Mutant p53 and Twist1 Co-Expression Predicts Poor Prognosis and Is an Independent Prognostic Factor in Breast Cancer

Purpose The basic helix-loop-helix transcription factor (bHLH) transcription factor Twist1 plays a key role in embryonic development and tumorigenesis. p53 is a frequently mutated tumor suppressor in cancer. Both proteins play a key and significant role in breast cancer tumorigenesis. However, the regulatory mechanism and clinical significance of their co-expression in this disease remain unclear. The purpose of this study was to analyze the expression patterns of p53 and Twist1 and determine their association with patient prognosis in breast cancer. We also investigated whether their co-expression could be a potential marker for predicting patient prognosis in this disease. Methods Twist1 and mutant p53 expression in 408 breast cancer patient samples were evaluated by immunohistochemistry. Kaplan-Meier Plotter was used to analyze the correlation between co-expression of Twist1 and wild-type or mutant p53 and prognosis for recurrence-free survival (RFS) and overall survival (OS). Univariate analysis, multivariate analysis, and nomograms were used to explore the independent prognostic factors in disease-free survival (DFS) and OS in this cohort. Results Of the 408 patients enrolled, 237 (58%) had high mutant p53 expression. Two-hundred twenty patients (53.9%) stained positive for Twist1, and 188 cases were Twist1-negative. Furthermore, patients that co-expressed Twist1 and mutant p53 (T+P+) had significantly advanced-stage breast cancer [stage III, 61/89 T+P+ (68.5%) vs. 28/89 T-P- (31.5%); stage II, 63/104 T+P+ (60.6%)vs. 41/104 T-P- (39.4%)]. Co-expression was negatively related to early clinical stage (i.e., stages 0 and I; P = 0.039). T+P+ breast cancer patients also had worse DFS (95% CI = 1.217–7.499, P = 0.017) and OS (95% CI = 1.009–9.272, P = 0.048). Elevated Twist1 and mutant p53 expression predicted shorter RFS in basal-like patients. Univariate and multivariate analysis identified three variables (i.e., lymph node involvement, larger tumor, and T+P+) as independent prognostic factors for DFS. Lymph node involvement and T+P+ were also independent factors for OS in this cohort. The total risk scores and nomograms were reliable for predicting DFS and OS in breast cancer patients. Conclusions Our results revealed that co-expression of mutant p53 and Twist1 was associated with advanced clinical stage, triple negative breast cancer (TNBC) subtype, distant metastasis, and shorter DFS and OS in breast cancer patients. Furthermore, lymph nodes status and co-expression of Twist1 and mutant p53 were classified as independent factors for DFS and OS in this cohort. Co-evaluation of mutant p53 and Twist1 might be an appropriate tool for predicting breast cancer patient outcome.


INTRODUCTION
Breast cancer (BC) is a heterogeneous disease that is classified into different subtypes depending on the expression of the estrogen and progesterone receptors and the amplification of human epidermal growth factor receptor 2 (HER2/ERBB2) (1). In particular, molecular profiling has divided BC into five subtypes: luminal A, luminal B, HER2+, and basal-like and triple-negative breast cancer (TNBC) (2). There are no effective targeted drugs for TNBC due to the lack of estrogen receptor (ERa) or HER2 overexpression. TNBC is one of the most heterogeneous breast cancer subtypes, accounting for 15-20% of all breast cancer. It has poor clinical and pathological features and is considered more aggressive than other BC subtypes, such as luminal A and luminal B (3). Therefore, it is important to identify the molecules and signaling pathways related to TNBC progression. Recent studies demonstrated that the epithelial-tomesenchymal transition (EMT) process promotes tumor invasion and metastasis in different solid tumor types (4)(5)(6). Thus, it is important to identify the transcription factors involved in EMT of TNBC progression (7). EMT is a process characterized by the loss of cell-to-cell adhesion and tight cell junctions, and increased motility and invasion. Twist1 is a major regulator of EMT in breast cancer (8). We previously revealed that Twist1 was overexpressed in breast cancers with higher node status and clinical stage and positively associated with EMT in breast cancer (9).
Mutation of the tumor suppressor gene p53 is found in various cancers, including breast, melanoma, bladder, and colon, particularly in tumors resistant to endocrine therapy or radiotherapy (10)(11)(12)(13). Altered p53 protein is prevalently associated with TNBC, and loss of p53 function has been linked to the induction of EMT. Recently, several studies demonstrated that p53-regulated EMT is a critical process in cancer metastasis and chemotherapy resistance. Chang et al. (14) showed that p53 regulates EMT by activating the microRNA miR-200c. Kim et al. (15) demonstrated that p53 loss-of-function or mutation promotes EMT by derepressing Snail. Most noteworthy, van Nes et al. (16) found that Twist1 overexpression is correlated with invasive lobular carcinoma, which is consistent with p53 changes in breast cancer and establishes a mechanistic link between Twist1, p53, and tumor progression. However, studies on the relationship between mutant p53 and Twist1 and their combined role in determining the prognosis and survival of breast cancer patients are scarce. In the present study, we investigated the clinical significance and the combined detection value of mutant p53 and Twist1 in breast cancer patients. We report that mutant p53 and Twist1 coexpression could represent a feasible and effective marker for predicting patient prognosis and survival in breast cancer.

Patients
A total of 408 breast cancer patients treated at the Cancer Hospital of Shantou University Medical College (SUMC) between 2011 and 2013 were enrolled in this study. None of the patients received any treatment before surgery. Clinicopathological data, including age, menopausal status, tumor size, nodal status, TNM stage, histology, and morphology, were collected. All samples were fixed in buffered formalin and embedded in paraffin for immunohistochemistry (IHC). The DFS and the OS of the patients were quantified. This study was approved by the Medical Ethics Committee of the Cancer Hospital of Shantou University Medical College.

Kaplan-Meier Analysis
The free online clinical database, Kaplan-Meier Plotter (http:// kmplot.com), was used to analyze the correlation between Twist1 co-expression with wild-type or mutant p53 and prognosis for recurrence-free survival in patients suffering from breast cancer.

Statistical Analysis
All data were analyzed using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL). The counting data are expressed as the rate using Pearson's c 2 test. Spearman was used for correlation analysis. Survival curves were assessed using the Kaplan-Meier method, and the data were compared using the log-rank test. Univariate and multivariate analyses were used to determine the impact of the variables on patient survival. Two-sided P-values < 0.05 were considered statistically significant.
As shown in Table 4, patients with T+P+ had higher Ki67, Ecadherin, and VEGF-C expression than the T-P-group (P < 0.001). There were no statistically significant differences between the T+P-and T-P+ groups in terms of age, menopausal status, tumor size or clinical stage.

Univariate and Multivariate Survival Analysis for Predicting DFS and OS
We next assessed the prognostic value of Twist1 with or without p53 mutation in breast cancer using the Kaplan-Meier plotter. The results demonstrated that elevated Twist1 expression combined with mutant p53 predicted shorter RFS for basal-like breast cancer patients (P = 0.027) ( Figure 2G); however, there was no difference for the OS (P = 0.18) ( Figure 3G). There were no statistical differences in RFS and OS for the other groups of patients (Figures 2 and 3).
Importantly, the multivariate analysis demonstrated that the T+P+ group had a worse DFS (95% CI = 1.217-7.499, P = 0.017) and OS (95% CI = 1.009-9.272, P = 0.048) than the T-P-group, but the T+P-and T-P+ groups were not statistically different from the T-P-group. Univariate and multivariate analysis identified three variables (high node stage, larger tumor size, and Twist1 and mutant p53 co-expression) as independent risk factors for DFS and node involvement and T+P+ as independent risk factors for OS in breast cancer. Therefore, total risk scores and nomograms were considered suitable for predicting the DFS and OS of breast cancer patients ( Figure 5).

DISCUSSION
Twist1 is a master transcription factor that induces migration and invasion and promotes EMT in various cancer cells (20)(21)(22). Wild-type p53 is a transcription factor that promotes cell cycle arrest, DNA repair, cellular senescence, and apoptosis (23). p53 inhibits cancer cell proliferation and metastasis; however, mutant TP53 has a transforming function. Previous studies have demonstrated that mutant p53 is overexpressed and accumulates to high levels in cancer cells, and is highly correlated with EMT (24). Moreover, Twist1 overexpression can inhibit oncogene-induced premature senescence by blocking key regulators of the p53 and Rb-dependent pathways (25). Numerous studies have shown that there is a regulatory relationship between Twist1 and p53. For instance, Shiota et al. (26) suggested that Twist1 suppresses the DNA-binding activity of p53. Moreover, Kogan-Sakin et al. (27) revealed that Twist1 might be upregulated following p53 mutation in cancer cells.     However, there are no reports on the combined detection of Twist1 and mutant p53 at the protein level and its prognostic impact on breast cancer patients. We analyzed the expression patterns of mutant p53 and Twist1 in breast cancer to explore the role of these molecular markers in breast carcinogenesis, their influence on prognosis, and clinical practicability in breast cancer. Our study showed that mutant p53 expression was positively associated with invasive ductal carcinoma (IDC). Mutant p53 was highest in the HER2+ breast cancer subtype, followed by the luminal B and TNBC subtypes. Twist1 positivity was significantly higher in TNBC patients. Consistent with previous studies (9, 28), we further confirmed that high Twist1expression was associated with larger tumor size, higher node involvement, and shorter DFS. Most importantly, we found, for the first time, that Twist1 was positively correlated with mutant p53, E-cadherin, and Ki67. The association of mutant p53 with Twist1 in clinical tissues was supported by the work of Yang-Hartwich et al. (29), who showed that wild-type p53 promoted Twist1 protein degradation and inhibited EMT, maintaining the epithelial phenotype. Interestingly, a preclinical study by Li et al. (30) found that Twist1 had noteworthy impacts on p53 and p21 induction, which were similar to our current results. Maestro et al. (31) found that Twist regulated p53 indirectly by modulating the ARF/MDM2/p53 pathway. Similarly, Piccinin et al. (32) found that Twist1 binds to the p53 C terminus through the Twist box to inactivate p53 in mesenchymal tumors. Conversely, Twist promotes the reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells by inhibiting the p53 pathway (33). Taken together, these findings suggest that a mutual regulation mechanism might exist between mutant p53 and Twist1. However, the clinical significance of coexpression of two proteins has not been clearly defined.
In this study, we also investigated the relationship and clinical significance of mutant p53 and Twist1. Our results suggested that co-overexpression of mutant p53 and Twist1 in breast cancer  was significantly associated with larger tumor size, greater lymph node involvement, and more advanced TNM stage. Coexpression of mutant p53 and Twist1 (T+P+ group) was significantly higher in TNBC, followed by HER2-enriched, suggesting the aggressiveness of T+P+ tumors. Previously, Twist 1 overexpression was shown to be associated with poor prognosis in breast cancer (20,34). Similarly, abnormal p53 expression has been widely accepted as a poor prognostic factor in breast cancer (35). Moreover, Sanambar et al. (36) found that p53 mutation detected by immunohistochemistry could effectively predict the prognosis of breast cancer patients. In our study, regression analysis was used to assess the role of mutant p53 by immunohistochemistry. As expected, the patients in the T+P+ group presented the worst DFS and OS by univariate analysis. Furthermore, our results demonstrated that coexpression of Twist1 and mutant p53 (T+P+) is an independent prognostic factor for both DFS and OS in breast cancer patients using multivariate analysis. Similarly, Kaplan-Meier analysis using the Kaplan-Meier plotter database demonstrated that co-expression of Twist1 and mutant p53 predicted shorter RFS in patients with basal-like breast cancer. However, due to the small sample size of the database, we did not find a statistical difference between Twist1 with mutant or wildtype p53 and RFS and OS in other subtypes.
A nomograph was established based on the survival analysis, and risk scores for DFS and OS time were calculated according to the Cox regression coefficient. Internal validation patients were randomly selected from the total population, and the verification confirmed that the risk score was related to DFS and OS time, showing the nomogram's reliability. However, the weakness is the lack of validation queues in our research. Future research should enroll more cases to further verify the nomogram.
This study is the first report showing that co-expression of Twist1 and mutant p53 could be used to evaluate treatment efficacy and prognosis in breast cancer patients. The relationship between the two proteins needs further study, such as the relationship between the specific mutation site of the p53 gene and breast cancer and the interactive regulatory mechanism of mutant p53 and Twist1. Using small molecular inhibitors that inhibit the expression of Twist1 and mutant p53 may be one strategy for targeted treatment of TNBC (37).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Cancer Hospital of Shantou University Medical College. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
Conception and design: G-JZ and X-LW. Administrative support: G-JZ. Provision of study materials or patients: G-JZ. Collection and assembly of data: Y-QZ. Data analysis and interpretation: Y-QZ and FZ. All authors contributed to the article and approved the submitted version.