated from online sources of textmining, experiments, and databases. The threshold of the confidence score was set at 0.7 (high).

FIGURE 5

Identification of key proteins involved in PI3K/AKT/mTOR pathway over-activation, apoptosis suppression, EMT, and p53 degradation in cancerous tissues of cervical cancer patients, in comparison with the paracancerous tissues. (A) WB images that are representative of all samples tested. (B) Densitometry quantification of the WB results from the experiment performed in A. The ratio of BAX:BCL2 is calculated to highlight the apoptosis activity. *p < 0.05. **p < 0.01. *** p < 0.001.

FIGURE 6

Identification of proteins involved in PI3K/AKT/mTOR pathway over-activation, apoptosis suppression, and EMT in cervical carcinoma cell lines SiHa and HeLa, in comparison with the non-malignant cervical epithelial cell line HCerEpic. (A) WB images that are representative of all replicates of the experiment. (B) Densitometry quantification of the WB results from the experiment performed in A. The ratio of BAX:BCL2 is calculated to highlight the apoptosis activity. *p < 0.05. **p < 0.01. ***p < 0.001.