Cancer Letters

Cancer Letters

Volume 391, 10 April 2017, Pages 59-73
Cancer Letters

Original Article
ERK2-ZEB1-miR-101-1 axis contributes to epithelial–mesenchymal transition and cell migration in cancer

https://doi.org/10.1016/j.canlet.2017.01.016Get rights and content

Highlights

  • miR-101, down-regulated by EGF, targets RHOA, RAC1, ZEB1, and ZEB2.

  • Activation of ERK2-Zeb1-miR-101-1 axis is crucial for EMT and cell migration.

  • miR-101 is a common regulator of both EMT-TFs and actin cytoskeleton modulators.

  • ERK2-Zeb1-miR-101-1 axis is active in cell lines and studied tumor tissues.

Abstract

Regulation of metastasis continues to remain enigmatic despite our improved understanding of cancer. Identification of microRNAs associated with metastasis in the recent past has provided a new hope. Here, we show how microRNA-101 (miR-101) regulates two independent processes of cellular metastasis by targeting pro-metastatic upstream regulatory transcription factors, ZEB1 and ZEB2, and downstream effector-actin modulators, RHOA and RAC1, providing a single target for therapeutic intervention. Further, we depict how down-regulation of miR-101 by extracellular signal-regulated kinase-2 (ERK2) is vital for MAP kinase pathway induced cellular migration and mesenchymal transition. Importantly, EKR2 induced expression of ZEB1 seems essential for down-regulation of miR-101-1 and induction of EMT. Given the role of EMT in metastasis, we also observe a significant correlation between miR-101 expression and lymph node metastasis; and identify the ERK2-ZEB1-miR-101-1 pathway active in breast cancer tissues, with an apparent clinicopathological implication.

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)

  • S. Ansieau et al.

    Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence

    Cancer Cell

    (2008)
  • S. Aznar et al.

    Rho GTPases: potential candidates for anticancer therapy

    Cancer Lett.

    (2004)
  • R. Manso et al.

    The RHOA G17V gene mutation occurs frequently in peripheral T-cell lymphoma and is associated with a characteristic molecular signature

    Blood

    (2014)
  • L. Brenan et al.

    Phenotypic characterization of a comprehensive set of MAPK1/ERK2 Missense mutants

    Cell Rep.

    (2016)
  • S. Lamouille et al.

    Molecular mechanisms of epithelial–mesenchymal transition. Nature reviews

    Mol. Cell Biol.

    (2014)
  • R. Kalluri et al.

    The basics of epithelial–mesenchymal transition

    J. Clin. Invest.

    (2009)
  • M. Bacac et al.

    Metastatic cancer cell

    Annu. Rev. Pathol.

    (2008)
  • A. Moustakas et al.

    Signaling networks guiding epithelial–mesenchymal transitions during embryogenesis and cancer progression

    Cancer Sci.

    (2007)
  • L.J. Talbot et al.

    Epithelial–mesenchymal transition, the tumor microenvironment, and metastatic behavior of epithelial malignancies

    Int. J. Biochem. Mol. Biol.

    (2012)
  • C. Lamaze et al.

    Regulation of receptor-mediated endocytosis by Rho and Rac

    Nature

    (1996)
  • T.S. Jou et al.

    Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases

    J. Cell Biol.

    (1998)
  • R. Perona et al.

    Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins

    Genes Dev.

    (1997)
  • T.R. Faruqi et al.

    Rac1 mediates STAT3 activation by autocrine IL-6

    Proc. Natl. Acad. Sci. U.S.A.

    (2001)
  • J. Lu et al.

    MicroRNA expression profiles classify human cancers

    Nature

    (2005)
  • J.M. Friedman et al.

    The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2

    Cancer Res.

    (2009)
  • S. Varambally et al.

    Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer

    Science

    (2008)
  • M. Smits et al.

    Down-regulation of miR-101 in endothelial cells promotes blood vessel formation through reduced repression of EZH2

    PloS One

    (2011)
  • J.T. Li et al.

    MiRNA-101 inhibits breast cancer growth and metastasis by targeting CX chemokine receptor 7

    Oncotarget

    (2015)
  • S. Manvati et al.

    MiR-101 induces senescence and prevents apoptosis in the background of DNA damage in MCF7 cells

    PloS One

    (2014)
  • L. Xiaoping et al.

    CPEB1, a histone-modified hypomethylated gene, is regulated by miR-101 and involved in cell senescence in glioma

    Cell Death Dis.

    (2013)
  • L. Wang et al.

    c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma

    Hepatology

    (2014)
  • C. Lin et al.

    miR-101 suppresses tumor proliferation and migration, and induces apoptosis by targeting EZH2 in esophageal cancer cells

    Int. J. Clin. Exp. Pathol.

    (2014)
  • R. Wang et al.

    MiR-101 is involved in human breast carcinogenesis by targeting Stathmin1

    PloS One

    (2012)
  • Cited by (0)

    View full text