Original ArticleERK2-ZEB1-miR-101-1 axis contributes to epithelial–mesenchymal transition and cell migration in cancer
Graphical abstract
Introduction
Metastasis is a major cause of cancer-related deaths and is driven by a highly mobile and invasive character of the cancer cells to spread to distant organs [1]. Acquisition of these properties involves reversible and well-regulated process of epithelial–mesenchymal transition (EMT). The major events of EMT and metastasis include disassembly of cell–cell junctions and apical-basal polarity; gaining of front-rear polarity through restructuring of the cytoskeleton; improved cell motility; repression of epithelial markers and activation of mesenchymal genes [2], [3]. Metastatic cancer cells are also multi-drug resistant and acquire stem cell-like properties [4], [5]. Extracellular matrix and many secreted soluble factors regulate metastasis through several signaling pathways and reprogram a less motile, non-invasive cell to a metastatic state [2], [6], [7]. A common integration point of the metastasis-inducing signaling involves a dynamic reorganization of cytoskeleton to increase the mobility and invasive capacity of cells to invade adjacent and distant tissues. The dynamics of actin cytoskeleton, controlled by Rho family proteins [8], [9], [10] and their activated signaling cascade, contributes to EMT and oncogenic transformation [11], [12], [13], [14] in response to the growth factors and oncogene-induced signalings. Studies have shown in EMT and metastasis a hand-in-hand role of the upstream regulatory signaling through TGF, EGF, Wnt, the transcription factors (EMT-TFs) - ZEB1/2, Snail, Slug and Twist1/2; and the downstream effectors, such as Rho-GTPases, MMPs [2]. Despite the growing knowledge about these molecular players, a comprehensive understanding of the controls of the expression of transcription factors and cytoskeleton modulators in EMT signaling and metastasis of a cancer cell is lacking.
While global alterations in expression of miRNA have been documented in tumors [15], [16], the role, however, of miRNA in regulating upstream signalings and downstream effectors of EMT and metastasis, is less understood. We unravel here the role of miR-101, one of the microRNAs down-regulated in several cancers [17], [18], [19], [20], [21], [22], the restoration of which in cells results in inhibiting proliferation either by inducing apoptosis or senescence [23], [24], [25], [26], [27]. The tumor suppressor activity of miR-101 has been correlated with its inhibitory potential for expression of EZH2, STMN1, COX2, MCL1, POMP, Lin-28B, HMGA2, CXCR7 genes [17], [19], [25], [27], [28], [29], [30], [31]. Ectopic expression of miR-101 has also been shown to enhance the sensitivity of cancer cells to radiation, cisplatin and etoposide [23], [32], [33], [34]. However, it is not clear if miR-101 modulated the dynamics of actin cytoskeleton or EMT signaling to affect the process of metastasis; and if its expression was regulated by EMT inducers.
We ascertain here the role of down-regulation of miR-101 in EGF-mediated or independently induced EMT; and establish how an ectopic expression of miR-101 obstructed cell migration and EMT signaling by inhibiting RHOA, RAC1, ZEB1, and ZEB2 expression. We further demonstrate that ERK2 dependent increased expression of ZEB1 was necessary for EGF-driven downregulation of miR-101-1 instead of miR-101-2. Finally, the active existence of ERK2-ZEB1-miR-101-1 axis was confirmed in a representative set of sporadic breast tumors. Overall, the observations made here showed the converging role of miR-101 as a common regulator for the two independent processes of transcription factors and actin modulators in metastasis.
Section snippets
Materials and methods
Details of the reagents and the techniques used, generation of stable cell lines in culture and patient details, protocols for RNA isolation, quantitative real-time PCR, reporter assays for target confirmation and Western blotting, are described in the Supplementary Information. Other methods used are described below as well as in the Supplementary Information.
Down-regulation of miR-101 is crucial for epidermal growth factor (EGF) induced EMT
EGF-induced stimulation of A549 cells for 24 and 48 h exhibited down-regulation of miR-101 (Fig. 1A) along with an increased expression of vimentin, a mesenchymal marker, and a concomitant reduction in expression of the epithelial marker, E-cadherin (Fig. 1B). The EGF stimulation also resulted in an increase in the expression of stem-cell markers, EMT-inducing transcription factors, metalloproteinase MMP9 (Fig. S1A) and reorganization of the cytoskeleton to form actin stress fibers (Fig. 1C).
Discussion
The growing knowledge, about the regulators of epithelial–mesenchymal transition (EMT) and actin cytoskeletal modulators, in the process of metastasis in cancer, has yet to provide a comprehensive understanding. An identification of a common control of these two independent processes could be one such step to target the regulator, and in containing both EMT and cell migration together. Here we demonstrated that EGF-driven downregulation of miR-101, by activating ERK2-ZEB1 mediated
Authors contributions
K.C.M. and R.N.K.B. conceived the experiments, wrote the manuscript. K.C.M. conducted the majority of experiments. S.M. and S.K.S. performed some experiments. K.P. provided essential bioinformatics expertise. S.K.A. contributed to the editing of the manuscript. G. A. provided sporadic breast cancer samples.
Acknowledgments
We would like to thank Amitabha Bandyopadhyay (IIT-Kanpur, India), Mutsuhiro Takekawa (Nagoya University, Japan), Dr. John Blenis (Weill Cornell Medicine, USA), Manohar Ratnam (Wayne State University, USA), Dr. Li Ma (MD Anderson Cancer Centre, USA), Dr. Sagar Sengupta (NII, India), Jong-In Park (Medical college of Wisconsin, USA) and Gary Bokoch for sharing knockdown and expression vectors used in this study. This work was partly supported by DST, UGC, and DBT. K.C.M. and S.K.S thanks CSIR for
References (67)
- et al.
Molecular networks that regulate cancer metastasis
Seminars Cancer Biol.
(2012) - et al.
The epithelial–mesenchymal transition generates cells with properties of stem cells
Cell
(2008) Rho family proteins: coordinating cell responses
Trends Cell Biol.
(2001)- et al.
Rac and Cdc42 are potent stimulators of E2F-dependent transcription capable of promoting retinoblastoma susceptibility gene product hyperphosphorylation
J. Biol. Chem.
(1998) - et al.
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway
Cell
(1995) - et al.
MicroRNAs in cancer: biomarkers, functions and therapy
Trends Mol. Med.
(2014) - et al.
MiR-101 downregulation is involved in cyclooxygenase-2 overexpression in human colon cancer cells
Exp. Cell Res.
(2009) - et al.
MicroRNA-101 exerts tumor-suppressive functions in non-small cell lung cancer through directly targeting enhancer of zeste homolog 2
J. Thorac. Oncol.
(2011) - et al.
MicroRNA-101 suppresses tumor cell proliferation by acting as an endogenous proteasome inhibitor via targeting the proteasome assembly factor POMP
Mol. Cell
(2015) - et al.
ERK2 but not ERK1 induces epithelial-to-mesenchymal transformation via DEF motif-dependent signaling events
Mol. cell
(2010)