Inhibition of p38 mitogen-activated protein kinase alters microRNA expression and reverses epithelial-to-mesenchymal transition

Abstract

Acquired chemoresistance and epithelial-to-mesenchymal transition (EMT) are hallmarks of cancer progression and of increasing clinical relevance. We investigated the role of miRNA and p38 mitogen-activated protein kinase (MAPK) signaling in the progression of breast cancer to a drug-resistant and mesenchymal phenotype. We demonstrate that acquired death receptor resistance results in increased hormone-independent tumorigenesis compared to hormone-sensitive parental cells. Utilizing global miRNA gene expression profiling, we identified miRNA alterations associated with the development of death receptor resistance and EMT progression. We further investigated the role of p38 MAPK in this process, showing dose-dependent inactivation of p38 by its inhibitor RWJ67657 and decreased downstream ATF and NF‑κB signaling. Pharmacological inhibition of p38 also decreased chemoresistant cancer tumor growth in xenograft animal models. Interestingly, inhibition of p38 partially reversed the EMT changes found in this cell system, as illustrated by decreased gene expression of the EMT markers Twist, Snail, Slug and ZEB and protein and mRNA levels of Twist, a known EMT promoter, concomitant with decreased N‑cadherin protein. RWJ67657 treatment also altered the expression of several miRNAs known to promote therapeutic resistance, including miR‑200, miR‑303, miR‑302, miR‑199 and miR‑328. Taken together, our results demonstrate the roles of multiple microRNAs and p38 signaling in the progression of cancer and demonstrate the therapeutic potential of targeting the p38 MAPK pathway for reversing EMT in an advanced tumor phenotype.

Figure 1

Death receptor resistance promotes hormone independent tumori-genesis. MCF-7 and MCF-7TN-R cells were injected in the mammary fat pads of female ovariectomized mice and measured for tumor formation (^*p<0.05).

Figure 2

Clustering analysis of microRNA expression profiles of MCF-7 and MCF-7TN-R cells. MCF-7 and MCF-7TN-R have distinctive microarray expression patterns, with samples of same cell lines clustered together. (A) Upregulated miRNA relative to MCF-7. (B) Downregulated miRNA relative to MCF-7. Trees on the left are gene clusters. Red color indicates upregulation and green color indicates downregulation.

Figure 3

RWJ67657 inhibits p38 signaling. (A) MCF-7TN-R cells were treated with increasing concentrations of RWJ67657 (0–10 μM) for 6 h and harvested for western blot analysis with anti-phospho-p38 or anti-phospho-ATF2 antibodies. (B) MCF-7TN-R cells were transfected with Gal4-CHOP and Gal4-luciferase for 6 h followed by treatment with increasing concentrations of RWJ67657 (0–10 μM). The following day cells were harvested for luciferase assay. Data points and error bars represent the mean ± SEM of three independent experiments in triplicate (^**p<0.01). (C) MCF-7TN-R cells were transfected with NF-κB-luciferase for 6 h and pre-treated with increasing concentrations of RWJ67657 (0–10 μM) followed by treatment with or without TNF (1.0 ng/ml) overnight. The following day cells were harvested for luciferase activity. Data points and error bars represent the mean ± SEM of three independent experiments in triplicate.

Figure 4

Inhibition of p38 blocks clonogenic survival and suppresses tumor growth. (A) MCF-7TN-R cells were plated for clonogenic survival assay, treated with increasing concentrations of RWJ67657 (0–10 μM) and allowed to grow until visible colonies appeared. Cells were then harvested, stained and counted for clonogenic survival. Data points and error bars represent the mean ± SEM of three independent experiments in triplicate (^**p<0.01, ^***p<0.001). (B) MCF-7TN-R cells (5×10⁵) were injected s.c. into immune-compromised mice. The mice treated with vehicle control or RWJ67657 (60 mg/kg) for 9 days. Tumor volume was monitored biweekly after palpable tumor formation (^*p<0.05).

Figure 5

Inhibition of p38 reverses epithelial-to-mesenchymal transition. (A) mRNA gene expression of the EMT markers Twist, Snail, Slug and ZEB2 were quantified using qPCR in MCF-7TN-R cells following treatment with RWJ67657 (10 μM) for 24 h. Mean values ± SEM of three independent experiments in duplicate are reported (^**p<0.01, ^*p<0.05). (B) RT-PCR analysis of Twist or GADPH (control) expression and western blot analysis of Twist and N-cadherin expression following treatment with indicated concentrations of RWJ67657 (0–10 μM) for 24 h. Blots shown are representative of three independent experiments.

Figure 6

Clustering analysis of microRNA expression profiles of RWJ67657. MCF-7 and MCF-7TN-R have distinctive microarray expression patterns, with samples of same cell lines clustered together. Trees on the left are gene clusters. Red color indicates upregulation and green color indicates downregulation.