YWHAZ-Mediated Modulation of Tumor Differentiation Impact on the Long-Term Prognosis in Esophageal Squamous Cell Carcinoma Patients Following Chemoradiotherapy
Yue-chenXu1,2
YingZhang3
Hong-yanWang1
Chief physician
FanWang1✉Emailwangfan1965@126.com1
A
Department of RadiotherapyThe first affiliated hospital of Anhui Medical University230022Hefei China2Institute of Radiation Medicine, School of Basic Medical SciencesAnhui Medical University230032Hefei China
3Department of PathologyThe first affiliated hospital of Anhui Medical University230022Hefei China
Yue-chen Xu1, 2, Ying Zhang3, Hong-yan Wang1, Fan Wang1
1 Department of Radiotherapy, The first affiliated hospital of Anhui Medical University, Hefei China, 230022.
2 Institute of Radiation Medicine, School of Basic Medical Sciences, Anhui Medical University, Hefei China, 230032.
3 Department of Pathology, The first affiliated hospital of Anhui Medical University, Hefei China, 230022.
ADDRESS FOR CORRESPONDECE:
Corresponding author name:
Fan Wang, Chief physician, Professor, Department of Radiotherapy, The first affiliated hospital of Anhui Medical University, Hefei China, 230022, E-mail: wangfan1965@126.com.
ABSTRACT
Objective
To investigate the correlation between the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) in esophageal squamous cell carcinoma (ESCC) tissues and tumor progression, and to analyze the mediating effect of YWHAZ on the association between the degree of tumor differentiation and prognosis in esophageal cancer patients.
Method
Expression of YWHAZ gene between 33 cancers and normal, overall survival (OS) between groups with low YWHAZ expression and high YWHAZ expression were analyzed by bioinformatic analysis with TCGA-GTEx dataset, comparison of YWHAZ expression between esophageal cancer (ESCA) and normal subjects, correlations between expression of YWHAZ and different cancer stages, survival in patients with ESCA were analyzed by bioinformatic analysis with TCGA-ESCA dataset. Then an immunohistochemical assay was used to detect the expression rate of YWHAZ in 75 cases with ESCC after chemoradiotherapy. Correlation between the expression of YWHAZ and tumor progression, including OS and progression-free survival (PFS), were analyzed using the rank sum test or chi-square test. Cox regression, general linear model (GLM) and mediation analyses were used to analyze the effect of YWHAZ expression on the long-term prognosis of ESCC patients.
Results
Bioinformatic analysis with TCGA-GTEx dataset found expression levels of YWHAZ in BRCA, CESC, COAD, LIHC, LUAD, LUSC, OV, PAAD, READ, STAD, THYM were higher than those in normal subjects (P < 0.05). OS in KIRP, LGG, LIHC, LUAD, PAAD and UVM with high expression of YWHAZ were shorter than those with low expression of YWHAZ(P < 0.05-0.0005), while it was longer in KIRC with high expression of YWHAZ than that with low expression of YWHAZ(P = 0.048). Expression level of YWHAZ was higher in ESCA than that in normal subjects in TCGA data (P < 0.0001), and it was higher in ESCC than that in esophageal adenocarcinoma (P < 0.0001). It was higher in Asian ESCA patients than that in Caucasian and Africa-american ESCA patients (P < 0.0001). There were no differences about expression level of YWHAZ among different cancer stages (P > 0.05). OS and PFS of ESCA in patients with low expression of YWHAZ were the same as patients with high expression of YWHAZ (P > 0.05), but effect of YWHAZ expression level interacted with tumor grade on ESCA patients survival differed from each other (P = 0.023). The expression rate of YWHAZ in carcinoma tissues with ESCC (0.59 vs 0.49) was significantly higher than that in anastomotic stomas (z = 2.692, P = 0.007). High expression of YWHAZ was a risk factor for long-term recurrence/disease progression (HR = 3.166, 95% CI: 1.623–6.176, P = 0.001) and death (HR = 6.321, 95% CI: 2.925–13.658, P < 0.001) in patients. GLM analysis found expression of YWHAZ interacted with tumor differentiation grade negatively correlated with OS in ESCC (B=-6.058, 95%CI:-9.233~-2.893, x2 = 14.076, P < 0.001). YWHAZ expression served as a mediator of the association between the grade of tumor differentiation and OS or PFS in ESCC patients (P < 0.0001). The indirect effect (mediating role) of YWHAZ expression accounted for 62.15% of the total effect of the grade of tumor differentiation on OS, and accounted for 47.39% of the total effect of the grade of tumor differentiation on PFS.
Conclusion
High expression of YWHAZ, which mediates the grade of tumor differentiation, is a risk factor for long-term recurrence/disease progression and death in patients with ESCC after chemoradiotherapy.
Key words:
esophageal squamous cell carcinoma
YWHAZ
tumor differentiation grade
prognosis
OS
and PFS
A
A
1 Background
The 14-3-3 protein family is a group of proteins present in all eukaryotes, containing seven subtypes of β, ε, γ, η, ζ, σ, and θ, including 153 isomers of 48 classes with molecular weights of about 25-30KD[1]. All of these do exist and function in some form in the human body[2]. The biological functions of 14-3-3 proteins have always been the focus of many studies. Studies have found that 14-3-3 proteins play an important role in many processes, such as inflammation, cell cycle regulation, cell migration, cell differentiation, and apoptosis[3]. 14-3-3 proteins, often expressed in tumor tissues, and also have been found to be closely related to tumors, which act as either oncogenes[4] or tumor suppressor genes[5, 6]. By binding to various ligands, they regulate the carcinogenesis and development of tumors[7–9] and even affect their therapeutic effect in cancer patients[10]. Recent studies have found that 14-3-3ζ protein, which, according to the HUGO gene nomenclature committee (HGNC), also known as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), acts as an oncogene to promote tumor development, and its high expression promotes the occurrence of tumors[11, 12], and even leads to tumor metastasis and chemotherapy resistance[13].
Esophageal squamous cell carcinoma (ESCC), one of the major causes of cancer related death worldwide[14], was also associated with YWHAZ[15]. Studies reported elevated expression of YWHAZ in ESCC, and the expression level of YWHAZ was positively correlated with lymph node metastasis and recurrence of ESCC[16]. It was also found that downregulation of YWHAZ signaling could inhibit the proliferation and migration of ESCC cells[17], and some studies proposed that YWHAZ might be a better target for anti-cancer therapy in the near future[18, 19]. Therefore, YWHAZ may play an important role in ESCC.
With the gradual increasing interest in oncology research, the grade of tumor differentiation has become a recognized risk factor affecting carcinogenesis and tumor development, which is closely related to prognosis[20]. Poor efficacy caused by chemotherapy and radiation resistance is also a major problem in the treatment of ESCC [21, 22]. Whether the close relationship between YWHAZ and ESCC progression is related to the grade of tumor differentiation, and whether the expression of YWHAZ influences the prognosis of ESCC patients is not yet fully elucidated. Therefore, it is of urgent practical significance to explore the correlation between YWHAZ and the ESCC prognosis. YWHAZ may be an important indicator for evaluating the long-term prognosis of ESCC patients and for optimizing the treatment regimen .
2 Materials and methods
2.1 Bioinfromatic analysis
Bioinformatic analysis with TCGA-GTEx and TCGA (https://xenabrowser.net/datapages/?host=https%3A%2F%2Ftoil.xenahubs.net) dataset were used for comparing expression of YWHAZ gene between cancers and normal subjects, exploring correlations between expression of YWHAZ and different cancer stages, survival in patients with TCGA-ESCA (https://ualcan.path.uab.edu/cgi-bin/TCGA, 184 cases with ESCA including 95 cases with ESCC and 89 cases with esophageal adenocarcinoma, 11 normal subjects).
2.2 General Information
A
Seventy-five ESCC patients including 63 males and 12 females admitted to the First Affiliated Hospital of Anhui Medical University between May 2017 and March 2018 were retrospectively recruited. All patients were diagnosed with ESCC by pathological testing after surgery, and all received chemoradiotherapy after surgery. The age range varied from 44 to 82 yrs, and the median age was 64 yrs. Body mass index (BMI) ranged from 14.57 to 27.68 kg/m2 and the median BMI was 19.21 kg/m2. Tumour, Node, Metastasis (TNM) staging showed that there were two cases (2.7%) in stage 1, 41 cases (54.7%) in stage 2, 29 cases (38.6%) in stage 3, and three cases (4%) in stage 4. According to the grade of tumor differentiation, patients were divided into three groups: 4 cases (5.3%) with high differentiation (G1), 51 cases (68.0%) with medium differentiation (G2), and 20 cases (26.7%) with low differentiation (G3). The general clinical and pathological data of ESCC patients was shown in Table 1. A
This study was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. P2021-10-22).Indicators | Total | Male | Female | P |
|---|---|---|---|---|
Age(years) | 64.00 (57.00-71.50) | 64.00(56.25-70.00) | 67.50(61.50–73.00) | 0.281 |
BMI(kg/m2) | 19.21 (18.26–21.25) | 19.03(18.12–20.93) | 20.62(18.93–22.92) | 0.076 |
Tumor lump length (cm) | 4.00 (3.00-4.50) | 4.00(3.00-4.65) | 3.25(3.00–4.00) | 0.144 |
Tumor lump width (cm) | 2.50 (2.00–3.00) | 2.65(2.00-3.15) | 2.00(2.00-2.50) | 0.242 |
Tumor lump height (cm) | 1.00 (0.80–1.48) | 1.00(1.00-1.50) | 0.75(0.53–1.23) | 0.011 |
Metastatic lymph nodes | 0.00 (0.00–2.00) | 0.50(0.00-2.75) | 0.00 (0.00–2.00) | 0.348 |
TNM staging (1/2/3/4) | 2(2.7)/41(54.7)/29(38.6)/3(4.0) | 2(3.2)/31(49.2)/27(42.8)/3(4.8) | 0(0.0)/10(83.3)/2(16.7)/0(0.0) | 0.214 |
Differentiation(G1/G2/G3) | 4(5.3)/51(68.0)/20 (26.7) | 4(6.3)/44(69.8)/15(23.9) | 0(0.0)/7(58.3)/5(41.7) | 0.371 |
Recurrence / Metastasis (yes/not) | 47(62.7)/28(37.3) | 41(65.1)/22(34.9) | 6(50.0)/6(50.0) | 0.507 |
Death (yes/not) | 54(72.0)/21(28.0) | 45(71.4)/18(28.6) | 9(75.0)/3(25.0) | 1.000 |
2.3 Treatment Options
1.
2.3.1 Chemotherapy regimen: The combination chemotherapy regimen of cisplatin and fluorouracil was adopted, fluorouracil 500 mg/m2 intravenous infusion on day 1–5, cisplatin 20 mg/m2 intravenous infusion on days 1–5, and 3 weeks for 1 cycle of chemotherapy, for a total of 4 cycles. During chemotherapy, systemic adverse reactions were carefully observed, and appropriate symptomatic treatment such as antiemetic, gastric protection, and increasing leucocytes were administered in time.
2.
2.3.2 Radiotherapy regimen: 6MV-X-ray treatment was adopted, with a single dose of 2 Gy, 5 times a week, and a total dose of (50–60) Gy. During radiotherapy, adverse reactions were carefully observed and appropriate symptomatic treatment was provided.
2.4 Pathological specimen collection and detection
2.4.1 Pathological specimens: 75 cases of cancer tissue (4 slices per case, a total of 300 slices), 44 cases of anastomotic stoma (4 slices per case, a total of 176 slices), and a total of 476 slices were stained, and immunohistochemical experiments were performed.
2.4.2 Result interpretation:
Interpretation criteria for staining results: YWHAZ was mainly expressed in the cytoplasm, and fine particles staining with yellow or brownish yellow were defined as positive cells. The percentage of positive cells in all cells throughout the field of view was calculated as the expression rate. Three different fields of view were observed in each slice, and the results were averaged.
2.4.3 Instruments and experimental reagents: The immunohistochemical instrument used was a conventional microtome, and microscope from the First Affiliated Hospital of Anhui Medical University. The YWHAZ kit (Catalog No: 67698-1-Ig) were purchased from Wuhan Sanying Biotechnology Co., Ltd.
2.5 Patient follow-up
Overall survival (OS) and progress free survival (PFS) of the patients were recorded via telephone follow-up. OS was from postoperative period to death or the last follow-up, and PFS was from postoperative period to disease progression.
2.6 Statistical methods
SPSS software (version 26.0) and R version 4.0.4 were used for the statistical analysis. Online database were used for bioinformatic analyses. Rank sum tests were used to compare measurement data, which were presented as M(P25-P75) and the chi-square test was used to compare categorical data, which were presented as incidence or percentage between groups. The intercept point value was calculated using ROC curve, and the correlation between the measurement data was analyzed using linear correlation. Cox regression and general linear model (GLM) were used to analyze the risk factors affecting the long-term prognosis of ESCC patients, and the mediating effect of YWHAZ on the grade of tumor differentiation affecting the long-term prognosis of ESCC patients was analyzed by intermediary analysis (Process method). The P-value was < 0.05 to consider statistically significant.
3 Results
3.1 Exploration of YWHAZ expression in cancers and correlations with tumor stage and survival with TCGA-GTEx and TCGA dataset by Bioinformatic analysis
Results of bioinformatic analysis with TCGA-GTEx dataset showed expression levels of YWHAZ in BRCA, CESC, COAD, LIHC, LUAD, LUSC, OV, PAAD, READ, STAD, THYM were higher than those in normal subjects (P < 0.05)(Fig. 1). OS in KIRP, LGG, LIHC, LUAD, PAAD and UVM with high expression of YWHAZ were shorter than those with low expression of YWHAZ (P < 0.05-0.0005), while it was longer in KIRC with high expression of YWHAZ than that with low expression of YWHAZ(P = 0.048)(Fig. 2). Expression level of YWHAZ was higher in ESCA than that in normal subjects in TCGA data (P < 0.0001), and it was higher in ESCC than that in esophageal adenocarcinoma (P < 0.0001). It was higher in Asian ESCA patients than that in Caucasian and Africa-american ESCA patients (P < 0.0001). Although expression levels of YWHAZ in ESCA with different stages (stage1, stage2, stage3, stage4) were all higher than that in normal subjects (P < 0.0001), there were no differences about expression level of YWHAZ among different cancer stages (P > 0.05). OS and PFS of ESCA in patients with low expression of YWHAZ were the same as patients with high expression of YWHAZ (P > 0.05), but effect of YWHAZ expression level & tumor grade on ESCA patients survival differed from each other (P = 0.023) (Fig. 3).
Fig. 1
Dot plot of the YWHAZ gene expression profile across 33 tumor samples and paired normal tissues
We use log2(TPM + 1) for log-scale with matched TCGA normal and GTEx data. Each dots represent expression of samples. T, tumor; N, normal. * P < 0.05.
ACC, Adrenocortical carcinoma; BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, Cholangio carcinoma; COAD, Colon adenocarcinoma; DLBC, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LAML, Acute Myeloid Leukemia; LGG, Brain Lower Grade Glioma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; MESO, Mesothelioma; OV, Ovarian serous cystadenocarcinoma; PAAD, Pancreatic adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; STAD, Stomach adenocarcinoma; TGCT, Testicular Germ Cell Tumors; THCA, Thyroid carcinoma; THYM, Thymoma; UCEC, Uterine Corpus Endometrial Carcinoma; UCS, Uterine Carcinosarcoma; UVM, Uveal Melanoma.
Fig. 2
Overall survival between groups with low YWHAZ expression and high YWHAZ expression in 7 cancers
A-G, Overall survival analysis between groups with low YWHAZ expression and high YWHAZ expression in KIRC, KIRP, LGG, LIHC, LUAD, PAAD, UVM. HR, hazard ratio.
KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LGG, Brain Lower Grade Glioma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; PAAD, Pancreatic adenocarcinoma; UVM, Uveal Melanoma.
Fig. 3
YWHAZ expression in ESCA and correlations with tumor stage and survival with TCGA-ESCA dataset
A, Comparison of YWHAZ expression levels between ESCA and normal; B, Comparison of YWHAZ expression levels among ESCA, esophageal adenocarcinoma and normal; C, Comparison of YWHAZ expression levels in ESCA among Asian population, Africa-american populations and Caucasian populations; D, Expression levels of YWHAZ in ESCA with different stages (stage1, stage2, stage3, stage4) and normal; E, OS of ESCA with low expression of YWHAZ and high expression of YWHAZ; F, PFS of ESCA with low expression of YWHAZ and high expression of YWHAZ; G, Effect of YWHAZ expression level & tumor grade on ESCA patients survival. ESCA, esophageal cancer; ESCC, esophageal squamous cell carcinoma. TCGA, the cancer genome atlas.
3.2 Comparison of YWHAZ expression between esophageal cancer tissue and anastomotic stoma
The expression rate of YWHAZ in ESCC tissue [0.59(0.51–0.73)] was significantly higher than that in anastomotic stoma [0.49(0.31–0.68)] (z = 2.692, P = 0.007).
3.3 Correlation between YWHAZ expression in ESCC tissues with grade of tumor differentiation and clinical stage
3.3.1 Calculation of the cut-off value of YWHAZ expression rate
Defining the YWHAZ expression rate as the test variable and group as the state variable (0 = anastomotic stoma, 1 = cancer tissue), the results of receiver operating characteristic (ROC) curve showed that the cut-off value of the YWHAZ expression rate was 0.548 (AUC = 0.648, p = 0.007; sensitivity was 62.7%, specificity was 70.5%). Therefore, in this study, a YWHAZ expression rate > 0.548 was defined as the high YWHAZ expression group, and a YWHAZ expression rate ≤ 0.548 was defined as the low-normal expression group.
3.3.2 Comparison of YWHAZ expression among different tumor differentiation grade
The results of the chi-square test showed that there were significant differences in the percentage of high expression rate of YWHAZ among groups with different grades of tumor differentiation (high differentiation-G1: 0/4, 0%; medium differentiation-G2: 30/51, 58.8%; low differentiation-G3: 17/20, 85.0%; x2 = 10.809, P = 0.002), and the worse the grade of tumor differentiation, the higher the percentage of high expression rate of YWHAZ (rs=0.717, P = 0.001). The results of the rank sum test showed that the expression rate of YWHAZ among the groups with different grades of differentiation was clearly different (z = 15.284, P < 0.001), as shown in Fig. 4.
Fig. 4
Comparison of YWHAZ expression among groups with different tumor differentiation grade
G1, high differentiation; G2, medium differentiation; G3, low differentiation.
3.3.3 Association of YWHAZ expression with characteristics of tumor
Chi-square test showed that the proportions of YWHAZ expression among groups with different TNM stages (T stage, N stage, and M stage) were not significantly different (P > 0.05). Non-parametric tests indicated that the width of tumor lump in the YWHAZ high-expression group was significantly higher than the low-normal expression group (P = 0.003), and the length and height of the tumor lump and the number of lymph node metastases between the two groups were the same (all P > 0.05), as shown in Table 2. Correlation analysis revealed a significant positive correlation between YWHAZ expression rate and width of tumor lump (r = 0.324, P = 0.005), but there were no significant correlations between YWHAZ expression rate and length (r = 0.145, P = 0.216) or height (r=-0.183, P = 0.115) of the tumor lump. There was also a significant positive correlation between the YWHAZ expression rate and the number of metastatic lymph nodes (r = 0.287, P = 0.012).
Staging | Low-normal expression of YWHAZ (n = 28) | High expression of YWHAZ (n = 47) | x2/z | P | |
|---|---|---|---|---|---|
TNM | 1 | 1 (50.0) | 1 (50.0) | 1.373 | 0.819 |
2 | 17 (41.5) | 24 (58.5) | |||
3 | 9 (31.0) | 20 (69.0) | |||
4 | 1 (33.3) | 2 (66.7) | |||
T | T1 | 1 (33.3) | 2 (66.7) | 2.850 | 0.398 |
T2 | 2 (16.7) | 10 (83.3) | |||
T3 | 23 (41.8) | 32 (58.2) | |||
T4 | 2 (40.0) | 3 (60.0) | |||
N | N0 | 17 (44.7) | 21 (55.3) | 2.370 | 0.502 |
N1 | 6 (28.6) | 15 (71.4) | |||
N2 | 5 (35.7) | 9 (64.3) | |||
N3 | 0 (0.0) | 2 (100.0) | |||
M | M0 | 27 (37.5) | 45 (62.5) | 0.000 | 1.000 |
M1 | 1 (33.3) | 2 (66.7) | |||
Tumor lump (cm) | length | 3.7(3.0-4.5) | 4.0(3.0-5.5) | 1.754 | 0.079 |
width | 2.0(2.0-2.5) | 3.0(2.0–4.0) | 3.009 | 0.003 | |
height | 1.1(1.0-1.5) | 1.0(0.8–1.5) | 0.584 | 0.559 | |
Number of Metastatic lymph nodes | 0 (0–1.0) | 1.0 (0–3.0) | 1.346 | 0.178 | |
3.4 Effect of expression of YWHAZ on long-term prognosis of patients with ESCC
3.4.1 Comparison of long-term survival between different YWHAZ groups
The 1-year, 3-year and 5-year survival rates in the 75 patients with esophageal cancer were 77.3% (58/75), 53.3% (40/75), and 25.0% (18/72), respectively. The 1-year survival rates (100.0% vs 63.8%), 3-year survival rates (96.4% vs 27.7%), and 5-year survival rates (56.0% vs 8.5%) were significantly different between groups with low normal expression and high YWHAZ expression (P < 0.001), as shown in Table 3. OS in patients with low-normal expression of YWHAZ [4.8(4.2–5.5)] (yrs) was significantly longer than that in patients with high expression of YWHAZ [1.5(0.8-4.0)] (yrs) (z = 5.663, P < 0.001). There was a significant negative correlation between the YWHAZ expression rate and OS (r=-0.843, P < 0.001).
Follow-up time | Low-normal expression of YWHAZ (n = 28) | High expression of YWHAZ (n = 47) | x2 | P | |
|---|---|---|---|---|---|
1-year | Survival | 28(100.0) | 30(63.8) | 13.096 | <0.001 |
Death | 0(0.0) | 17(36.2) | |||
3-year | Survival | 27(96.4) | 13(27.7) | 33.341 | <0.001 |
Death | 1(3.6) | 34(72.3) | |||
5-year | Survival | 14(56.0) | 4(8.5) | 19.629 | <0.001 |
Death | 11(44.0) | 43(91.5) | |||
3.4.2 Survival analysis between groups with different expression of YWHAZ and groups with different grade of tumor differentiation (univariate Cox regression analysis)
Defining OS as the time variable and death (0 = no, 1 = yes) as the state variable, the results of univariate Cox regression showed that the cumulative survival rate of patients with high YWHAZ expression was significantly lower than that in the low-normal expressing group (HR = 7.259, P < 0.001), as shown in Fig. 5A. The cumulative survival rate among high differentiation, medium differentiation, and low differentiation differed from each other (P < 0.001), it was significantly lower in the low differentiation group than that in the high differentiation group (HR = 17.671, P = 0.006), as shown in Fig. 5B. After defining YWHAZ expression (1 = high expression of YWHAZ, 0 = low-normal expression of YWHAZ) and grade of tumor differentiation as covariables, the results of multivariate Cox regression showed that high expression of YWHAZ was still a risk factor for long-term prognosis (death) in patients with ESCC (HR = 6.321, 95% CI: 2.925–13.658, P < 0.001).
Fig. 5
Cox survival analysis between groups with low-normal expression and high expression of YWHAZ or different grades of tumor differentiation in ESCC
A: Cox survival analysis between groups with low-normal expression and high expression of YWHAZ in ESCC; B: Cox survival analysis between groups with different grades of tumor differentiation in ESCC; ESCC, esophageal squamous cell carcinoma.
3.5 Effect of YWHAZ expression in ESCC on long-term recurrence/metastasis after radiotherapy and chemotherapy
3.5.1 Comparison of recurrence or metastasis rates between groups with different YWHAZ expression
Recurrence/metastasis rates in patients with high YWHAZ expression (66.0%, 31/47) were not significantly different from those in patients with low-normal YWHAZ expression (57.1%, 16/28) (P > 0.05). PFS of patients with ESCC in high expression of YWHAZ group [1.20(0.60-3.00)] (year) was significantly lower than that in low-normal expression of YWHAZ group [4.10(3.00–5.00)] (year) (z = 5.308, P < 0.001). Correlation analysis showed that there was a significant negative correlation between the YWHAZ expression rate and PFS (r=-0.765, P < 0.001).
3.5.2 Multivariate Cox regression analysis of PFS in patients with ESCC
Defining PFS as the time variable, recurrence/metastasis as the state variable (0 = no, 1 = yes), and YWHAZ expression (1 = high expression of YWHAZ, 0 = low-normal expression of YWHAZ), degree of tumor differentiation (0 = medium-high differentiation, 1 = low differentiation), TNM stage, age, sex, and BMI as covariables, results of multivariate Cox regression indicated that high expression of YWHAZ (HR = 3.166, 95% CI: 1.623–6.176, P = 0.001), and low tumor differentiation (HR = 3.450, 95% CI: 1.451–8.205, P = 0.005) were risk factors for disease progression.
3.6 Mediating role of YWHAZ on the association of tumor differentiated grade with long-term prognosis in patients with ESCC
Defining OS (0 = live, 1 = death) as the dependent variable, age, sex, TNM stages, grade of tumor differentiation and expression of YWHAZ as independent variables, results of GLM showed YWHAZ expression level interacted with tumor differentiation grade negatively correlated with OS in ESCC (B=-6.058, 95%CI:-9.233~-2.893, x2 = 14.076, P < 0.001) (Fig. 6). Defining grade of tumor differentiation as an independent variable, OS or PFS as the dependent variable, YWHAZ expression as the mediator, and results of Process v4.0 (Bootstrap method) showed that YWHAZ expression served as a mediator of the association between the grade of tumor differentiation and OS in patients with ESCC (P < 0.001). The indirect effect (mediating role) of YWHAZ expression on the grade of tumor differentiation on OS was − 1.1378 (-1.5622 ~ -0.7023), accounting for 62.15% (-1.1378/-1.8306) of the total effect of the grade of tumor differentiation on OS. YWHAZ expression also served as a mediator of the association between the grade of tumor differentiation and PFS in patients with ESCC (p < 0.001). The indirect effect (mediating role) of YWHAZ expression on the grade of tumor differentiation on PFS was − 0.8630 (-1.2651 ~ -0.4957), accounting for 47.39% (-0.8630/-1.8788) of the total effect of the grade of tumor differentiation on PFS.
Fig. 6
Nomogram of risks for OS with expression of YWHAZ in ESCC (GLM)
BMI, body mass index; ESCC, esophageal squamous cell carcinoma; GLM, general linear model; OS, overall survival; Pr, probability.
4. Discussion
Bioinformatic analysis with TCGA-GTEx dataset found expression levels of YWHAZ in some kinds of caners were higher than those in normal subjects. OS in only 6 cancers correlated with expression of YWHAZ. Although there are few studies on the role of YWHAZ in ESCA, results of bioinformatic analysis showed expression levels of YWHAZ in TCGA-ESCC and esophageal adenocarcinoma were all higher than that in normal subjects(P < 0.0001), and it was higher in ESCC than that in esophageal adenocarcinoma(P < 0.0001). Expression level of YWHAZ in ESCA in Asian population was higher than that in Africa-american populations (P < 0.0001) or Caucasian populations (P < 0.0001). Results of this study which were the same as those with bioinformatic analysis in TCGA data also indicated that the expression of YWHAZ in ESCC was significantly overexpressed in the secretome derived from ESCC cell lines compared to normal cell-derived secretome[23]. Immunohistochemical analysis of 77 pairs of ESCC tissues by Tong et al [16] revealed that YWHAZ was highly expressed in ESCC (P < 0.05), and Cox regression analysis showed that YWHAZ was a risk factor for disease progression in ESCC patients (HR = 1.89, P = 0.030). The vector containing YWHAZ was transfected into ESCC cells for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression of Sphingosine-1-phosphatereceptor 2 (S1PR2) was decreased, and there was a significant negative correlation between S1PR2 (r=-0.233, P = 0.031) and YWHAZ. Tumor cell proliferation and migration were significantly accelerated compared to those without YWHAZ treatment.. These results suggest that YWHAZ may promote the growth and metastasis of ESCC by inhibiting S1PR2 signal transduction. Similar results were reported in a study on YWHAZ in lung cancer. Immunohistochemical analysis by Deng et al,[24] showed that the expression of YWHAZ in non-small cell lung cancer (NSCLC) was significantly elevated compared to normal lung tissue (P < 0.001). Upon downregulation of YWHAZ in NSCLC cell lines, the migration (P < 0.001) and invasion (P < 0.001) abilities of lung cancer cells attenuated. In conclusion, the high expression of YWHAZ in NSCLC promotes tumor progression. In our study, it was also found that the expression rate of YWHAZ was higher in ESCC tissues (P = 0.59) than that in the normal tissues (0.49) (P = 0.007). The YWHAZ expression increased with the grade of tumor differentiation (rs=0.717, P = 0.001). The expression rate of YWHAZ was also positively correlated with the lump width of ESCC tissues and the number of metastatic lymph nodes. These results were in concordance with those of Tong et al., which suggested the expression level of YWHAZ not only served as a biomarker protein in ESCC tissue, but also suggested the higher the expression level of YWHAZ was, the worse the differentiation grade was and the worse the progression stage of cancer was.
Many researchers working in the field of ESCC have hoped to find biomarkers that could predict the long-term survival of patients. Zhong et al[25] found that keratin pearls (KP) was important indicators for the degree of differentiation of ESCC. In this study, 3,069 patients with high KP and 7,144 patients with low KP were enrolled in the Kaplan-Meier survival analysis. Patients with high KP had significantly longer OS than those patients with low KP, and KP was a protective factor for long-term death in ESCC patients (HR = 0.872, P < 0.001). They found that the 3-yr, 5-yr and 10-yr survival predicted by KP levels were in good concordance with the actual observed values, indicating that KP could be used to predict the prognosis of ESCC patients. There was not even a single study in the literature on the correlation between YWHAZ and the prognosis of ESCC patients. However, a previous study by Deng et al,[24] showed that the expression of YWHAZ was significantly negatively correlated with OS in NSCLC patients (P < 0.001). Further, Cox regression analysis also suggested that high expression of YWHAZ was a risk factor for death in NSCLC patients (univariable: HR = 2.374, P < 0.001; multivariable: HR = 1.748, P = 0.012). In addition, the results from Liu et al. [26] and Qin et al. [27] also suggested that YWHAZ could be used as a biomarker to diagnose and evaluate the prognosis of the gastric cancer patients. Although there were no differences about expression level of YWHAZ among different cancer stages (stage1, stage2, stage3, stage4) (P > 0.05) and prognosis (OS and PFS) of in ESCA between low expression of YWHAZ and high expression of YWHAZ (P > 0.05) by bioinformatic analysis (P > 0.05). In our study, we found that YWHAZ expression was significantly correlated with the prognosis of ESCC patients. The 1-yr (100%), 3-year (96.4%), and 5-year (56.0%) survival rates of the patients in the low YWHAZ expression group were significantly higher than those in the high YWHAZ expression group (63.8%, 27.7%, and 8.5%, respectively) (P < 0.001). Furthermore, the OS of patients in the low YWHAZ expression group (4.8 yrs) was significantly longer than that in the high YWHAZ expression group (1.5 yrs). High expression of YWHAZ was an independent risk factor for survival in ESCC patients (HR = 7.259, P < 0.001), with a 6-fold increased risk of death in patients with high expression of YWHAZ compared to those with low YWHAZ expression. YWHAZ expression was not only able to influence the OS of ESCC patients but also the recurrence and metastasis of ESCC. Further analysis showed a significant negative correlation between the YWHAZ expression rate and PFS (r=-0.765, P < 0.001). The PFS of patients with low YWHAZ expression (4.1 yrs) was significantly longer than that of patients with high YWHAZ expression (1.2 yrs) (P < 0.001). High expression of YWHAZ was also a risk factor for recurrence or metastasis in the ESCC patients (HR = 3.166, P = 0.005), with a 2-fold increased risk. In conclusion, YWHAZ expression was significantly correlated with survival, recurrence, and metastasis of ESCC patients, and this could be used as an indicator to predict the prognosis of ESCC patients.
Expression of YWHAZ was significantly correlated with ESCC progression, and prognosis in ESCC patients. However, these findings were not able to establish whether it directly or indirectly affects the prognosis of ESCC patients. It is well known that the grade of tumor differentiation is an important indicator for measuring the invasiveness of the malignant degree of tumors and also directly impacts the prognosis of tumor patients[28]. Whether the effect of high YWHAZ expression on the prognosis of ESCC patients was associated with the grade of tumor differentiation remains unclear. In our study, YWHAZ expression level interacted with tumor differentiation grade negatively correlated with OS in ESCC by GLM analysis, furthermore results of Process analysis (Bootstrap method) showed that the expression of YWHAZ mediated the effect of tumor differentiation grade on the prognosis of ESCC patients as an indirect risk factor, accounting for 62.15% and 47.39% of the total effect of tumor differentiation on OS and PFS, respectively (P < 0.001). Results of bioinformatic analysis about effect of YWHAZ expression level & tumor grade on ESCA patients survival which was insistent with this study also revealed survival probability in ESCA with high expression of YWHAZ plus G3 was the lowest while it was the highest in ESCA with low/medium expression of YWHAZ plus G1(P = 0.023), suggesting expression of YWHAZ interacted with tumor differentiation grade and affected the prognosis of ESCA patients. In conclusion, YWHAZ plays a mediating role in the prognosis of ESCC patients.
A
In the recent years, the mechanism by which YWHAZ is involved in the development of ESCC has been investigated. Zhang et al.[17] transfected the vector expressing d PR (PRDI-BF1 and RIZ) domain protein 5 (PRDM5) into ESCC cells, and found that the expression of both YWHAZ and protein kinase B (Akt) was decreased. Also, there was a significant attenuation observed in ESCC cells in terms of cellular proliferation and migration. In contrast, after knockdown of PRDM5 in ESCC cells, the expression of YWHAZ/Akt increased, and cell proliferation and migration were accelerated, suggesting that PRDM5 inhibited these biological processes via the YWHAZ/Akt signaling pathway. This also indicated that the high expression of YWHAZ could promote the progression of ESCC. Studies on the mechanism of action of YWHAZ in other tumors have found similar results. Chen et al[29] determined the CircCDK17, miR-1294, and YWHAZ expression in cervical cancer tissues using qRT-PCR and Western Blot techniques. Upon silencing CircCDK17 expression in cervical cancer cells, the expression of miR-1294 and YWHAZ decreased. In addition, the ability of cells to migrate and invade was inhibited and apoptosis was accelerated. Therefore, the low expression of CircCDK17 may promote the apoptosis of tumor cells by inhibiting the miR-1294/YWHAZ signaling pathway. Seo et al.[30] also found in the head and neck cancer patients (n = 472) that patients with low YWHAZ expression had significantly longer OS as compared patients with high YWHAZ expression group (P < 0.01). RNAi-mediated inhibition of YWHAZ in laryngeal cancer cell lines (Hep-2) led to the suppression of colony formation, and an increase in the cell population in the G/M phase of the cell cycle suggested inhibition of cell proliferation via premature induction of senescence.. Although there are contradictory reports as well, e.g. Liu et al.[31] reported that the knockdown of YWHAZ in muscle-invasive bladder cancer i.e., MIBC cell lines (T24 and 5637), promotes tumorigenesis both in vitro and in vivo, as inhibition of YWHAZ accelerates migration and invasiveness of cells, promotes cell cycle progression, and promotes tumor growth in a mouse model.Despite the fact that the YWHAZ protein was significantly correlated with the progression of ESCC and the prognosis of patients, the current study did not further explore the molecular mechanism by which YWHAZ mediates cell proliferation and metastasis in ESCC. This is indeed a significant gap, as understanding the underlying pathways could provide valuable insights into disease progression and identify potential treatment targets. Additionally, the sample size was small, and further large cohort screening was performed to establish the representation of the results, which may have been limited.
5. Conclusion
In conclusion, the YWHAZ expression in ESCC tissues by bioinformatic analysis and this study were significantly increased, and positively correlated with the grade of tumor progression. The high expression of YWHAZ plays a mediating role in the effect of tumor differentiation on the prognosis of patients with ESCC, leading to shorter OS and PFS. YWHAZ may be of great significance in the diagnosis, treatment, and may prove to be an important biological indicator for the diagnosis and prognosis of the disease.
List of abbreviation
Abbreviation
Full name
ACC
Adrenocortical carcinoma
Akt
Protein kinase B
BLCA
Bladder Urothelial Carcinoma
BMI
Body Mass Index
BRCA
Breast invasive carcinoma
CESC
Cervical squamous cell carcinoma and endocervical adenocarcinoma
CHOL
Cholangio carcinoma
COAD,
Colon adenocarcinoma
DLBC
Lymphoid Neoplasm Diffuse Large B-cell Lymphoma
ESCA
Esophageal Cancer
ESCC
Esophageal Squamous Cell Carcinoma
Fig
Figure
GBM
Glioblastoma multiforme
HGNC
HUGO gene nomenclature committee
HNSC
Head and Neck squamous cell carcinoma;
KICH
Kidney Chromophobe
KP
Keratin Pearls
KIRC
Kidney renal clear cell carcinoma
KIRP
Kidney renal papillary cell carcinoma
LAML
Acute Myeloid Leukemia
LGG
Brain Lower Grade Glioma
LIHC
Liver hepatocellular carcinoma
LUAD
Lung adenocarcinoma
LUSC,
Lung squamous cell carcinoma
MESO
Mesothelioma
NSCLC
Non-small cell lung cancer
OS
Overall Survival
OV
Ovarian serous cystadenocarcinoma
PAAD
Pancreatic adenocarcinoma
PCPG
Pheochromocytoma and Paraganglioma
PFS
Progress Free Survival
PRAD
Prostate adenocarcinoma
PRDM5
PR (PRDI-BF1 and RIZ) domain protein 5
qRT-PCR
Quantitative Real-time Polymerase Chain Reaction
READ
Rectum adenocarcinoma
SARC
Sarcoma
S1PR2
Sphingosine-1-phosphatereceptor 2
SKCM
Skin Cutaneous Melanoma
STAD
Stomach adenocarcinoma
Tab
TGCT
Testicular Germ Cell Tumors
THCA
Thyroid carcinoma
THYM
Thymoma
TNM
Tumor Node Metastasis
UCEC
Uterine Corpus Endometrial Carcinoma;
UCS
Uterine Carcinosarcoma
UVM
Uveal Melanoma
WB
Western Blot
YWHAZ
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta
Declarations
Ethics approval and consent to participate
A
All individuals and/or their parents provided informed consent to participate in this study and approval was provided by Medical Research Ethics Committee of The First Affiliated Hospital of Anhui Medical University. Reference Number: PJ 2021-10-22Consent for publication
All authors and subjects agreed to publish the article in the journal.
Availability of data and materials
The datasets generated and/or analysed during the current study are not publicly available due the manuscript contains the subject's individual information but are available from the corresponding author on reasonable request.
λ Competing interests
The authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A
Funding
This project was supported by University Natural Science Research Project of Anhui Province (Award Number: KJ2016A341), provincial teaching research foundation in Anhui (Grant No. 2019jyxm0994).
A
Author Contribution
All authors have participated in the study and have read and approved the manuscript, agreed to be accountable for the content of this work. YC X: Data curation, Methodology, Pathological test, statistical analysis, Writing-Original draft preparation. Y Z: Pathological test, HY W: Methodology, Funding acquisition, statistical analysis, F W: Project administration, Conceptualization, Writing - review & editing.
Acknowledgements
Thanks to all the authors who contributed to this article, and thanks to University Natural Science Research Project of Anhui Province funding for this research.
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