Activin and TGFβ regulate expression of the microRNA-181 family to promote cell migration and invasion in breast cancer cells

Highlights

• Activin and TGFβ induce miR-181 expression in human breast cancer cells.

• Activin and TGFβ induce miR-181 in a broad range of cancer cell types.

• miR-181 modulation does not affect activin and TGFβ tumor suppressive effects.

• miR-181 is required for migration and invasion in breast cancer.

• miR-181 is a novel target for activin.

Abstract

MicroRNA-181 (miR-181) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. While miR-181 is often overexpressed in human tumors, a direct role for this miRNA in breast cancer progression has not yet been characterized. In this study, we found this miRNA to be regulated by both activin and TGFβ. While we found no effect of miR-181 modulation on activin/TGFβ-mediated tumor suppression, our data clearly indicate that miR-181 plays a critical and prominent role downstream of two growth factors, in mediating their pro-migratory and pro-invasive effects in breast cancer cells miR-181 acts as a metastamir in breast cancer. Thus, our findings define a novel role for miR-181 downstream of activin/TGFβ in regulating their tumor promoting functions. Having defined miR-181 as a critical regulator of tumor progression in vitro, our results thus, highlight miR-181 as an important potential therapeutic target in breast cancer.

Introduction

The TGFβ ligands are multitasking cytokines that play important roles in embryonic development, cell proliferation, motility, invasion and apoptosis, extracellular matrix production and modulation of immune function [1], [2], [3], [4], [5]. TGFβ, the founding member of this family, and its receptors are expressed everywhere in the body and deregulation of the TGFβ signaling pathways has been implicated in multiple human diseases [6]. TGFβ plays a dual role in cancer: it limits proliferation in epithelial cells and early-stage cancer cells, whereas in late stage cancer, it accelerates cancer progression and metastasis [2], [7], [8], [9], [10], [11]. In the cancer niche, TGFβ can be produced and secreted into the extracellular environment by both cancer cells and host cells, such as lymphocytes, macrophages and dendritic cells. In cancer patients, high levels of TGFβ at tumor sites correlate with high histological grade, risk of metastasis, poor response to chemotherapy, and poor patient prognosis [8]. TGFβ interacts with and signals through two transmembrane serine/threonine kinase receptors (TβRI/ALK5 and TβRII), which then activate the Smad family of transcription factors (Smad2 and 3) [1], [2], [12].

Another member of the family, activin was initially isolated from gonadal fluid [13], [14] based on its ability to induce FSHβ secretion and regulate the anterior pituitary function [15], [16], [17]. Activin was later shown to regulate cell growth, apoptosis and differentiation in a variety of tissues, including breast cancer [4], [18], [19], [20], [21]. Similar to TGFβ, activin initiates its signaling through ligand binding to the activin type II receptors at the cell surface, leading to the recruitment and phosphorylation of the type I receptor (ALK4) [19], [22]. The activated ALK4 in turn phosphorylates the two intracellular Smad2 and Smad3, the main activin/TGFβ downstream mediators and further lead to their association with the common partner Smad4 [23].

Activin and TGFβ signaling is not limited to the canonical Smad pathway, as they have also been reported to transduce their signal through non-Smad signaling pathways [2], [11], [18], [19], [22], [23], [24], [25]. While the role of TGFβ in mammary gland and breast cancer has been well characterized, the role and function of activin in this tissue remain largely unknown. In breast tissue, activin and its receptors are expressed during lactation [26] and activin was suggested to participate in mammary epithelium development [27]. In breast cancer, activin can act as a tumor suppressor by inducing cell growth arrest [18], [28], apoptosis [29] and by inhibiting telomerase activity [30], [31]. However, even though circulating levels of activin have been correlated to bone metastasis in breast cancer [32] and that inhibiting activin was shown to prevent cancer-induced bone destruction in vivo [33], a direct role for activin in promoting breast cancer cell invasion and metastasis has yet to be demonstrated.

MicroRNAs (miRNAs) are a novel class of small non-coding RNAs which have eluded researchers for decades stealthily regulating many of the major biological processes in eukaryotic cells by regulating their target genes post transcriptionally. In the past decade, our understanding of miRNA has grown tremendously from an observed oddity in worms [34] to the establishment of a fully recognized new class of regulatory molecules. They are a novel class of small (19-25nt) non-coding RNAs which play important roles in development. Bioinformatics approaches suggest that miRNAs represent 1% all human genes and yet over a third of the transcriptome is regulated by these miRNA [35]. It clearly became apparent that miRNA play central and critical role in human diseases, including cancer. Half of the known miRNAs are located on fragile sites of the chromosomes suggesting that they could play major roles in cancer [36]. Cancer-specific chromosomal rearrangement studies have shown that half of the breakpoints coincide with fragile chromosomal sites [37]. Half of the miRNA-encoding genes are located in chromosomal regions that are altered during tumorigenesis [38]. Both TGFβ and activin have been shown to regulate miRNAs in vitro [39], [40] although very little work has been done on the latter regulation. The role of miRNAs in the progression of breast cancer (BC) is emerging only recently. Several miRNAs have been implicated in several steps of breast cancer progression (reviewed in [2]). For instance miR-31 has been shown to target several genes involved in breast cancer metastasis [41] and miR-200 has been shown to target ZEB2, a transcription factor involved in EMT [42]. We also recently found TGFβ-mediated down regulation of miR-584 to be critical for breast cancer cell actin skeleton reorganization and cell motility [43].

In this study, we identified miR-181 as a potent regulator of activin and TGFβ signaling in human breast cancer. We found miR-181 to be Smad2/3-dependent downstream target of TGFβ/activin signaling. Furthermore, our data demonstrate that activin, like TGFβ, acts as a potent inducer of cell migration and cell invasion in human breast cancer cells, thus, highlighting a novel function for this growth factor in cancer cells. Moreover, we also found miR-181 to be required for activin/TGFβ-mediated cell migration and invasion, as silencing miR-181 expression significantly antagonize these growth factors pro-invasive effects. Interestingly, while significantly blocking activin/TGFβ-induced cell migration and invasion, modulation of miR-181 endogenous levels did not altered activin and TGFβ tumor suppressive effects in cancer cells, highlighting the therapeutic potential of small antagonists to this microRNA for breast cancer treatment.