A novel PAK4-CEBPB-CLDN4 axis involving in breast cancer cell migration and invasion

https://doi.org/10.1016/j.bbrc.2019.02.070Get rights and content

Highlights

  • PAK4 and CLDN4 expression are positively correlated in human breast cancer.

  • Knockdown of PAK4 significantly inhibits breast cancer cell migration and invasion by downregulating CLDN4.

  • CEBPB binds to CLDN4 promoter and regulates CLDN4 transcription in breast cancer cells.

  • PAK4 enhances CEBPB phosphorylation on Thr235.

Abstract

Claudin-4 (CLDN4), a crucial member of tight junction proteins, is aberrantly expressed in breast cancer cells and contributes to cell migration and invasion. However, the mechanisms controlling CLDN4 expression in breast cancer are poorly understood. Here, we reported that CLDN4 expression correlated positively with p21-activated kinase 4 (PAK4) expression in human breast cancer tissues. Knockdown of PAK4 in MDA-MB-231 and ZR-75-30 cells suppressed CLDN4 expression and significantly inhibited cell migration and invasion. Conversely, restoration of CLDN4 expression in PAK4-knockdown cells reversed the inhibition of migration and invasion. We identified CCAAT/enhancer-binding protein β (CEBPB) as a novel transcriptional regulator of CLDN4 and confirmed that CEBPB bound to the −1093 to −991 bp region of the CLDN4 promoter. Importantly, we found that PAK4 enhanced CEBPB phosphorylation on Thr-235. In summary, we showed that PAK4-mediated CEBPB activation upregulated CLDN4 expression to promote breast cancer cell migration and invasion. Our results might contribute to understanding the mechanisms of CLDN4 regulation and suggest PAK4-CEBPB-CLDN4 axis as a potential therapeutic target for breast cancer.

Introduction

Breast cancer is one of the most common malignant tumors and is the principal cause of cancer-related mortality in women worldwide [1,2]. Most breast cancer-related deaths are caused by tumor metastasis which influences the survival and prognosis [3,4]. Therefore, it is urgent to identify novel biomarkers and explore the key mechanisms involved in tumor metastasis, which may be helpful in designing new therapeutic strategies for breast cancer.

Claudin4 (CLDN4), a vital member of tight junction proteins, appears to be elevated at the protein level in variety of human carcinomas [[5], [6], [7], [8]]. It has been reported that CLDN4 promoted ovarian cancer cell invasion through activating MMP2 [9]. A recent study showed that CLDN4 reinforced proliferation, invasion, and EMT in gastric cancer cells [10]. Consistent with these findings is a report that expression of CLDN4 in breast cancer lead to an increase in invasion, motility and cell survival, which are important for metastasis [11]. Moreover, silence of CLDN4 in MCF-7 breast cancer cells resulted in a significant reduction of cell migration [12]. These studies indicate that CLDN4 plays a crucial role in tumor metastasis. However, underlying mechanisms of CLDN4 regulation in breast cancer cells remain to be further identified.

P21-activated kinase 4 (PAK4), a classical serine/threonine protein kinase, has an essential role in cell signaling and participates in a variety of cellular processes [13,14]. Overexpression of PAK4 has been demonstrated in most human cancer cells, as well as in clinical samples [15]. High PAK4 activity is linked with many features of tumorigenesis, such as increased cell survival, migration and invasion [16,17]. It has been shown that activated-PAK4 predicted worse prognosis in breast cancer and promoted tumorigenesis through activation of PI3K/AKT signaling [18]. Our previous work reported that nuclear PAK4 promoted bone metastasis of ERα-positive breast cancer cells by targeting LIFR [19]. Nevertheless, no previous studies have explored the relationship between PAK4 signaling and CLDN4 in breast cancer.

In the present study, we revealed that CLDN4 was required in PAK4-mediate promotion of cell migration and invasion in breast cancer. Furthermore, we identified CCAAT/enhancer-binding protein β (CEBPB) as a novel transcriptional regulator of CLDN4. We also found that PAK4 enhanced CEBPB phosphorylation on Thr-235. Our findings uncover a novel mechanism by which PAK4 facilitates breast cancer cell migration and invasion via upregulation of CEBPB–CLDN4 signaling.

Section snippets

Clinical tissue samples

Breast cancer tissues were obtained from patients with pathologically verified breast cancer who underwent surgery at the First Affiliated Hospital of China Medical University. None of the patients had received chemotherapy or radiotherapy before surgery. Clinical information was collected from the accompanying medical records. This study was approved by the Research Ethics Committee of China Medical University.

Immunohistochemical (IHC) staining

The paraffin-embedded specimens were sliced into 4-μm-thick sections,

PAK4 and CLDN4 expression are positively correlated in human breast cancer

To evaluate the relationship between PAK4 and CLDN4 in human breast cancer tissues, we analyzed a breast cancer mRNA dataset from The Cancer Genome Atlas (TCGA). Interestingly, analysis of the TCGA dataset revealed that PAK4 mRNA expression correlated positively with CLDN4 mRNA expression in the 1097 breast cancer tissues (Pearson's coefficient test, r = 0.3451, p < 0.001; Fig. 1A), and this was confirmed by IHC staining of PAK4 and CLDN4 protein in 135 breast cancer specimens. Consistent with

Discussion

Claudins are the major components of tight junctions, and aberrant expression of these proteins, especially CLDN4, has been shown to contribute to tumor development [23]. Previous studies have demonstrated that CLDN4 expression was associated with aggressive metastatic activity in breast cancer [7]. Recently, increasing numbers of studies have indicated that PAK4 contributed to the development and progression of breast cancer [18,19]. However, the relationship between PAK4 and CLDN4 in breast

Conflicts of interest

The authors declare no potential conflicts of interest.

Acknowledgment

This research was supported by Grants from the National Natural Science Foundation of China Nos. 31571457, 31371424, 31771553. 81872044. 81602564.

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