TPPP3 Associated with Prognosis and Immune Infiltrates in Head and Neck Squamous Carcinoma

Tubulin polymerization promoting protein family member 3 (TPPP3) is a kind of protein that can mediate the dynamics and stability of microtubules. However, the correlations of TPPP3 between prognosis and immune infiltrates in different tumors are still unclear. The analysis of TPPP3 expression was performed via Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) website. We also used GEPIA to assess the impact of TPPPT3 on clinical outcomes. The related pathways involved in TPPP3 were analyzed by gene-set enrichment analysis (GSEA), and the correlation between TPPP3 and immune infiltration was studied by Tumor Immune Estimation Resource2.0 (TIMER 2.0). The TPPP3 expression was significantly reduced in head and neck squamous carcinoma (HNSC) compared to adjacent tissues. In addition, the low expression of TPPP3 in HNSC was significantly associated with prognosis. The pathways closely related to the low expression of TPPP3 are “Antigen Processing and Presentation,” “Primary Immunodeficiency,” and so on. The TPPP3 expression was negatively correlated with the level of CD8+ T cell, B cell, and myeloid dendritic cell infiltration in HNSC. The TPPP3 expression is closely related to multiple immunomarkers in CD8+ T cell and Myeloid dendritic cells. These data indicate that TPPP3 is associated with multiple cancers and involves multiple immune-related pathways, and TPPP3 is associated with immune infiltration levels. Besides, the TPPP3 expression may help regulate tumor-associated CD8 + T cells, DC cells in HNSC. We conclude TPPP3 can be considered as a biomarker for predicting head and neck squamous cell carcinoma prognosis and immune infiltration.


Introduction
Head and neck squamous carcinoma (HNSC) is one of the most frequent tumors in Southeast Asia and southern China. The mechanism of HNSC development is complex and involves the alteration of polygenic and multisignaling pathways [1]. During this process, viral factors, environmental factors, and genetic factors affect tumor-related gene regulation and abnormal expression [2][3][4][5]. The early diagnosis of HNSC is difficult due to the hidden physiological position of HNSC, and it is prone to lymph node and distant metastasis. Therefore, screening biomarkers are helpful for the diagnosis and pathological indicators of tumorigenesis and development.
TPPP3, also known as TPPP/p20, is a protein-coding gene located on chromosome 16 that was first reported in 2006. TPPP3 is a brain-specific protein homologous to TPPP/p25, expressed in many human cells and organs, which could induce tubulin polymerization and microtubule (MT) bundling [6]. Microtubules are the main components of mitotic spindles, which control all aspects of cell division and chromosome separation. Studies have shown that microtubule dynamics change in cancer cell division and are associated with the development of chromosomal instability, anaplasia, and drug resistance [7]. Many kinds of tumor drugs exert their anticancer effect by acting on microtubules and microtubule-related proteins. The classical anticancer drugs such as vincristine and paclitaxel are widely used in the clinic, so the further exploration of microtubules and tubulin is of great significance to the study of tumor prevention and treatment.
A growing number of researchers in recent years have begun to explore the relationship between TPPP3 and tumors. Studies suggest that reduced TPPP3 can lead to abnormal mitoses, such as the formation of multipolar spindles and chromosome segregation errors, leading to HeLa apoptosis [8]. However, the potential role of TPPP3 in HNSC development or metastasis remains unknown.
In the tumor microenvironment, immune and stromal cells are two major types of nontumor components. The extent of tumor immune infiltration and stromal cells has significant value for tumor diagnosis and prognosis evaluation. This study provides a comprehensive analysis of TPPP3 expression in cancer databases and its relationship to the prognosis of cancer patients. Then, we performed pathological verification with clinical specimens. In addition, we also examined the relationship between TPPP3 and tumorinfiltrating immune cells in HNSC through the Tumor Immunity Estimation Resource 2.0(TIMER 2.0). This report clarifies the important role of TPPP3 in HNSC and provides evidence for the relationship between TPPP3 and tumorimmune cell infiltration interactions.

TPPP3 mRNA Expression Levels in Different Types of Human Cancer.
To understand the differences in TPPP3 expression between human tumor and nontumor tissues, we utilized the Oncomine database to analyze the expression levels of TPPP3 in multiple cancer types and different tumors and normal tissues. Comparing to normal tissues, the study demonstrated that TPPP3 observed lower expression in bladder cancer, brain cancer, breast cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, and sarcoma and high expression in gastric cancer (Figure 1(a)). To more accurately assess TPPP3 expression in human cancers, we used RNA-seq data from 31 malignancies in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) to examine the TPPP3 expression. Figure 1(b) shows the differential expression of TPPP3 between tumors and normal tissues. The TPPP3 expression was significantly decreased in HNSC, bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIPC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC). However, compared with adjacent normal tissues, the TPPP3 expression was significantly higher in cholangiocarcinoma (CHOL) and kidney renal clear cell carcinoma (KIRC).

TPPP3 Gene Set Enrichment Analyses.
To discover the underlying mechanism of TPPP3 and HNSC, Gene Set Enrichment Analysis (GSEA) was utilized to obtain the TPPP3-related gene collection based on the actual overall trend analysis and compare enrichment analysis such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Consequently, the enrichment of 100 functional gene sets was obtained. The pathways closely related to the low expression of TPPP3 are "Antigen Processing and Presentation," "Primary Immunodeficiency," "RIG I Like Receptor Signaling Pathway," "Endometrial Cancer," and "P53 Signaling Pathway." The pathways associated with TPPP3 high expression are "Parkingson Disease," "Oxidative Phosphorylation," and "Phenylalanine Metabolism" (Figures 2(h)-2(o)). "Translation repressor activity mRNA regulatory element-binding," "Negative regulation of regulated secretory pathway," "Negative regulation of regulated secretory pathway," "Amino acid betaine metabolic process," "Protein kinase a regulatory subunit binding," and "Site of DNA damage" are the six most enriched items in GO related to the low expression of TPPP3 (Figures 2(p)-2(t)). The GO items associated with TPPP3 high expression are "Nuclear envelope reassembly," "High density lipoprotein particle clearance," "High density lipoprotein particle remodeling," "Reverse cholesterol transport," and "Negative regulation of lipase activity" (Figures 2(u)-2(y)).

TPPP3 Expression Is Associated with Immune Infiltration
Levels in HNSC. Tumor-infiltrating lymphocytes are independent risk factors affecting tumor prognosis [9]. Therefore, we analyzed by TIMER2.0 whether the TPPP3 expression was associated with immune infiltration levels in HNSC. The results show that the TPPP3 expression has a certain correlation with the infiltration of CD8 + T cells, B cells, and myeloid dendritic cells ( Figure 3).

Analysis of the Correlation between TPPP3 Expression
and Immune Cell Genes. To gain insight into the intrinsic link between TPPP3 and related immune cells, we applied the TIMER 2.0 database to analyze the correlation between TPPP3 genes in HNSC related immune infiltrating cells. We obtained results on the correlation between TPPP3 expression and marker genes of tumor infiltrationassociated immune cells, including CD8+T cells (CD8A and CD8B), B cell (CD19 and CD79a), and Myeloid dendritic cell (HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, CD1C, NRP1, and ITGAX). The results manifested that TPPP3 expression levels were negatively correlated with most of the immune marker genes in HNSC ( Figure 4). In the high-level immune infiltration mode of HNSC, TPPP3 has an intense correlation with CD8 + T cell marker and DC markers. These results further indicate that there is a certain relationship between TPPP3 and DC penetration. Promote dendritic cells to regulate cytotoxic CD8 + T cell responses and exert anticancer effects [10,11]. Whether TPPP3 is a key factor in mediating CD8 + T cell, DC, and tumor metastasis need further research.

Low Expression of TPPP3 in Clinical Specimens of NPC.
In the present research, the expression of TPPP3 in nasopharyngeal carcinoma (NPC) tissues was detected in 57 cases of NPC and 30 cases of normal tissue by immunohistochemistry, and the relationship between TPPP3 and clinicopathological features of NPC was analyzed at the same time. The results showed that the expression of TPPP3 in NPC was significantly lower than that in normal nasopharyngeal tissue; the difference was statistically significant ( Figure 5, Table 1). But there was no significant correlation with clinicopathological features ( Table 2).

Discussion
A growing number of researchers have been exploring the relationship between TPPP3 and tumor development in recent years. According to the Zhou et al.'s study, knockdown of TPPP3 inhibits cell proliferation of HeLa cells while inducing cell cycle arrest [8]. Li et al. demonstrated that TPPP3 was highly expressed in nonsmall cell lung cancer, and that the high expression of TPPP3 was positively correlated with clinical stage, tumor volume, lymph node metastasis, and poor prognosis [12]. Ye et al. also revealed that TPPP3 was highly expressed in colorectal cancer and was associated with colorectal cancer progression and poor prognosis, and that interfering with TPPP3 expression suppressed tumor cell proliferation, migration, and invasion, and increased apoptosis [13]. Although TPPP3 has not been extensively studied, according to the available data, it is found that the expression level of TPPP3 was increased in some tumors and correlated with tumor proliferation. The expression of TPPP3 is upregulated in many tumors, and experimental studies have confirmed its oncogenic effect. We carried out a pan-cancer analysis of TPPP3, which indicates that TPPP3 has a different expression trend with other tumors in HNSC. However, no relevant studies were found in head and neck squamous carcinoma. Therefore, it is necessary to carry out detailed research on TPPP3 in HNSC.
In this article, we first performed a pan-cancer analysis of TPPP3 expression. The Oncomine database and GEPIA were used to analyze the mRNA expression data of 31 tumors to check the expression level and prognosis of TPPP3 in different types of tumors. According to the results of the Oncomine database, we found that TPPP3 was lower expressed in breast cancer, head and neck cancer, kidney cancer, ovarian cancer, and sarcoma compared to normal tissues, while higher expression in gastric cancer. Furthermore, GEPIA demonstrated that compared with neighboring normal tissues, the expression of TPPP3 in HNSC, BLCA, BRCA, KICH, KIRC, LUAD, and LUSC was significantly reduced, while the expression of TPPP3 in CHOL and KIRC was significantly higher. We found that using different databases, TPPP3 expression levels  differed among different tumor types, which may be due to the data collection method and the different biological characteristics of TPPP3. Interestingly, in these databases, we found consistency in TPPP3 expression in head and neck squamous cell carcinoma. In addition, the analysis of survival data indicates that low levels of TPPP3 expression are associated with a poor prognosis for HNSC.
In this research on the mechanism of TPPP3, we selected the transcript data of 70 cases HNSC in the TCGA database and performed GSEA analysis on these datasets. The pathways closely related to the low expression of TPPP3 are "Antigen Processing and Presentation," "Primary Immunodeficiency," "RIG I Like Receptor Signaling Pathway," "Endometrial Cancer," and "P53 Signaling Pathway." Results   BioMed Research International also indicate that TPPP3 may be related to the mechanism of tumor immunity. Therefore, we shifted the research focus of TPPP3 to research on tumor immune infiltration. Our results indicate that TPPP3 expression is correlated with multiple immune cell infiltration levels in head and neck squamous cell carcinoma. There is a negative correlation between the expression of TPPP3 and the infiltration levels of CD8+T cells and B memory cells. However, TPPP3 expression is positively correlated with DC cells. Moreover, the correlation between TPPP3 expression and immune cell marker genes suggests that TPPP3 is regulating the immunological role of HNSC. Further, the genetic markers of CD8+ T cells and myeloid dendritic cells are weakly correlated with TPPP3 expression. These results reveal a potential regulatory role of TPPP3 in tumor-associated myeloid dendritic cellmediated T cell toxic effects.
In addition, to further understanding of TPPP3 expression in head and neck tumors, we also performed immunohistochemistry on clinical NPC specimens and validated tumor and normal tissue TPPP3 mRNA expression in two GEO NPC datasets. The results we obtained are consistent with those of Oncomine and GEPIA in that TPPP3 expression is significantly reduced in the HNSC. But there is no significant correlation with clinicopathological features. This study for the first time analyzed the expression and prognosis of TPPP3 in head and neck squamous cell carcinoma, unexpectedly, in contrast to the expression in other tumors. We are as well the first to reveal the link between TPPP3 and immune infiltration. This study also has some shortcomings, such as we still need to perform in vitro and in vivo experiments on TPPP3 for cellular function studies as well as pathway studies.   Expression level (log2 TPM)  TPPP3  TPPP3  TPPP3   TPPP3   TPPP3   TPPP3   TPPP3   TPPP3  TPPP3   TPPP3  TPPP3  TPPP3   2  reference gene sets for GSEA. Cut-off criteria were defined as p < 0:05, FDR < 0:05, and enrichment score ðESÞ > 0:6.

Materials and Methods
4.6. TIMER Website Analysis. TIMER2.0 (http://timer .cistrome.org) [18] is an interactive web server for systematically estimating the abundance of immune infiltration in tumors from the TCGA database. We performed a genetic module to analyze the correlation between TPPP3 expression and immune infiltration abundance in HNSC, including CD4+ T cells, CD8+ T cells, B cells, neutrophils, Monocyte, Macrophages, Common lymphoid progenitor, NK cell, Endothelial cell, Mast cell, Tregs, and dendritic cells, via gene modules. These genetic markers have been cited in previous researches [19,20]. The website provides both TIMER and CIBERSORT immune infiltration estimations.   7 BioMed Research International expression levels were compared between the tumor group and normal tissues in these two datasets.
4.9. Statistical Analysis. The mRNA expression between NPC and normal group was compared, respectively, using t-test. The correlation of TPPP3 expression was evaluated by Spearman's correlation and statistical significance. p values < 0.05 were considered statistically significant.

Conclusions
All in all, the TPPP3 expression is significantly reduced in HNSC, and the low expression of TPPP3 is associated with CD8 + T cell and myeloid dendritic cell immune infiltration levels. Therefore, thus, TPPP3 can serve as a potential prognostic indicator for HNSC patients and may play an important role in immune cell infiltration. Overall survival DFS: Disease-free survival.

Data Availability
The data used in this study are from open public databases, and how to obtain them has been explained in the manuscript.

Disclosure
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Conflicts of Interest
The authors declare no conflict of interest.