Clinicopathological and Prognostic Role of STAT3/p-STAT3 in Breast Cancer Patients in China: A Meta-Analysis

In order to explore the important factors in the diagnosis of breast cancer in China, meta-analysis of previous studies was performed to understand the association between STAT3/p-STAT3 and breast cancer. Information about STAT3/p-STAT3 expression and clinical data about breast cancer in China in particular were gathered from PubMed, Web of Science, CNKI and WanFang databases. RevMan 5.3 and STATA 14.0 were used to analyze the occurrence, development and metastasis of breast cancer for 2818 patients in 18 studies. STAT3/p-STAT3 expression was higher in breast cancer tissue than in normal ones (OR = 7.48, 95% CI = 5.64–9.94), in highly differentiated breast cancer tissue than in lowly differentiated cancer tissues (OR = 2.13, 95% CI = 1.53–2.98), in III/IV stage breast cancer than in I/II stage breast cancer (OR = 3.58, 95% CI = 2.44–5.25), and in tissue with lymphatic metastasis than in normal tissues (OR = 3.72, 95% CI = 2.59–5.35), respectively. Thus, the expression of STAT3/p-STAT3 plays a clinicopathological and prognostic role in the diagnosis and treatment of Chinese breast cancer patients.


1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 211198, P. R. China. 2 School of International Pharmaceutical Business, China Pharmaceutical University, Nanjing, 211198, P. R. China. 3 School of Life Science and Technology, China Pharmaceutical University, Nanjing, 211198, P. R. China. 4 Nanjing First Hospital, China Pharmaceutical University, Nanjing, 211198, P. R. China. 5 Foreign Languages Department, China Pharmaceutical University, Nanjing, 211198, P. R. China. 6 School of Pharmacy, Guangxi Medical University, Nanning, 530021, P. R. China. 7 The Department of Dermatology, The First People's Hospital of Changzhou, Changzhou, 213003, P. R. China. Correspondence and requests for materials should be addressed to F.C. (email: cfcpu72@163. com) or W.Y. (email: ywy@cpu.edu.cn) Screen and excerpt. All studies were brought into this research by two researchers independently according to the exclusion and inclusion criteria, with the full text being acquired with the extracted data inside. After all the data were crosschecked, divergence would be discussed and the third researcher would give some references. The extracted data included characteristic data, the focused type, numbers of treatment and control groups and the expression level of STAT3 or p-STAT3 in treatment and control groups, age groups, tissue types, TNM stages, tumor sizes and the states of lymphatic metastasis. Quality assessment. Quality assessment was performed by two researchers separately, with differences being resolved through discussion. We referred to the Cochrane evaluation clauses: (1) Random sequence generation; (2) Allocation concealment; (3) Blind method; (4) Incomplete outcome data; (5) Non-selective ending report; (6) Without other bias source. Each coincident item gives one point. Studies with scores ≥3 were assigned as high-quality studies.

Statistical analysis.
Extracted data were used to analyze the correlation between the expression of STAT3 or p-STAT3 and the focused type, the numbers of treatment and control groups, the expression level of STAT3 or p-STAT3 in treatment and control groups, age groups, tissue types, TNM stages, tumor sizes and the states of lymphatic metastasis. All data in literatures were combined to obtain a value of OR (Odds Ratio) and 95% CI (Confidential Interval). For the results of χ 2 test, when P < 0.1 and I² > 50%, supra presence included significant heterogeneity, using the random effects model by choosing the model option in the Review Manager 5.3 software when generating the forest plots. Then, regression was analyzed by drawing funnel plot and Egger's test. The analysis was performed using STATA statistical software version 14.0.  The results of STAT3 expression and analysis. Correlation between STAT3/p-STAT3 expression level and breast cancer occurrence. 13 studies reported the correlation between STAT3 expression level and breast cancer occurrence, 6 on p-STAT3 and 7 on STAT3, 1362 cases for breast cancer tissues and 773 for normal tissues. No substantial heterogeneity existed with each group (P = 0.30, I 2 = 14%), and we performed the meta-analysis  www.nature.com/scientificreports www.nature.com/scientificreports/ using the random-effects model. STAT3/p-STAT3 expression level in breast cancer tissues was higher than that in normal ones (OR = 7.48, 95% CI = 5.64-9.94). In the subgroup analysis, we achieved a consistent result (STAT3: OR = 8.81, 95% CI = 5.18-15.00; p-STAT3: OR = 7.13, 95% CI = 5.13-9.92). The results demonstrated that the STAT3/p-STAT3 expression level in breast cancer tissue was higher than that in normal ones (Fig. 2).

Results
The correlation of STAT3/p-STAT3 expression level and histological differentiation. 13 studies reported the correlation of STAT3/p-STAT3 expression level and histological differentiation, with data inside being used to analyze the difference between low-differentiated and high-differentiated cases. In the 13 reports, 395 cases were high-differentiated and 750 were low-differentiated. There was no substantial heterogeneity in each group (P = 0.25, I 2 = 27%), and the random-effects model was chosen. The result showed that the STAT3/p-STAT3 expression level in high-differentiated cases was higher than that in low-differentiated cases (OR = 2.13, 95% CI = 1.53-2.98). In the subgroup analysis, we achieved a consistent result (STAT3: OR = 1.83, 95% CI = 1.23-2.72; p-STAT3: OR = 2.34, 95% CI = 1.18-4.67). The analytical results were stable, as shown in Fig. 3.

The correlation of STAT3/p-STAT3 expression level and breast cancer TNM stages.
15 studies reported the correlation of STAT3/p-STAT3 expression level and breast cancer TNM stages. We regarded stages I and II as early stage (involving 1271 cases), III and IV as late stage (involving 509 cases). We used the random-effects model and there was no evident heterogeneity inside (P = 0.11, I 2 = 32%). The results showed that the STAT3/p-STAT3 expression level in breast cancer of the late stage was much higher than the early stage (OR = 3.58, 95% CI = 2.44-5.25). In the subgroup analysis, we achieved a consistent result (STAT3: OR = 3.37, 95% CI = 1.98-5.73; p-STAT3: OR = 3.88, 95% CI = 2.44-5.25), as shown in Fig. 4.
The correlation of STAT3/p-STAT3 expression level and breast cancer lymphatic metastasis. 11 studies reported the correlation of STAT3/p-STAT3 expression level and breast cancer lymphatic metastasis, including 1249 patients of lymphatic metastasis and 350 patients of normal condition. The random-effects model was used and no evident heterogeneity inside (P = 0.14, I 2 = 32%). The results showed that the STAT3/p-STAT3 expression level of lymphatic metastasis patients is evidently higher than that for other patients (OR  Considering the studies on the relationship between STAT3/p-STAT3 and survival prognosis of breast cancer patients are rare, and the papers did not use a uniformed statistical method, it's very difficult to generate statistical graphs. The results were shown in the Table 3. www.nature.com/scientificreports www.nature.com/scientificreports/ Discussion JAK-STAT signal pathway is of prime importance for STAT3 phosphorylation 10 . When receptors are stimulated by some special cytokines or growth factors, tyrosine kinase (JAKs) and Src tyrosine kinase coupled with these receptors would phosphorylate STAT3. Moreover, some environmental factors like smoke and UV radiation also phosphorylates STAT3 through tyrosine kinases like Src and ABL independently of receptors 5 .
The gene expression products controlled by STAT3 have multiple functions, like the growth and proliferation of cells, angiogenesis and immunosuppression. P-STAT3 could improve the occurrence of cancers by inducing different kinds of genes controlling cell proliferation to express abnormally. MYC 11 , cyclin D1/D2 12 , BCL-XL 13  Currently, abundant studies have reported that the STAT3 or p-STAT3 expression has close connection with the occurrence, differentiation, TNM stages and lymphatic metastasis of breast cancer. However, on the one hand, simplex research samples are scarce and have no statistical significance; on the other hand, the results of each research are different. So we performed this meta-analysis to search and screen researches which are satisfactory, and to make our analysis statistically significant.     www.nature.com/scientificreports www.nature.com/scientificreports/ The breast cancer patients in our research were Chinese. The results showed that STAT3 or p-STAT3 expression in breast cancer tissues was much higher than that in normal ones, indicating a positive correlation between STAT3 or p-STAT3 overexpression and the occurrence of breast cancer. In addition, our research found a higher STAT3 or p-STAT3 expression level in breast cancer cells which kept the characteristics of rapid proliferation, less differentiation and lymphatic metastasis. The STAT3 expression difference has not been found between patients of different ages or tumor sizes. Above all, STAT3/p-STAT3 expression could induce the occurrence of breast cancer; in breast cancer cells, STAT3 or p-STAT3 overexpression could also predict rapid proliferation, the late stage of TNM and the possibility of lymphatic metastasis.
As for survival the relationship between STAT3 (or p-STAT3) expression and patients' survival prognosis, the outcomes of five studies in Table 3 were not in full accord, but most studies showed the trend that the overexpression of STAT3 (or p-STAT3) was associated with reduced OS, indicating the expression of STAT3/p-STAT3 plays a prognostic role in Chinese breast cancer patients.
There are still many limitations to our analysis. First of all, the inclusive researches are mainly focused on the patients in China, with insufficient persuasion for more massive ethnic groups. Secondly, the difference of inclusive research quality could also affect the reliability of our analysis. Thirdly, the operation methods and evaluation criteria were different in inclusive researches, bringing the potential indeterminacy.
In conclusion, the occurrence of breast cancer has a close correlation with STAT3/p-STAT3 overexpression and phosphorylation. Also, the STAT3/p-STAT3 expression level in tumor tissue could indicate the deteriorating condition, meaning that STAT3/p-STAT3 could be an important target for various cancers. More studies remain to be undertaken for the target STAT3/p-STAT3 protein.