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

Accepted Manuscript ERK2-ZEB1-miR-101-1 Axis contributes To Epithelial-Mesenchymal Transition and Cell Migration in Cancer Kailash Chandra Mangalhara, Siddharth Manvati, Sunil Kumar Saini, Kalaiarasan Ponnusamy, Gaurav Agarwal, Suresh K. Abraham, Rameshwar N.K. Bamezai PII:

S0304-3835(17)30039-3

DOI:

10.1016/j.canlet.2017.01.016

Reference:

CAN 13202

To appear in:

Cancer Letters

Received Date: 2 November 2016 Revised Date:

6 January 2017

Accepted Date: 11 January 2017

Please cite this article as: K.C. Mangalhara, S. Manvati, S.K. Saini, K. Ponnusamy, G. Agarwal, S.K. Abraham, R.N.K. Bamezai, ERK2-ZEB1-miR-101-1 Axis contributes To Epithelial-Mesenchymal Transition and Cell Migration in Cancer, Cancer Letters (2017), doi: 10.1016/j.canlet.2017.01.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ERK2-ZEB1-miR-101-1 Axis contributes To Epithelial-Mesenchymal Transition and Cell

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Migration in Cancer

Kailash Chandra Mangalharaa, Siddharth Manvatib, Sunil Kumar Sainia, Kalaiarasan Ponnusamyb, Gaurav Agarwalc, Suresh K. Abrahama, Rameshwar N. K. Bamezaia*

School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi-110067, India.

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School of Biotechnology, Jawaharlal Nehru University, New Delhi, Delhi-110067, India.

Department of Endocrine & Breast Surgery, Sanjay Gandhi Post-Graduate Institute of Medical

Sciences (SGPGIMS), Lucknow-226014, India. *

National Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru

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University, New Delhi, Delhi-110067, India. E-mail: [email protected]

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Abstract Regulation of metastasis continues to remain enigmatic despite our improved understanding of

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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

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target for therapeutic intervention. Further, we depict how down-regulation of miR-101 by

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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,

Highlights

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with an apparent clinicopathological implication.

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

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

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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

Keywords: microRNA; miR-101; Breast Cancer; EMT-TFs; Cytoskeleton; Epidermal Growth Factor

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1.

Introduction

Metastasis is a major cause of cancer-related deaths and is driven by a highly mobile and

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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

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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;

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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-

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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.

2. 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 4

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confirmation and Western blotting, are described in the Supplementary Information. Other methods used are described below as well as in the Supplementary information.

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2.1 Breast Cancer samples

Thirty-seven primary breast tumors and an equal number of adjacent normal tissues were surgically resected at the Department of Endocrine & Breast Surgery, Sanjay Gandhi

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Postgraduate Institute of Medical Sciences, Lucknow, with the approval of the medical ethics committee. All clinical data and tissue samples were obtained after obtaining informed consent,

Supplementary Information.

2.2 Phalloidin staining

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according to institutional ethics review board procedures. The patient details are provided in

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The cells grown in culture on glass coverslips were fixed with 3.7% formaldehyde/1XPBS. After two washes in 1XPBS, cells were incubated with 1X-Alexa-488 conjugated phalloidin (Abcam) and imaged with Andor Spinning Disk Confocal Microscope. Details of the protocol are

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provided in the Supplementary Information.

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2.3 Cell migration assay

Cells were cultured in 6 well plates in respective media containing 0.5% FBS to sub-confluence. Wounds were inflicted on the growing monolayers of sub-confluent cells with a 10µl plastic pipette tip. The wounds were imaged at two-time points of 0hr and after 20hrs. Average distance of the wounds inflicted was calculated in five microscopic fields and converted into the percent

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wound distance after 20hrs. Experiments were repeated thrice. Details of the protocol are provided in the Supplementary Information.

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2.4 Zymography

MMP9 activity was measured by zymography. In brief, cells were cultured in serum-free medium with and without treatment. The conditioned medium was subjected to 0.1% gelatin-

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SDS-PAGE at 4°C, followed by incubation at 37°C in 50mM Tris (pH-7.4), 10mM CaCl2 buffer for 40 hrs. After staining and destaining, gels were imaged with a gel documentation system and

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gelatinolytic activity was quantified by densitometry. The experiments were repeated twice for MMP activities. Details of the protocol are provided in the Supplementary Information.

2.5 Dual Luciferase Reporter Assay

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The luciferase assay was carried out for miRNA target confirmation; where, cells were plated in 24 well plate and transfected with pGL3-3’UTR luciferase reporter, pcDNA6.2-miR-101 expressing vector in absence or presence of miR-101 inhibitor-oligos. Renilla luciferase plasmid

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was used in these as an internal control. Luciferase activity of cells was assayed using dual luciferase assay kit (Promega) after 48hrs of transfection. For miR-101 promoter assays,

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indicated cells, plated in 24 well plates were transfected with pGL4.28-miR-101-1-luc or pGL4.28-miR-101-2-luc reporter vectors, along with pcDNA3-ZEB1, together with Renilla luciferase plasmid as an internal control. Wherever transduction was required, indicated cells were infected after six hours of transfection of reporter and internal control vectors. Details of plasmid is provided in the Supplementary Information.

2.6 Bioinformatics analysis of miRNA target genes 6

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Target genes of miR-101 were identified using online target prediction algorithm, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/miRretsys-self.html),

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miRNA target information from fifteen different prediction algorithms, including miRanda,

algorithms,

were

used

for

functional

annotation

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TargetScan, and miRWalk. Putative target genes of miR-101 (1,151), identified by three of these clustering

using

DAVID

v6.7

(https://david.ncifcrf.gov/), yielding 285 functional annotation clusters for miR-101 target, of

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which 76 clusters were highly enriched (enrichment score≥1.0). The functional cluster analysis was analyzed along with data on known and predicted associations between individual miR-101

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target genes that were enriched for regulation of cell migration and EMT, using Protein-Protein Interaction (PPI) modeling. The PPI of cell mobility proteins was retrieved from Human Protein Reference Database (HPRD) [35]. Cytoscape V4.0 was used for constructing and visualization of the network of PPI and miR-101 targets [36]. The Hubs were identified using in-house PERL

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programming language, and the modules were constructed using MCODE [37]. Important molecules from the module proteins were predicted by subjecting the module proteins for microRNA target prediction, using miRanda, miRDB, PICTAR, PITA, RNAhybrid, miRMap,

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miRWalk, miRNAMap and TargetScan.

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2.7 Molecular subtype association and subtype analysis

Prognostic gene expression analysis was performed with published genomic data sets on breast cancer patients categorized in the six molecular subtypes [38; 39], using bc-GenExMiner v3.2 [40; 41]. A gene expression map was determined by molecular subtype predictors (single sample predictors [SSPs] and/or subtype clustering models [SCMs]). A gene expression table was also generated for robust classification, distributing for each subtype the proportion of patients with

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low, intermediate, and high gene expression; by splitting the gene expression values to form three equal groups. Gene expression data of all the molecular subtypes, along with relapse-free and overall survival information was analyzed. To analyze the prognostic value of miR-101, the

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patient samples were divided into two groups based on the median expression of miR-101.

2.8 Statistics

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The statistical significance of the difference between mean values was tested using Student’s t-

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test. Data was presented as means ± SEM of at least three independent experiments.

3. Results

3.1 Down-regulation of miR-101 is crucial for Epidermal Growth Factor (EGF) induced EMT

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EGF-induced stimulation of A549 cells for 24 and 48 hrs exhibited down-regulation of miR-101 (Figure 1A) along with an increased expression of vimentin, a mesenchymal marker, and a

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concomitant reduction in expression of the epithelial marker, E-cadherin (Figure 1B). The EGF stimulation also resulted in an increase in the expression of stem-cell markers, EMT-inducing

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transcription factors, metalloproteinase MMP9 (Figure S1A) and reorganization of the cytoskeleton to form actin stress fibers (Figure 1C). These features were observed in addition to the activation of MAPK pathway by an increased pERK1/2 expression. EGF treatment of miR101 overexpressing cells (Figure S1B) depicted a significantly reduced effect of EGF on vimentin, E-cadherin and the expression of other EMT markers (Figure 1D, 1E); besides the reorganization of filamentous actin to stress fibers (Figure 1F), which confirmed the role of miR101 in EGF-induced EMT and the feature of cellular migration in A549 cells. 8

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Next, the epithelial to mesenchymal transition was studied under over-expressed and silenced states of miR-101 in A549 to examine the independent role of miR-101 in the process of EMT. Same approach was replicated in MDA-MB-231 cells (Figure S2A). The ectopic expression of

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miR-101 resulted in a cuboidal morphology, presence of cortical arrangement of actin with less stress fibers (Figure 2A), and increased expression of E-cadherin at both RNA (Figure 2B) and protein level in both A549 and MDA-MB-231 cells (Figure 2C). Whereas, the cells silenced for

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miR-101 depicted an elongated fibroblast-like morphology with the rearrangement of actin cytoskeleton from cortical to a stress fiber pattern (Figure 2A), decreased expression of E-

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cadherin and increased vimentin expression (Figure 2B, 2C). Expression of E-cadherin was undetectable in MDA-MB-231 at all studied condition. The miR-101 over-expressing cells also showed a lower migration potential (Figure 2D, S2B) and activity of secreted metalloproteinase, MMP9, in culture medium (Figure 2E); along with a decrease in the levels of EMT-TFs and the

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stem cell markers (Figure S2C), when compared to the vector transduced cells. Whereas, the cells silenced for miR-101 had the reverse effect. Since EMT is known to provide chemoresistance, we checked the effect of cisplatin, a commonly used drug in chemotherapy, in miR-

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101 over-expressing A549 and MDA-MB-231 cells; and observed a 20% and 40% increased sensitivity, respectively (Figure 2F). The feature of sensitivity was reversed and resistance to

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cisplatin observed when cells were silenced for miR-101 (Figure 2F), confirming the role of miR-101 expression in inhibiting EMT as well as the migration potential; and increasing the chemosensitivity towards cisplatin.

3.2 miR-101 targets pro-metastatic genes involved in remodeling of cytoskeleton and induction of EMT

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An unbiased in-silico analysis (explained in Material and Methods section) suggested that miR101 could regulate a vast network of genes involved in the induction and establishment of EMT and cell migration. The key regulators of the cell migration process targeted by miR-101 and the

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network based on miR-101 target prediction along with Protein-Protein Interaction (PPI) results identified two hubs (key regulators), RAC1 and RHOA, which influenced a high number of proteins with the degree of 93 and 67, respectively (Figure S3A). Retrieving of all the proteins

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involved in EMT pathway database as potential targets of has-miR-101 [42], confirmed RAC1 and RHOA as predominant molecules (Figure S3B). Three modules (data not shown) were

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identified after subjecting the PPI network of miR-101 targets for module analysis; which showed presence of ZEB1 and ZEB2 in first module with highest score of 6.5, the highest as compared to other genes in the module with 68 nodes and 150 edges (Figure S3C). These two transcription factors, ZEB1 and ZEB2, were confirmed by 8 prediction programs as targets when

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module genes were analyzed for microRNA target prediction. Further analysis established the involvement of RAC1, RHOA, besides ZEB1, ZEB2 in cell migration and EMT processes, as

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targets of miR-101.

The direct interaction of these genes and the modulation of their expression with miR-101 was

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established experimentally by cloning 3’UTRs of the genes in pGL3 vector (Figure 3A) and cotransfecting cells either with control or miR-101 expressing vector, in absence or presence of miR-101 inhibitor-oligos. The co-expression of miR-101 decreased luciferase activity by 40% to 60%; which was reversed by co-transfection of miR-101 inhibitor (Figure 3B), indicated that miR-101 binds to the selected target gene 3’UTR and regulates their expression. Similar results were observed under EGF stimulation in reporter vector transfected cells. EGF stimulation of

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these cells increased the luciferase activity of the reporter vectors which was overridden by miR101 overexpression (Figure S3D).

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Further, the overexpression of miR-101 in A549 cells repressed the endogenous mRNA levels of RHOA, RAC1, ZEB1, and ZEB2; whereas, silencing of miR-101 resulted in an increase in their expression (Figure 3C). These observations were replicated in MDA-MB-231 cells as well.

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Protein expression of the two representative genes, RHOA and ZEB1, also showed a decreased and increased expression in miR-101 overexpressed and silenced cells, respectively (Figure 3D),

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confirming that miR-101 played a role in downregulating the expression of the genes associated with remodeling of cytoskeleton and induction of EMT.

Next, to examine if the down-regulation of these targets played a critical role in miR-101 mediated inhibition of EMT and cellular migration, we overexpressed RHOA and ZEB1 in miR-

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101 overexpressing A549 and MDA-MB-231 cells. Overexpression of UTR-less RHOA and ZEB1 in miR-101 overexpressing cells, rescued the expression of E-cadherin and vimentin mRNA (Figure 4A), validated by protein levels of vimentin (Figure 4B). RHOA overexpression,

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in A549 as well as MDA-MB-231 cells, also rescued the migration potential (Figure 4C). The migration potential, however, showed only a marginal but non-significant effect in A549 cells

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(Figure 4Ci); which could be seen significantly only in MDA-MB-231 cells when transfected with Zeb1 (Figure 4Cii). miR-101 driven downregulation of MMP9 activity (Figure 4D) along with cortical organization of F-actin (Figure 4E) apparently depended on RHOA; since ZEB1 overexpression had no impact. Collectively, our results suggested that RHOA, RAC1, and ZEB1, ZEB2, are part of an important mechanism underlying the tumor suppressive function of miR101

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3.3 miR-101-1 is a major contributor of mature miR-101 expression and is regulated by ERK2 signaling

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After establishing that EMT-induced signaling down-regulated miR-101 expression, and miR101 negatively regulated EMT and cellular migration by downregulating key transcription factors and cytoskeleton modulators, it was pertinent to resolve which of the two miR-101 genes

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in humans, miR-101-1 (intergenic at chromosome 1p31.6) and miR-101-2 (intronic at chromosome 9p24.1), contributed to the formation of mature miR-101. The recent identification

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of the promoters of the two genes [25] allowed to measure their activities experimentally and showed that miR-101-1 promoter activity was significantly higher than that of miR-101-2 promoter (Figure 5A). The observations made matched with the high expression of the precursor of miR-101-1 in lung-derived - A549, H1299; and breast tissue-derived - MCF7 and MDA-MB231 cells (Figure 5B), suggesting that miR-101-1 gene contributed predominantly to mature miR-

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101 expression in cells from diverse tissue origins. A computational analysis of miR-101-1 promoter with MetInspector identified the transcription factor binding sites (Table S1); the

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pathway enrichment analysis of which showed enrichment of TGF-β, WNT, and ERK1/2 related signaling pathways (Table S2). This incidentally was in conformity with our observation of miR-

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101 being down-regulated by EGF.

The involvement of ERK1/2 in regulation of miR-101 expression was established experimentally after the treatment of A549 cells with PD98509 (MEK1/2 inhibitor)(Figure S4A), which resulted in an increased expression of miR-101 (Figure S4B). Pre-treatment of A549 cells with PD98509 before EGF treatment (Figure 5Ci), subdued the growth factor driven downregulation effect on miR-101 expression (Figure 5Cii); whereas, pretreatment of p38 and JNK inhibitor had no

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significant effect (Figure S4C). This confirmed the role of ERK1/2 in EGF-mediated downregulation of miR-101. Further, to understand and delineate the role of ERK1/2, stable knockdown cells of ERK1 and ERK2 using shRNAs were generated in A549 and MDA-MB-231

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cells (Figure 5Di). Interestingly, ERK2 silenced cells had high expression of mature miR-101 as compared to the vector (control) transduced cells (Figure 5Dii). This increased expression of

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miR-101 correlated with an increased RNA level of the precursor of miR-101-1 (Figure 5E).

Further confirmation of the role of EKR2 in regulating miR-101-1 expression was obtained by

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overexpressing a catalytically active mutant of ERK2 (ERK2-CA) in A549 and MDA-MB-231 cells (Figure 5Fi), observing a decrease in expression of the mature miR-101 (Figure 5Fii). The results obtained for precursor miR-101-1 in A549 and MDA-MB-231 cells further confirmed the results of ERK2 knockdown studies (Figure 5G). Overall, these outcomes suggested the role of

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ERK2 in downregulation of miR-101-1 expression by transcriptional regulation.

3.4 ERK2 downregulates miR-101 via ZEB1 protein expression

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In order to further investigate the molecular basis of ERK2 mediated down-regulation of miR101, knockdown studies were designed with bioinformatically predicted transcription factors,

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ZEB1, c-MYC and ELK1, which could bind miR-101-1 promoter in ERK2-CA transduced cells. Knockdown of c-MYC and ELK1 did not prevent the reduction in miR-101 levels with the constitutively expressing ERK2-CA expression (Figure 6A), suggesting that c-MYC & ELK1 genes are not the major contributors of ERK2 mediated down-regulation of miR-101. However, ZEB1 knockdown in ERK2-CA transduced cells not only increased the expression of miR-101 (Figure 6A) but also had a significant effect on the expression of vimentin at both mRNA (Figure 5SA) and protein (Figure 6B) levels. Thus, the role of ZEB1 was evident in ERK2-CA driven 13

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miR-101 expression and EMT induction in both A549 and MDA-MB-231 cells. Further validation of the role of ZEB1 in ERK2 mediated down-regulation of miR-101 was carried out in the overexpression and knockdown studies, using shERK2 and ERK2-CA transduced cells.

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Overexpression of ZEB1 in ERK2 knockdown cells not only decreased the promoter activity (Figure 6C) of miR-101-1 gene but also decreased the expression levels of mature miR-101 (Figure 6D) along with restoration of the expression of vimentin (Figure 6E). Whereas,

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knockdown of ZEB1 in ERK2-CA transduced cells increased the promoter activity of miR-101-1 gene (Figure 6F) which correlated with the increased expression of mature miR-101 (Figure 6G)

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and decreased expression of vimentin (Figure 6H). Additionally, the overexpression of ZEB1 seemed essential for ERK2-WT transduced cells to mimic the ERK2-CA overexpression condition, suggesting the requirement of an active EKR2 driven ZEB1 expression for miR-101 down-regulation. Further overexpression and knockdown of ZEB1, in both A549 and MDA-MB-

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231 cells, resulted in the decrease (Figure S5B) and an increase (Figure S5C) of miR-101 expression. ZEB1 overexpression also decreased the promoter activity of miR-101-1 (Figure S5D), which correlated with decreased expression of miR-101. Collectively, these results

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suggested that EMT-TF, ZEB1, downregulated miR-101 expression by regulating miR-101-1

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promoter and acted downstream to EKR2 for the down-regulation of miR-101.

3.5 miR-101 downregulation is essential for ERK2 induced EMT

In view of the importance of ERK2 in RAS-induced EMT [43] and our results, we hypothesized that miR-101 down-regulation might be important for ERK2 induced EMT. To test this hypothesis, first, we checked the expression of EMT markers and cellular migration potential of ERK2 knockdown cells. ERK2 knockdown increased the expression of epithelial marker, E-

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cadherin and lowered the expression of vimentin at both protein (Figure 7A) and mRNA levels (Figure 7B), along with the decreased expression of ZEB1. ERK2 silenced cells also had lower migration potential as compared to vector transduced cells (Figure 7C). Interestingly, these cells

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had lower expression of all the four miR-101 target genes, RHOA, RAC1, ZEB1 and ZEB2 (Figure S6A). To assess the role of miR-101 in increased epithelial character of shERK2-A549 cells, the cells were transduced with miR-101 silencing lentiviruses to down-regulate miR-101

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expression in the background of ERK2 knockdown (Figure S6B). Silencing of miR-101 in shERK2 cells reduced the expression of E-cadherin and increased the expression of vimentin at

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both protein (Figure 7D) and mRNA (Figure 7E) levels and also increased the migration potential of cells in scratch assay as compared to shERK2 cells (Figure 7F). miR-101 knockdown also increased the mRNA expression of its target genes (Figure S6C). Together, these results suggested that miR-101, operating downstream to ERK2 signaling, was essential to

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ERK2 mediated induction of EMT.

3.6 The ERK2-miR-101 axis is active in cancer cell lines and sporadic breast cancer tumors

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Finally, the role of ERK2 activated ZEB1-miR-101-1 axis was examined for its activity in paired cancer cell lines of two different tissue origin and with differential potential of EMT (Figure

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S7A). While comparing the activity of EKR2-ZEB1-miR-101 pathway in MCF7 and MDA-MB231 (breast tumor cell lines with a higher EMT potential in the latter) and A549 and H1299 (lung cancer cell lines with a higher EMT potential in the latter), MDA-MB-231 and H1299 depicted a high expression of phospho-ERK1/2 as compared to MCF7 and A549 counterparts (Figure 8A). Interestingly, the lower phosphorylation ERK1/2 correlated with a higher expression of miR-101 in both MCF7 and A549 cells, when compared to high phosphor-ERK1/2 expressing cells

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(Figure 8B). Altogether, the results depicted the presence of ERK2-ZEB1-miR-101 target gene axis in cancer cell lines derived from at least two different tissue origins in this study.

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To confirm our in-vitro observation of tumor cell lines expressing high level of phosphorERK1/2, we did protein extraction from a representative set of human sporadic breast tumor tissues and checked the expression of phospho-ERK1/2. Interestingly, expression levels of

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phosphorylated ERK1/2 were high in tumor samples as compared to normal tissues (Figure 8C), especially in advanced-stage-samples, correlating with the presence of lower expression of miR-

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101 (Figure 8D). We also observed downregulation of miR-101 expression in 37 tumor samples when compared to 37 adjoining normal tissue samples (Figure 8E), as reported earlier [28; 44]. Importantly, the expression of miR-101 was significantly lower in node extension positive as compared to node-negative tumors (Figure 8F). We also observed a significant decrease in the expression of pre-miR-101-1 in tumor tissues (Figure 8G), which correlated with the expression

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of miR-101 in advanced stage tumors (Figure 8H). There was no change in precursor-miR-101-2 expression (Figure S7.B). An analysis of five different molecular subtypes of breast cancer,

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basal-like, HER2-enriched, luminal A, luminal B, and normal breast-like (details in Materials and Methods), differing in the expression of components of ERK pathway, also showed

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significantly lower expression of miR-101 in HER2-E, basal-like, and luminal B subtypes; 50% of HER2-E, 45% of basal-like and 43% of luminal B expressed the lowest of miR-101 (Figure 8I), consistent with our experimental in vitro data of down-regulation of miR-101 with activeERK signaling (Figure 8J). Moreover, miR-101 expression depicted a significant negative correlation with the mRNA expression of MAPK1 and MAP2K1 (Figure S7D-G), supporting the role of ERK signaling in downregulation of miR-101 in-vivo. The low miR-101 expression was also significantly associated with both high tumor recurrence (Figure 8K) and decreased overall 16

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survival (Figure 8L). Altogether, these results suggested that the decreased expression of miR101 contributed predominantly from miR-101-1 gene, correlated strongly with increasing nodal

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positivity in breast cancer patients with less favorable outcome. Discussion

The growing knowledge, about the regulators of epithelial-mesenchymal transition (EMT) and

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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

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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 transcriptional inactivation of miR-101-1 instead of 101-2 gene, validated in vitro and in vivo, was crucial for EGF-induced EMT and cell migration. Our results indicated that EGF need to overcome miR-101 functions in order to activate EMT and

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cell migration, as shown by suppression of the expression of mesenchymal and stem cell markers along with the expression of metalloproteinase and EMT-TFs, after ectopic expression of miR-

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101 in EGF-stimulated cells. The overexpression of miR-101 resulting in downregulation of mesenchymal marker, vimentin, along with EMT-TFs; reduced migration potential of cells and

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actin stress fibers, validated further the role of miR-101 in preventing the processes of EMT and cell migration. Reversal of these processes after silencing of miR-101 confirmed the conclusions drawn.

Identification and analysis of miR-101 target genes, using in-silico, PPI networking, and module approach, showed ZEB1, ZEB2, RHOA and RAC1 as important molecules to execute the suppression activity of miR-101 in EGF-induced EMT. ZEB1 and ZEB2, the EMT-TFs, have been reported with high expression at the invasive front of tumors, repressing E-cadherin 17

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expression [45; 46; 47; 48]; whereas, downregulation of ZEB1 in cells with active oncogenes has been shown to result in oncogene-induced senescence and apoptosis [49; 50] which was confirmed by us in an earlier report on the involvement of miR-101 in senescence [23]. Another

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complementary event to facilitate metastasis independently has been shown to implicate actin cytoskeleton rearrangement, of which the important regulators are Rho-GTPases [51; 52]. These are not only overexpressed in tumor tissues [53; 54], but also have various gain of function

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mutations [55; 56; 57]. Our results with ZEB1 and RHOA related rescue experiments in miR101 overexpressing cells showed the individual importance of both categories of targets. RHOA

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overexpression rescued the cell migration, actin cytoskeleton arrangement, and expression of metalloproteinase; whereas, ectopic ZEB1 expression reversed the expression of EMT markers only. These results were in conformity with the observations of dominant negative mutants of RhoGTPases reported to inhibit RAS-induced transformation and TGF induced EMT of

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fibroblasts and cancer cells, respectively [58; 59; 60], despite the presence of EMT-TFs. Our rescue experiments showed the individual importance of inhibition of actin modulators as well as EMT-TFs in growth factor-induced EMT and cell migration; suggesting that miR-101 apparently

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acted as a master regulator of both the processes with a therapeutic potential.

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To understand the regulation of expression of miR-101 by EGF, we used pharmacological inhibitors and knockdown and overexpression approaches; and established the role of MEK1/2ERK2 in EGF-mediated downregulation of miR-101. Although the importance of ERK2 in induction and establishment of EMT via the regulation of EMT-TFs is documented [43; 61; 62] however, regulation of differential expression of non-coding RNAs either by ERK1 or ERK2 has not been studied. We, for the first time, highlight the central role of microRNA in ERK2 mediated EMT and cellular migration processes based on i) knockdown of endogenous ERK2

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and overexpression of ERK2-CA, resulting in increased and decreased expression of miR-101, respectively; ii) the significant increase in mesenchymal characters and migration potential of shERK2 cells when silenced for miR-101. Further, knockdown of ZEB1 in ERK2-CA

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transduced cells and overexpression of ZEB1 in shERK2 negatively regulated miR-101-1 gene, which correlated with the expression of EMT markers. We did not find the role of MYC which complexes with EZH2, in ERK2 driven downregulation of miR-101 [25]. Altogether, our results

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of in-vitro studies showed the importance of EGF-activated MEK1/2-ERK2-ZEB1-miR-101-1 axis in regulation of EMT and cell migration and its potential as a therapeutic target. These

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conclusions were supported by a global analysis of published data set of breast cancer, showing a significant decrease in expression of miR-101 in HER2-E, Basal-like and Luminal-B molecular subtypes known to have high expression of components of MAPK pathway [63]. Further support was observed in an inverse correlation between miR-101 and MAPK1 (ERK2) and MAP2K1

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(MEK1). Role of MAPK pathway, incidentally, is well established in cancer and metastasis. Two third of the human cancers have deregulated activity and expression of various components of MAPK pathway [64], and have been found associated with lymph node extensions [65]. We

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also observed a significant decrease in expression levels of precursor-miR-101-1, which correlated with the expression of mature miR-101 in advanced stages of sporadic breast tumors.

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Interestingly, we observed a decrease in expression of miR-101 in lymph node positive sporadic breast tumors when compared to the expression in node negative tumors. Also, a negative correlation was observed in the expression of miR-101 and protein levels of pERK1/2 in a representative set of the studied tumor samples. In summary, we report identification of a novel pathway of ERK2-ZEB1-miR-101-1 that promotes cell migration and EMT; and presents with stem cell features along with resistance to

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cisplatin. The activation of this pathway was observed in cell lines as well as in studied sporadic breast tumors with high pERK1/2. We propose that the pathway could be cell autonomous in case of gain of function mutations of RAS or under recently identified ERK2 gain of function

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mutant conditions [66; 67]. Taken together, our results suggest the involvement of MAPK signaling as a critical regulator of miR-101 expression, validated in both in-vitro and in-vivo conditions, pointing towards a common control mechanism of miR-101 in repressing two

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independent processes of EMT signaling and cell migration during metastasis in cancers.

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5. 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

6. Acknowledgments

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sporadic breast cancer samples.

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We would like to thank Amitabha Bandyopadhyay (IIT-Kanpur, India), Mutsuhiro Takekawa (Nagoya University, Japan), Dr. John Blenis (Weill Cornell Medicine, USA), Manohar Ratnam

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(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 financial support. S.M. thanks, UGCDS Kothari Fellowship for financial support. 7. Conflict of interest

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The authors declare that they have no competing interests.

8. Supplementary Information

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Supplementary Information includes Supplementary Experimental Procedures, eight figures, and six tables. 9. Figure Legends

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Figure 1: EGF-induced down-regulation of miR-101 is crucial for EMT induction

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(A) qPCR of miR-101 expression in A549 cells treated with EGF (50ng/ml) for the indicated time points of 0hr, 24hr, and 48hr. Bar diagram depicts mean ± SEM of two independent experiments. (B) Western blot to show E-cadherin, vimentin, pERK1/2, ERK1/2 and actin in extracts of A549 cells stimulated with EGF (50ng/ml). (C) Phalloidin staining of filamentous actin in A549 cells with and without (control) EGF (50ng/ml for 24hrs). Scale bar indicate 10

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µm. (D) Western to show vimentin and actin in stably transduced A549 cells in different combinations with presence or absence of, vector-control, miR-101-expression vector, EGF (50ng/ml for 24 hrs). (E) Relative expression after qPCR quantification of E-cadherin, vimentin,

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CD44, ALDH1, MMP9, ZEB1, ZEB2, SNAIL1 and SNAIL2 mRNA in stably transduced A549

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cells; independently and with different combinations of, vector-control, miR-101-expression vector, with and without EGF (50ng/ml for 24 hrs). Bar diagram depicts mean ± SEM of two separate experiments. See also figure S1. (F) Phalloidin staining of filamentous actin in A549 cells stably transduced with vector-control and miR-101-expression vector, with and without EGF (50ng/ml for 24 hrs). Scale bar indicates 10 µm.

Figure 2: Expression of miR-101 negatively regulates EMT and migration capacity of cells

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(A) Phalloidin staining in (i) A549 and (ii) MDA-MB-231 cells stably transduced with vectorcontrol or miR-101-expression or miR-101-silencing vectors. Scale bar indicate 10 µm. (B) Relative expression after qPCR quantification of, E-cadherin and vimentin mRNA in (i) A549

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and (ii) MDA-MB-231 cells, stably transduced with vector control, miR-101-expression, and miR-101-silencing vectors. Bar diagram depicts mean ± SEM of two separate experiments. (C) Western blot to show E-cadherin, vimentin, and actin in lysates of (i) A549 and (ii) MDA-MB-

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231 cells, stably transduced with vector-control, miR-101-expression, and miR-101-silencing vectors. (D) Wound healing assay for migration potential of (i) A549 and (ii) MDA-MB-231

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cells, stably transduced with vector-control, miR-101-expression, and miR-101-silencing vectors. The % wound distance was plotted after 20 hrs of introduction of the wound. Bar diagram depicts mean ± SEM of three separate experiments. See also figure S2B. (E) Gelatin-substrate zymograph for secreted MMP9 activity of A549 cells stably transduced with vector-control,

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miR-101-expression, and miR-101-silencing vectors. Bar diagram (below) represents the arbitrary activity of MMP9. Bar diagram depicts mean ± SEM of two separate experiments. (F) Cell viability of (i) A549 and (ii) MDA-MB-231 cells stably transduced with vector-control,

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miR-101-expression, and miR-101-silencing vectors; and treated with 5 µM and 10 µM of cisplatin for 24 hrs. Cell viability was measured by MTT assay and represented as percent of

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control. Bar diagram depicts mean ± SEM of two independent assays.

Figure 3: ZEB1, ZEB2, RHOA, and RAC1 are direct targets of miR-101

(A) Schematic presentation of the predicted miR-101 binding sites within the 3’UTRs of (i) ZEB1, (ii) ZEB2, (iii) RHOA, and (iv) RAC1 with sequence complementarity. (B) Relative luciferase activity of reporter constructs harboring 3’UTR of the indicated genes upon

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transfection of either vector-control or miR-101-expression vector (pcDNA6.2-miR-101) with and without miR-101 inhibitor-oligos. Bar diagram depicts mean ± SEM of three independent assays. (C) Relative expression after qPCR quantitation of RHOA, RAC1, ZEB1, and ZEB2

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mRNA in (i) A549 and (ii) MDA-MB-231 cells, stably transduced with vector-control, miR101-expression or miR-101-silencing vectors. Bar diagram depicts mean ± SEM of two independent experiments. (C) Western to show RhoA, Zeb1, and actin in lysates of (i) A549 and

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(ii) MDA-MB-231 cells, stably transduced with vector-control, miR-101-expression, and miR-

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101-silencing vectors.

Figure 4: miR-101 inhibits EMT and cell migration by targeting EMT-TFs and cytoskeleton modulators (A) Relative expression after qPCR quantitation of E-cadherin, vimentin and MMP9 mRNA from total RNA preparation of (i) A549 and (ii) MDA-MB-231 cells, transduced independently

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with vector control, miR-101-expression vector, along with transfected UTR less RHOA or ZEB1 expression vector. Bar diagram depicts mean ± SEM of two independent experiments. (B) Western blot for vimentin, myc-tag for RHOA, flag-tag for ZEB1 and actin, using protein

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extracts of (i) A549 and (ii) MDA-MB-231 cells, transduced independently with vector control, miR-101-expression vector, along with transfected UTR less RHOA or ZEB1 expression vector.

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(C) Wound healing assay to assess the migration potential of (i) A549 and (ii) MDA-MB-231 cells, transduced independently with vector control, miR-101-expression vector, along with transfected UTR less RHOA or ZEB1 expression vector. The % wound distance was plotted after 20 hrs of introduction of the wound. Bar diagram depicts mean ± SEM of three separate experiments. (D) Gelatin-substrate zymograph for secreted MMP9 activity in (i) A549 and (ii) MDA-MB-231 cells, transduced independently with vector control, miR-101-expression vector, along with transfected UTR less RHOA or ZEB1 expression vector. Bar diagram in lower panel 23

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represents the arbitrary activity of MMP9. Bar diagram depicts mean ± SEM of two separate experiments. (E) Phalloidin staining in (i) A549 and (ii) MDA-MB-231 cells, transduced

RHOA or ZEB1 expression vector. Scale bar indicates 10 µm.

Figure 5: ERK2, not ERK1 downregulates miR-101

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independently with vector control, miR-101-expression vector, along with transfected UTR less

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(A) Promoter activity assay. Depicted cells were transfected either with miR-101-1-luc promoter or miR-101-2-luc promoter along with pRL-TK vector. After 48 hrs, firefly luciferase activity

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was measured, normalized with renilla luciferase activity and plotted as relative luciferase activity. Bar diagram depicts mean ± SEM of three independent assays. (B) Relative expression after qPCR quantitation of precursor-miR-101-1 and precursor-miR-101-2 from total RNA extract of depicted cells analyzed by 2-∆Ct method. Bar diagram depicts mean ± SEM of two

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independent experiments. (C) (i) Western to show pERK1/2 and actin; and (ii) Relative expression after qPCR quantitation of miR-101; using A549 cells stimulated with EGF in presence (for 24hrs) or absence of PD98059. Bar diagram depicts mean ± SEM of two separate

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experiments. (D) (i) Western to show ERK1/2 and actin; and (ii) Relative expression after qPCR quantitation of miR-101; using A549 and MDA-MB-231 cells, stably transduced with vector-

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control, shERK1, or shERK2 vectors. Bar diagram depicts mean ± SEM of two separate experiments. (E) qPCR quantitation of precursor-miR-101-1 and precursor-miR-101-2 in total RNA extract of (i) A549 and (ii) MDA-MB-231 cells, stably transduced with vector-control, shERK1, or shERK2 vectors. Bar diagram depicts mean ± SEM of two separate experiments. (F) (i) Western blot to show His-tagged-ERK2-CA, vimentin, and actin in protein extracts; (ii) qPCR quantitation of miR-101 in total RNA extract of A549 and MDA-MB-231 cells,

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transiently transduced with vector-control (pHAGE) and His-ERK2-CA encoding lentiviruses. Bar diagram depicts mean ± SEM of two separate experiments. (G) qPCR quantitation of precursor-miR-101-1 and precursor-miR-101-2 in total RNA extracts of (i) A549 and (ii) MDA-

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MB-231 cells, transiently transduced with vector-control (pHAGE) and His-ERK2-CA encoding lentiviruses. Bar diagram depicts mean ± SEM of two separate experiments.

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Figure 6: ZEB1 expression is necessary for ERK2 driven miR-101 downregulation

(A) Relative expression after qPCR quantitation of miR-101 in total RNA extract of (i) A549 and

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(ii) MDA-MB-231 cells, transiently transduced with vector (pHAGE) alone and His-ERK2-CA in combination with transduced shZEB1, or shMYC, or shELK1 lentiviruses. Bar diagram depicts mean ± SEM of two separate experiments. (B) Western blot to show pERK1/2, ERK1/2, vimentin and actin in protein extracts of (i) A549 and (ii) MDA-MB-231 cells, transiently

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transduced with vector (pHAGE) alone and His-ERK2-CA in combination with transduced shZEB1, or shMYC, or shELK1 lentiviruses. (C) Relative luciferase activity of miR-101-1 and miR-101-2-promoters in stably transduced A549 cells with vector-control and shERK2 with and

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without transfected pcDNA3-ZEB1-vector. Bar diagram depicts mean ± SEM of three independent assays. (D) Relative expression after qPCR quantitation of miR-101 in total RNA

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extract of cells transduced with vector-control and shERK2 with and without transfected pcDNA3-ZEB1-vector. Bar diagram depicts mean ± SEM of two separate experiments. (E) Western blot to show E-cadherin, vimentin, and actin in protein extracts of cells transduced with vector-control and shERK2 with and without transfected pcDNA3-ZEB1-vector. (F) Relative luciferase activity of miR-101-1-promoter and miR-101-2-promoter in transiently transduced A549 cells with vector-control (pHAGE) alone, and His-ERK2-CA with and without shZEB1

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transduction. Bar diagram depicts mean ± SEM of three independent assays. (G) qPCR quantitation of miR-101 expression in total RNA preparation of cells with vector-control (pHAGE) alone, and His-ERK2-CA with and without shZEB1 transduction. Bar diagram depicts

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mean ± SEM of two separate experiments. (H) Western blot to show E-cadherin, vimentin and actin in the protein extract of cells with vector-control (pHAGE) alone, and His-ERK2-CA with

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and without shZEB1 transduction.

Figure 7: Silencing of miR-101 in shERK2 cells results in increased mesenchymal

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characters

(A) Western blot to show E-cadherin, vimentin, ZEB1, EKR1/2 and actin in protein extract of (i) A549 and (ii) MDA-MB-231 cells stably transduced with vector-control and shERK2. (B) qPCR quantitation of E-cadherin and vimentin in total RNA extract of cells stably transduced with

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vector-control and shERK2. Bar diagram depicts mean ± SEM of two separate experiments. (C) Wound healing assay to assess the migration potential of A549 and MDA-MB-231 cells stably transduced for vector-control and shERK2. The plot shows the % wound distance after 20 hrs of

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introduction of wound. Bar diagram depicts mean ± SEM of three separate experiments. (D) Western blot to show E-cadherin, vimentin, pERK1/2, and actin in protein extract of A549 cells,

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stably transduced with vector-control, shERK2, with and without miR-101-silencing vector. (E) Relative expression after qPCR quantitation of E-cadherin and vimentin in total RNA extract of A549 cells, stably transduced with vector-control, shERK2, with and without miR-101-silencing vector. Bar diagram depicts mean ± SEM of two separate experiments. (F) Wound healing assay to access the migration potential of A549 cells, stably transduced with vector-control, shERK2, with and without miR-101-silencing vector. The plot shows the % wound distance after 20 hrs of

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introduction of wound. Bar diagram depicts mean ± SEM of three separate experiments. (G) Cell viability of A549 cells, stably transduced with vector-control, shERK2, with and without miR101-silencing vector; with and without the treatment of cisplatin (10µM for 24 hrs). Cell viability

depicts mean ± SEM of two independent assays.

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was measured by MTT assay and represented as percent of vector-control cells. Bar diagram

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Figure 8: ERK2-miR-101-1 axis is active in cell lines and breast tumor samples

(A) Western blot to show pERK1/2, ERK1/2, and actin in protein extracts of A549, H1299,

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MCF7, and MDA-MB-231 cells. (B) Relative expression after qPCR quantitation of miR-101 in total RNA extracts of A549, H1299, MCF7, and MDA-MB-231 cells; analyzed by 2-∆Ct method. Bar diagram depicts mean ± SEM of two independent assays. (C) Western blot to show pERK1/2 and ERK1/2 in protein extracts of normal and tumor pair samples of the depicted sporadic breast

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cancer stages. (D) qPCR quantitation of miR-101 in total RNA extract of tumor samples used in panel (C). (E) qPCR quantitation of miR-101 expression in total RNA extract of normal and tumor tissue pairs of sporadic breast cancer samples. Expression levels were analyzed by 2-∆Ct

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method. (F) Box plot for differential expression of miR-101 in normal and tumor samples with negative or positive nodal extension. Log2 fold expression of miR-101 is plotted. Bars represent

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the minimum to maximum distribution. (G) Box plot for precursor-miR-101-1 expression in normal and tumor tissues of studied sporadic breast cancer tissue samples analyzed by qPCR. Bars represent the minimum to maximum distribution. (H) Box plot for differential expression of pre-miR-101-1 in stage I+II vs stage III+IV. Bars represent the minimum to maximum distribution. (I) miR-101 expression profile output analysis using the data from Breast Cancer Gene-Expression Miner v4.0 (bc-GenExMiner v3.2). For each breast cancer subtype, the number

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of samples and percentage of samples with low, intermediate, or high miR-101 expression are indicated. (J) Box plot for miR-101 expression levels in different molecular subtypes. Global significant difference between groups is assessed by Welch's test. (K) Kaplan-Meier curve

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showing overall survival with high or low miR-101 expression (log rank p = 0.0017). (L) Kaplan-Meier curve showing recurrence-free survival with high or low miR-101 expression (log rank p = 0.0122). References

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