Original Article Mining Expression and Prognosis of TACSTD2 and SPP1 in Thyroid Carcinoma by Using Oncomine and Kaplan-Meier Plotter Databases

Int J Clin Exp Med 2019;12(9):11231-11240 www.ijcem.com /ISSN:1940-5901/IJCEM0094709

Original Article Mining expression and prognosis of TACSTD2 and SPP1 in thyroid carcinoma by using Oncomine and Kaplan-Meier plotter databases

Qing Chang1, Mingda You1, Cui Li2

1Department of Head and Neck Surgery, Tangshan Worker Hospital of Hebei Province, Tangshan 063000, Hebei Province, PR China; 2Department of Neurosurgery, Yanghe People’s Hospital, Suqian 223800, Jiangsu Province, PR China Received April 1, 2019; Accepted June 11, 2019; Epub September 15, 2019; Published September 30, 2019

Abstract: Objective: To further investigate TACSTD2 and SPP1 gene mRNA expression in thyroid carcinoma and their association with patients’ prognosis and clinical pathological characteristics by data mining of the Oncomine and Kaplan-Meier plotter databases. Methods: TACSTD2 and SPP1 mRNA expression data from thyroid carcinoma were retrieved from the Oncomine database and further analyzed by comparing tumor tissue with corresponding normal tissue. The prognostic significance of TACSTD2 and SPP1 mRNA in thyroid carcinoma were evaluated by the online survival analysis tool “Kaplan-Meier Plotter” (KM plotter) database. Fifty-one patients with thyroid carcinoma who received surgery were retrospectively analyzed. The TACSTD2 and SPP1 protein expression in cancer tissue and normal thyroid tissue were examined by immunohistochemical assay. The correlation between TACSTD2, SPP1 protein expression and patients’ clinicopathological characteristics were also evaluated. Results: Four analyses compared the TACSTD2 gene expression between thyroid cancer and normal tissue were included in the present work. The pooled results showed that TACSTD2 mRNA expression levels in thyroid carcinoma tissue was significantly higher than that of normal thyroid tissue (p=0.003). For SPP1, the combined analysis from 8 analyses demonstrated that SPP1 mRNA level in thyroid carcinoma was up regulated compared to normal tissue with statistical significance (p<0.05). A significant difference of overall survival (OS) between TACSTD2 high and low expression groups was found in thyroid carcinoma patients (HR=0.17, 95% CI: 0.04-0.74, p=0.0078). However, for the SPP1 gene the difference was not statistically different between high and low mRNA expression groups (HR=0.062, 95% CI: 0.23-1.71, p=0.35). TACSTD2 interacted with TGFBI, MAP3K11, RAB13, MMP2, CHD1, KRT19, KRTAP5-2, ZNF92, C1QBP, and CYP8B1, which are related to cell proliferation and cellular protein metabolic pathways. The protein coded by SPP1 interacted with FGF2, RUNX2, IGF1, INS, VEGFA, IL6, MMP13, PTH, SP7, and BMP2, which are related to epithelial cell to mesenchymal cell transition (EMT), cell proliferation and negative regulation of apoptotic processes. Immunohistochemical analysis showed that TACSTD2 positive expression was correlated with the tumor capsular invasion (p<0.05) and lymph node metastasis (p<0.05). SPP1 positive expression was correlated with lymph node metastasis (p<0.05). Conclusion: TACSTD2 and SPP1 mRNA expression were up-regulated in cancer tissue compared to normal thyroid tissue in thyroid carcinoma. High level TACSTD2 mRNA was associated with poor prognosis.

Keywords: Data mining, TACSTD2, SPP1, prognosis, thyroid carcinoma

Introduction ground and acquired environmental factors of the patients [3]. The genetic background in- Thyroid cancer is one of the most commonly cludes family history of malignant tumors, ge- diagnosed head and neck malignant tumors netic polymorphism of some metabolic enzymes [1]. Although the incidence of thyroid cancer [4-6]. The acquired factors are mainly related to accounts for only 5% of thyroid nodules and 1% radiation [7], abnormal iodine uptake and other of total solid malignant tumors, it is the most factors [8, 9]. Early thyroid cancer patients can common cancer in the endocrine system [2]. At be treated by surgery, and the prognosis is present, the molecular mechanism of thyroid good. The 5-year survival rate after operation cancer is not fully understood. Most studies can reach more than 90%, but the prognosis of believe that it is related to the genetic back- patients with advanced disease is poor. Th- TACSTD2 and SPP1 expression in thyroid carcinoma erefore, it is particularly important to explore interaction network for TACSTD2 and SPP1. The the prognostic factors of thyroid cancer. correlation between TACSTD2 and SPP1 mRNA expression and clinical pathological character- Transmembrane glycoprotein TACSTD2, also istics of the patients were evaluated by immu- known as Trop-2, is an important signal trans- nohistochemical assay. duction molecule regulating the growth of cancer cells [10]. Recent studies have shown that Data extraction from oncomine database and TACSTD2 is overexpressed in breast cancer, analysis non-small cell lung cancer, head and neck squamous cell carcinoma and other malignant Oncomine database (https://www.oncomine. solid tumors [11, 12]. It plays an important role org/) is a gene web-based database and inte- in the proliferation and invasion of tumor cells, grated data mining platform, in which data can and is associated with metastasis and poor be screened and mined according to need. prognosis of tumors. The SPP1 gene located on From the Oncomine database, we retrieved chromosome 4q13, is a secretory phosphory- TACSTD2 and SPP1 related data sets with the lated glycoprotein with various biological activi- following restrictions: (1) tumor type: thyroid ties. It can promote cell chemotaxis, adhesion, carcinoma; (2) tissue comparison: normal thy- migration, proliferation and induce angiogene- roid tissue vs thyroid carcinoma tissue; (3) data sis, promote the degradation of the extracellu- type: mRNA; (4) significance: P<0.0001; (5) Ge- lar matrix, mediate the escape of cancer cells, ne fold change: >2; (6) gene sequencing: the inhibit apoptosis of cancer cells, stimulate sig- first 10%. nal transduction pathways, and it is closely related to the migration and metastasis of can- Survival analysis by Kaplan-Meier plotter cer cells [13, 14]. The prognosis (overall survival, OS) of TACSTD2 However, the correlation between both TA- and SPP1 mRNA expression of thyroid carcino- CSTD2 and SPP1 expression and thyroid carci- ma patients was analyzed by Kaplan-Meier noma in patient prognosis is not completely plotter (www.kmplot.com). This database pro- clear. In our present work, we evaluated the vides the online survival analysis of lung can- correlation between TACSTD2, SPP1 expres- cer, ovarian cancer, gastric cancer and breast sion and thyroid carcinoma patient prognosis cancer. In order to evaluate prognostic signifi- by deep data mining of the Oncomine and cant of TACSTD2 and SPP1 expression in thy- Kaplan-Meier plotter databases. roid cancer patients’ survival, we divided the patient data into two cohorts according to the Material and methods median TACSTD2 and SPP1 mRNA expression (high versus low expression). The overall sur- Study design vival of the thyroid carcinoma patients were compared between TACSTD2 and SPP1 high We first analyzed the expression of TACSTD2 and low expression groups. and SPP1 mRNA in human normal thyroid tissue in BioGPS (http://biogps.org/goto=wel- Immunohistochemical assay come) database, and then analyzed the differ- ential expression of TACSTD2 and SPP1 mRNA Fifty one cases of thyroid carcinoma who re- between cancer tissue and normal tissue of ceived surgery were retrospectively analyzed. thyroid carcinoma patients in Oncomie (https: The TACSTD2 and SPP1 protein expression in //www.oncomine.org/) database of oncogene cancer tissue and normal thyroid tissue were expression chip. The expression of TACSTD2 examined by immunohistochemical assay. Pa- and SPP1 was divided into two groups, and the raffin sections were dewaxed and hydrated. prognosis of patients with TACSTD2 and SPP1 Antigen was repaired after soaking and rinsing high and low expression was evaluated through in distilled water. Sections were rinsed in PBS the Kaplan-Meier Plotter (http://kmplot.com/ and dripped with non-immune serum. After analysis/) database. The protein interaction da- incubation at room temperature for 10 min- tabase STRING (https://string-db.org/cgi/in- utes, the antigen was washed off. Antibody put.pl) was used to predict the protein-protein incubation was done at room temperature for

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Figure 1. TACSTD2 and SPP1 mRNA relative expression in thyroid tissue from human beings (A: TACSTD2 mRNA relative expression level was 5.20; B: SPP1 mRNA relative expression level was 8.10).

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Figure 2. TACSTD2 and SPP1 mRNA expression in multiple cancers comparing cancer tissue to normal tissue.

60 minutes. The negative control was replaced Results by PBS. After PBS was washed, biotin-labeled antibodies were dripped on and incubated at TACSTD2 and SPP1 mRNA relative expression room temperature for 10 minutes. PBS was rinsed and dripped with Streptomyces antibiot- TACSTD2 and SPP1 mRNA relative expression ic-peroxidase solution, incubated at room tem- level of normal thyroid tissue from human be- perature for 30 minutes. After PBS wash, DAB ings were screened from the BioGPS database color developer was applied, and sealed with (http://biogps.org/#goto=welcome). The rela- neutral gum. tive TACSTD2 and SPP1 mRNA expression levels were 5.20 and 8.10 from normal thyroid tis- Statistical analysis sue in human beings, Figure 1.

Data was analyzed by Stata 11.0 software._ The TACSTD2 and SPP1 mRNA expression in mul- measurement data was expressed by x ± s and tiple cancers compared by student-t test. The counted data were expressed by rate and compared by χ2- Four hundred forty-nine and 431 studies were test. A proportional hazard ratio was used for included in the Oncomine database from mul- survival analysis through a log-rank test and tiple types of cancers, comparing cancer ver- survival curve. P<0.05 was considered statisti- sus normal tissue for TACSTD2 and SPP1 mRNA cally different. expression, respectively (Figure 2). Four analy-

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Figure 3. TACSTD2 and SPP1 mRNA expression in thyroid carcinoma comparing cancer tissue to normal tissue.

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Figure 4. The survival curve of thyroid carcinoma patients according to high and low TACSTD2 and SPP1 mRNA expression (A: TACSTD2 mRNA expression; B: SPP1 mRNA expression). ses compared the TACSTD2 gene expression CSTD2 interacted with TGFBI, MAP3K11, RA- between thyroid cancer and normal tissue [15- B13, MMP2, CHD1, KRT19, KRTAP5-2, ZNF92, 18]. The pooled results showed that TACSTD2 C1QBP, and CYP8B1, etc. (Figure 5). Gene mRNA expression level in thyroid carcinoma ontology (GO) analysis showed that most of the tissue was significant higher than that of nor- above proteins were related to cell proliferation mal thyroid tissue (p<0.05), Figure 3. For SPP1, and cellular protein metabolic processes. The the combined analysis from 8 analysis (3 stud- protein coded by SPP1 interacted with FGF2, ies) [15-17] demonstrated that SPP1 mRNA RUNX2, IGF1, INS, VEGFA, IL6, MMP13, PTH, level in thyroid carcinoma was up regulated SP7, BMP2 and etc. (Figure 6). Most of these compared to normal tissue with statistical dif- proteins are related to the epithelial cell to ference (p<0.05), Figure 3. mesenchymal cell transition (EMT), cell proliferation and negative regulation of apoptotic TACSTD2 and SPP1 mRNA expression and processes. prognosis Immunohistochemical analysis The prognostic significance of TACSTD2 and The positive expression rate of TACSTD2 in can- SPP1 mRNA expression and thyroid carcinoma cer tissue was significantly higher than that of patient prognosis were analyzed by Kaplan- normal thyroid tissue (62.7% vs 15.7%, p<0.05). Meier Plotter. A significant difference of overall For SPP1, the positive expression rate in thy- survival (OS) between TACSTD2 high and low roid cancer tissue and normal tissue were expression groups was found in thyroid carci- 68.6% and 23.5%, respectively, with significant noma patients (HR=0.17, 95% CI: 0.04-0.74, statistical difference (p<0.05), Table 1. p=0.0078). However, for the SPP1 gene the difference was not statistically different be- TACSTD2 and SPP1 expression and patient tween high and low mRNA expression groups clinicopathological features (HR=0.062, 95% CI: 0.23-1.71, p=0.35), Figure 4. TACSTD2 positive expression was correlated with tumor capsular invasion (p<0.05) and TACSTD2 and SPP1 protein-protein interaction lymph node metastasis (p<0.05). SPP1 positive networks expression was correlated with lymph node metastasis (p<0.05). However, TACSTD2 and The possible protein-protein interaction of TA- SPP1 expression was not correlated with age, CSTD2 and SPP1 were analyzed through the gender, pathology type nor prognostic index (all STRING database. The results showed that TA- p>0.05), Table 2.

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Figure 5. Protein-protein interactions of TACSTD2.

Discussion in length and encodes 323 amino acids [21]. TACSTD2 protein is a phosphorylated glycopro- Thyroid cancer accounts for more than 90% of tein with a single transmembrane region on the human endocrine gland malignant tumors [19]. cell surface. It contains an EGF-like repeat The International Organization for Cancer esti- region, a thyroglobulin-like repeat region and a mates that in 2010, there were about 44,670 transmembrane region. There are serine and new cases of thyroid cancer worldwide and tyrosine phosphorylation sites and a PIP2 bind- about 1,690 deaths from this disease. Globally, ing sequence at the carboxyl end. Recent stud- 122,800 new cases of differentiated thyroid ies have found that TACSTD2 is highly expressed cancer are diagnosed each year, the majority of in various epithelial cancers, but very low or not which occur in young adults, with annual in- expressed in normal epithelial tissues [11, 12, crease of about 4% [20]. 22]. Overexpression of TACSTD2 can promote the proliferation, and invasion of some cancer TACSTD2, tumor associated calcium signal tra- cells, including ovarian cancer. Conversely, si- nsducer 2, also known as Trop2 gene, is 1.8 Kb lencing the TACSTD2 gene can inhibit the gr-

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Figure 6. Protein-protein interactions of SPP1.

Table 1. TACSTD2 and SPP1 protein expression in thyroid sue. The median overall survival of carcinoma (n, %) thyroid carcinoma patients with ele- TACSTD2 SPP1 vated TACSTD2 levels were significant lower than that of lower TACS- Positive Negative Positive Negative TD2 level cases. This indicated that Cancer tissue (n=51) 32 (62.7) 19 (37.3) 35 (68.6) 16 (31.4) TACSTD2 mRNA level can potentially Normal tissue (n=51) 8 (15.7) 43 (84.3) 12 (23.5) 39 (76.5) be a biomarker for poor prognosis of Chi-square 15.83 20.87 thyroid carcinoma patients. P <0.001 <0.001 The SPP1 encoding protein is a che- mokine-like protein, rich in sialic owth of cancer cells [23]. It was found that the acid and secreting non-collagen type. It is a TACSTD2 was associated with a variety of regu- phosphorylated acidic glycoprotein containing latory factors and might play a role in activating arginine, glycine, and aspartic acid (RGD) resi- the ERK/MAPK pathway to drive the expression dues. SPP1 encoding protein is associated with of NF-kB, cyclin D1 and other proteins [24]. In the occurrence, development, metastasis and our work, we found that TACSTD2 mRNA expres- prognosis of various tumors [25, 26]. In our sion level in thyroid carcinoma tissue was sig- work, we found that SPP1 mRNA levels in thy- nificant higher than that of normal thyroid tis- roid carcinoma was up regulated compared to

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Table 2. Correlation between TACSTD2, SPP1 protein expression and patient clinical pathological feature TACSTD2 SPP1 N=51 Positive Negative χ2 P Positive Negative χ2 p (n=32) (n=19) (n=35) (n=16) Gender 1.49 0.22 1.73 0.19 Female 35 20 15 22 13 Male 16 12 4 13 3 Age (year) 1.15 0.28 2.52 0.11 <45 30 17 13 18 12 ≥45 21 15 6 17 4 Pathology 1.02 0.31 0.22 0.64 Papillary 36 21 15 24 12 Follicular 15 11 4 11 4 Tumor capsular invasion 4.19 0.04 4.09 0.04 Positive 20 16 4 17 3 Negative 31 16 15 18 13 Prognostic index 1.51 0.22 2.07 0.15 <66 38 22 16 24 14 ≥66 13 10 3 11 2 Lymph node metastasis 3.95 0.046 0.43 0.51 Positive 10 9 1 6 4 Negative 41 23 18 29 12 normal tissue with statistical differences. This [2] Sprøgel P and Storm HH. Thyroid cancer in indicated that SPP1 may play an important role Denmark during the period 1943-85. Cancer in the development of thyroid carcinoma. statistics number 23. Ugeskr Laeger 1989; 151: 507-11. Conclusion [3] Agate L, Lorusso L and Elisei R. New and old knowledge on differentiated thyroid cancer In our present work, we investigate TACSTD2 epidemiology and risk factors. J Endocrinol and SPP1 gene mRNA expression in thyroid car- Invest 2012; 35: 3-9. cinoma and their association with patient prog- [4] Barczyński M, Cichoń S and Trystuła M. [Risk nosis and clinical pathological outcome by data factors and surgical tactics in differentiated mining of the Oncomine and Kaplan-Meier plot- thyroid cancer]. Przegl Lek 1994; 51: 437-42. [5] Duntas L and Grab-Duntas BM. Risk and prog- ter databases. According to the present evi- nostic factors for differentiated thyroid cancer. dence, TACSTD2 and SPP1 mRNA expression Hell J Nucl Med 2006; 9: 156-62. levels were up-regulated in cancer tissue com- [6] Dal Maso L, Bosetti C, La Vecchia C and Fran- pared to normal thyroid tissue in thyroid carci- ceschi S. Risk factors for thyroid cancer: an noma. High level TACSTD2 mRNA was associ- epidemiological review focused on nutritional ated with poor prognosis. factors. Cancer Causes Control 2009; 20: 75- 86. Address correspondence to: Mingda You, Depart- [7] Albi E, Cataldi S, Lazzarini A, Codini M, Beccari ment of Head and Neck Surgery, Tangshan Worker T, Ambesi-Impiombato FS and Curcio F. Ra- Hospital of Hebei Province, No. 27 Wenhua Road, diation and thyroid cancer. Int J Mol Sci 2017; Lubei District, Tangshan Road, Tangshan 063000, 18. Hebei Province, PR China. E-mail: 389120620@ [8] Hong CM and Ahn BC. Factors associated with qq.com dose determination of radioactive iodine ther- apy for differentiated thyroid cancer. Nucl Med References Mol Imaging 2018; 52: 247-253. [9] Vaisman F, Carvalho DP and Vaisman M. A new [1] Siegel RL, Miller KD and Jemal A. Cancer sta- appraisal of iodine refractory thyroid cancer. tistics, 2018. CA Cancer J Clin 2018; 68: 7-30. Endocr Relat Cancer 2015; 22: R301-10.

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