Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2

https://doi.org/10.1016/j.bbrc.2020.07.058Get rights and content

Highlights

  • Forced expression of sequestosome 1 (p62) suppresses PR expression.

  • p62 accumulation and PR suppression correlate with poor prognosis.

  • p62 accumulation suppresses AGO2 expression.

  • AGO2 restores PR expression suppressed by p62 accumulation.

  • p62 negatively regulates PR expression by suppressing AGO2 expression.

Abstract

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

Introduction

Breast cancer is the most frequently diagnosed cancer in women worldwide, and the global incidence of breast cancer has been rising [1]. Based on the immunohistochemical expression of the following three established biomarkers: estrogen receptor (ESR1; ERα; hereafter referred as ERα), progesterone receptor (PGR; PR; hereafter referred to as PR), and human epidermal growth factor receptor 2 (ERBB2; HER2; hereafter referred to as HER2), breast cancer can be classified into four clinicopathological subtypes: luminal A, luminal B, HER2-enriched, and triple-negative. Each subtype exhibits distinct prognoses, rates of recurrence, and different treatment strategies [2]. Recent studies have demonstrated that hormonal and HER2 receptor status can be changed between primary and recurrent breast cancer or between primary tumors and metastatic lesions in patients with breast cancer, and that loss of receptor status is associated with poor prognosis [3,4]. However, the molecular mechanisms by which subtype discordance proceed are poorly understood.

Sequestosome 1 (SQSTM1; p62; hereafter referred to as p62) is a multifunctional adapter protein that participates in selective autophagy and acts as a central hub due to its ability to interact with key signaling proteins [5,6]. As a multifunctional protein, p62 is known to be linked to cancer. Abnormal p62 accumulation has been detected in various cancers, including breast cancer, suggesting the presence of a functional relationship between p62 accumulation and cancer progression [7]. High levels of p62 protein in epithelial cells are essential and sufficient for inducing hepatocellular carcinogenesis [8]. A recent study reported that p62 recruits a subset of RNA-binding proteins to extend the half-life of mRNAs of several pro-metastatic factors in melanoma cells [9].

In breast cancer, high expression of p62 is significantly correlated with advanced clinical stages as well as a high risk of distant metastases [10]. High p62 expression is also associated with the expression of epidermal growth factor receptor family, including HER2, and facilitates HER2-induced mammary tumorigenesis through multiple signaling pathways, including the nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (NRF2)-kelch-like ECH-associated protein 1 (KEAP1) pathways [11,12]. Moreover, p62 was shown to enhance breast cancer stem-like properties by stabilizing MYC mRNA [13]. These findings suggest that p62 plays a potential oncogenic role in tumor progression. However, the detailed mechanisms underlying p62-mediated breast cancer initiation and progression remain unclear, and the role of p62 in subtype discordance and patient prognosis is poorly characterized.

In this study, we aimed to elucidate the role of p62 in breast cancer progression and prognosis and its underlying mechanism, as well as explore its involvement in subtype discordance.

Section snippets

Cells

The human breast cancer cell lines BT-474 (ER-positive, PR-positive, HER2-positive) and MCF-7 (ER-positive, PR-positive, HER2-negative) were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37 °C in a 5% CO2 atmosphere in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (Biosera, Nuaillé, France). Stable cell lines were generated as described below. To construct a mammalian

Changes in gene expression in p62-overexpressing BT-474 cells

In order to analyze the role of p62 in breast cancer cells, we first established a p62-overexpressing BT-474-derived cell line (Fig. 1A). Initially, we started with clone 2 (Cl. 2; moderate expression), Cl. 6 (slight expression), and Cl. 8 (high expression). Later, however, we used Cl. 5 instead of Cl. 6 because the expression of extrinsic p62 in Cl. 6 was gradually suppressed. DNA microarray analysis revealed the upregulated genes such as ATP binding cassette subfamily A member 12 (ABCA12),

Discussion

In the present study, we showed that p62 plays a crucial role in the suppression of PR expression in breast cancer cells, and functions in part via AGO2 suppression. A previous study reported that interleukin-1 induces p62 expression and autophagy in breast cancer cells concomitant with the suppression of ERα and PR expression; however, it did not demonstrate a causal relationship between p62 and PR expression [22]. To the best of our knowledge, this is the first study to demonstrate the

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgments

The authors would like to thank Dr. Kana Miyahara (Department of Breast Oncology and Surgery, Tokyo Medical University) for carefully reviewing the clinical background on this article. We also thank Editage (www.editage.jp) for English language editing.

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