The Prognostic Significance of Anisomycin-Activated Phospho-c-Jun NH2-Terminal Kinase (p-JNK) in Predicting Breast Cancer Patients' Survival Time

Abstract

This study aims to investigate the prognostic significance of p-JNK in breast cancer patients receiving neoadjuvant chemotherapy (NACT) and analyze the relationship between anisomycin, p-JNK. A total of 104 breast cancer patients had NACT were enrolled in this study. The western blot and immunohistochemistry assays were used to determine the protein expressions of p-JNK in human breast cancer cell lines and patients' cancer tissues. The chi-square test and Fisher's exact test were adopted to gauge the associations between breast cancer and clinicopathological variables by p-JNK expression, whereas the univariate and multivariate Cox proportional hazards regression models were used to analyze the prognostic value of p-JNK expression. The Kaplan-Meier plots and the log-rank test were adopted to determine patients' disease-free survival (DFS) and overall survival (OS). Findings indicated that the p-JNK expression had prognostic significance in univariate and multivariate Cox regression survival analyses. Results of log-rank methods showed that: (1) the mean DFS and OS times in patients with high p-JNK expression were significantly longer than those in patients with low p-JNK expression (χ² = 5.908, P = 0.015 and χ² = 6.593, P = 0.010, respectively). p-JNK expression is a significant prognostic factor that can effectively predict the survival in breast cancer patients receiving NACT. Treatment with the JNK agonist anisomycin can induce apoptosis, lead to increased p-JNK expression and decreased p-STAT3 expression. Moreover, the p-JNK expression was inversely correlated with p-STAT3 expression.

Keywords: anisomycin; breast cancer; neoadjuvant chemotherapy; p-JNK; p-STAT3.

FIGURE 1

Expression of p-JNK in human breast cancer tissues.

FIGURE 2

Breast cancer patients’ DFS and OS. (A) Patients’ DFS for the p-JNK expression, assessed by the Kaplan-Meier analysis. (B) Patients’ of OS for the p-JNK expression, assessed by the Kaplan-Meier analysis.

FIGURE 3

Patients’ DFS and OS for the p-JNK expression, by molecular subtypes. (A) Patients’ DFS by molecular subtypes, assessed by the Kaplan-Meier analysis. (B) Patients’ OS by molecular subtypes, assessed by the Kaplan-Meier analysis. (C) Patients’ DFS by Luminal A subtype, assessed by the Kaplan-Meier analysis. (D) Patients’ OS by Luminal A subtype, assessed by the Kaplan-Meier analysis. (E) Patients’ DFS by Luminal B subtype, assessed by the Kaplan-Meier analysis. (F) Patients’ OS by Luminal B subtype, assessed by the Kaplan-Meier analysis. (G) Patients’ DFS by HER2-enriched subtype, assessed by the Kaplan-Meier analysis. (H) Patients’ OS by HER2-enriched subtype, assessed by the Kaplan-Meier analysis. (I) Patients’ DFS by triple-negative subtype, assessed by the Kaplan-Meier analysis. (J) Patients’ OS by triple-negative subtype, assessed by the Kaplan-Meier analysis.

FIGURE 4

Patients’ DFS and OS by lymph vessel invasion status. (A) Patients’ DFS without lymph vessel invasion by p-JNK expression, assessed by the Kaplan-Meier analysis. (B) Patients’ OS without lymph vessel invasion by p-JNK expression, assessed by the Kaplan-Meier analysis. (C) Patients’ DFS with lymph vessel invasion by p-JNK expression, assessed by the Kaplan-Meier analysis. (D) Patients’ OS with lymph vessel invasion by p-JNK expression, assessed by the Kaplan-Meier analysis.

FIGURE 5

Apoptosis induced by anisomycin in human breast cancer. (A) CCK-8 detected IC50 of human mammary epithelial cell line (184B5) and human breast cancer cell lines (MDA-MB-231, MDA-MB-436, BT549, and Hs578T). (B) Prior to flow cytometry, human mammary epithelial cell and breast cancer cells were conditioned with 0 μM or 0.2 μM anisomycin for 48 h. (C) Flow cytometry analyzed apoptosis of 184B5, MDA-MB-231, MDA-MB-436, BT549, and Hs578T.

FIGURE 6

Apoptosis induced by anisomycin in human mammary epithelial cell line and human breast cancer cell lines. (A–E) 184B5 (A), MDA-MB-231 (B), MDA-MB-436 (C), BT549 (D), and Hs578T (E) cells were treated with indicated concentration of anisomycin. After being treated with anixomycin for 48 h, western blot was adopted to examine PARP, procaspase3, cleaved caspase-3, and β-actin expressions.

FIGURE 7

Anisomycin activated JNK and inhibited the activation of STAT3. (A–E) 184B5 (A), MDA-MB-231 (B), MDA-MB-436 (C), BT549 (D), and Hs578T (E) cells were conditioned with anisomycin, concentration as indicated. After being treated with anixomycin for 48 h, western blot was adopted to examine STAT3, p-STAT3, p-JNK1/2/3, and -actin expression.