MicroRNA-206 suppresses TGF-β signalling to limit tumor growth and metastasis in lung adenocarcinoma

Cellular Signalling 50 (2018) 25–36

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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

MicroRNA-206 suppresses TGF-β signalling to limit tumor growth and metastasis in lung adenocarcinoma

T

Kathleen Watta,b, Daniel Newsteda,b, Elena Voorandb, Robert J. Goodinga,c,d, Adrianna Majewskib,d, Peter Truesdella,b, Binchen Yaob, Thomas Tuschle, Neil Renwickd,e, ⁎ Andrew W. Craiga,b, a

Cancer Biology & Genetics Division, Queen's Cancer Research Institute, Kingston, Canada Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada c Department of Physics, Queen's University, Kingston, Canada d Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada e HHMI Laboratory of RNA Molecular Biology, The Rockefeller University, New York, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lung cancer Metastasis microRNA miR-206 TGF-β Smad3

MicroRNA-206 (miR-206) has demonstrated tumor suppressive effects in a variety of cancers. Numerous studies have identified aberrantly expressed targets of miR-206 that contribute to tumor progression and metastasis, however, the broader gene-networks and pathways regulated by miR-206 remain poorly defined. Here, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumors to identify differentially expressed genes, and study the effects on tumor growth and metastasis. In H1299 tumor xenograft assays, stable expression of miR-206 suppressed both tumor growth and metastasis in mice. Profiling of xenograft tumors using small RNA sequencing and a targeted panel of tumor progression and metastasis-related genes revealed a network of genes involved in TGF-β signalling that were regulated by miR-206. Among these were the TGFB1 ligand, as well as direct transcriptional targets of Smad3. Other differentially expressed genes included components of the extracellular matrix involved in TGF-β activation and signalling, including Thrombospondin-1, which is responsible for the activation of latent TGF-β in the stroma. In cultured lung adenocarcinoma cells treated with recombinant TGF-β, ectopic expression of miR-206 impaired canonical signalling, and expression of TGF-β target genes linked to epithelial-mesenchymal transition. This was due at least in part to the suppression of Smad3 protein levels in lung adenocarcinoma cells with ectopic miR-206 expression. Together, these findings indicate that miR-206 can suppress tumor progression and metastasis by limiting autocrine production of TGF-β, and highlight the potential utility of TGF-β inhibitors for the treatment of lung adenocarcinomas.

1. Introduction Lung cancer is the leading cause of all cancer-related mortalities, resulting in 1.6 million deaths worldwide annually [1]. Non-small cell lung cancers (NSCLC) account for close to 85% of cases, with lung adenocarcinomas and squamous cell carcinomas making up the majority of these. Despite a growing understanding of driver mutations in NSCLCs, current therapies are still failing to halt the progression to metastatic disease, and the five-year survival of patients with the disease is very poor at around 15% [2]. In recent years, some improvements have been made in extending the survival of NSCLC patients with the use of immune checkpoint inhibitors like pembrolizumab,

compared to platinum-based chemotherapies. However, fewer than 30% of NSCLCs are positive for programmed cell death ligand-1 (PDL1), and < 50% of these patients respond to treatment [3]. This represents a critical gap in cancer therapy, where an improved understanding of molecular mechanisms linked to NSCLC progression and metastasis has the potential to identify new therapeutic targets and drive the development of more effective treatment strategies. Increasingly, studies of the molecular mechanisms of cancer metastasis have implicated microRNAs (miRNAs), a class of non-coding RNA that negatively regulate gene expression at a post-transcriptional level [4]. miRNAs are often dysregulated in disease states like cancer, leading to changes in target mRNA expression and activity of signalling

Abbreviations: NSCLC, non-small cell lung cancer; miRNAs, microRNAs; CSCs, cancer stem cells; EMT, epithelial-mesenchymal transition; TGF-β, transforming growth factor-β; BMPs, bone morphogenetic proteins; GDFs, growth differentiation factors; ECM, extracellular matrix ⁎ Corresponding author at: Cancer Biology & Genetics, Queen's University, CRI 315, 18 Stuart St., Kingston, Ontario K7L 3N6, Canada. E-mail address: [email protected] (A.W. Craig). https://doi.org/10.1016/j.cellsig.2018.06.008 Received 7 February 2018; Received in revised form 18 June 2018; Accepted 18 June 2018 Available online 20 June 2018 0898-6568/ © 2018 Published by Elsevier Inc.

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benefit of inhibiting TGF-β signalling in metastatic lung adenocarcinoma.

pathways. Disrupted miRNA regulatory networks can contribute to maintenance of cancer stem cells (CSCs), tumor recurrence, drug resistance, epithelial-mesenchymal transition (EMT), mesenchymal-epithelial transition, and drive metastasis in numerous cancer models [5–7]. Investigating the genes and pathways regulated by miRNA with tumor or metastasis-suppressive effects can provide useful insights into the mechanisms driving these processes and aid in the identification of new therapeutic targets. miRNA-206 (miR-206) is a member of the miR-1 sequence family of miRNAs that is most highly expressed in skeletal muscle. The miR-206 gene is located on chromosome 6 in a cluster with miR-133b, while other family members miR-1-1/miR-133a-2, as well as miR-1-2/miR133a-1 exist in paralogous clusters on separate chromosomes [8, 9]. At the onset of myogenesis, increased expression of miR-206 promotes myoblast differentiation from a more stem-like state into mature skeletal muscle [10]. A link between miR-206 and metastasis was first established in triple-negative breast cancer, where it was found that ectopic expression reduced metastasis in mice [11]. A growing number of studies continue to identify miR-206 as having tumor and metastasis suppressive effects when expressed in breast cancer, melanoma, rhabdomyosarcoma, medulloblastoma, and NSCLC [10, 12–19]. Expression of miR-206 in NSCLC has been shown to alter tumor growth, survival, EMT, and chemotherapy resistance [14, 15, 18, 20]. Several potential driver genes in NSCLC, including MET and KRAS, have been identified as direct targets of miR-206 [21]. However, most studies to date have focused on validating a limited number of direct targets. A more systematic analysis of tumor progression genes and pathways regulated by miR-206 is needed to understand the mechanism of its anti-tumor effects on NSCLCs, and identify targets for intervention. The TGF-β superfamily is composed of > 30 secreted factors, including TGF-β family ligands, and, bone morphogenetic proteins (BMPs), and growth differentiation factors (GDF), among others. TGF-β signalling affects a wide variety of cell types and cellular processes, and its role in cancer progression and metastasis is both pleiotropic, and highly context-dependent. In normal epithelium or premalignant lesions, TGF-β signalling can have a tumor-suppressive function by regulating cell proliferation, apoptosis, and differentiation. However, in advanced cancers where these pathways are defective, TGF-β signalling can be co-opted by tumor cells to drive processes such as EMT, cell invasion, angiogenesis, immune evasion, and modulation of the tumor microenvironment [22]. In the canonical pathway, binding of TGF-β1 ligand dimers to TGFBR2 triggers TGFBR1 activation and phosphorylation of Smad2 and Smad3, which translocate to the nucleus with Smad4 to regulate expression of hundreds of genes [22, 23]. This includes a number of miRNAs whose biogenesis and expression are regulated by TGF-β signalling via Smad proteins [24]. In lung adenocarcinoma, increased expression of TGF-β1 has been associated with poor survival and increased risk of pulmonary metastasis [25, 26], and treatments with TGF-β1 have been shown to increased metastasis in mouse models [27]. In A549 lung adenocarcinoma cells, TGF-β1 promotes EMT and resistance to chemotherapeutic drugs. Further to this, an EMT signature in NSCLC tumor samples is linked to poor prognosis [28]. However, the mechanisms that regulate TGF-β signalling in lung adenocarcinoma remain poorly defined. In this study, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumor xenograft models, and used a targeted panel of tumor progression and metastasis genes to identify pathways regulated by miR-206. Using this approach, we discovered that miR206 suppresses TGF-β expression, signalling, and target gene expression. These effects of miR-206 were explained by reduced Smad3 expression leading to loss of transcriptional activation of target genes, as well as suppression of genes involved in the activation of latent TGF-β in the stroma. The loss of TGF-β signalling likely explains the profound suppression of metastasis we observed with ectopic miR-206 in lung adenocarcinoma tumor xenografts. These findings functionally link miR-206 and TGF-β signalling, and highlight the potential therapeutic

2. Materials and methods 2.1. Cell culture and reagents Human lung adenocarcinoma cell lines were cultured at 37 °C in a 5% CO2 atmosphere. NCI-H1299 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (SigmaAldrich) supplemented with 10% fetal bovine serum (PAA Laboratories Inc.); A549 cells (kindly provided by Dr. Susan Cole, Queen's University) were grown as above supplemented with 5% fetal bovine serum. NCI-H1975 and HCC827 cells (American Type Culture Collection) were grown in RPMI medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum. The identity of all cell lines was verified by STR profiling using the GenePrint 10 System (Promega), carried out at The Center for Applied Genomics at the Hospital for Sick Children (Toronto, Canada). 2.2. Generation of cell lines with stable and inducible expression of miR-206 Stable expression of miR-206 was achieved using a pEZX-MR03 lentiviral construct encoding the human gene fragment containing the precursor miR-206 (pre-miR-206), and a GFP reporter. Control cell lines were produced using a pEZX-MR03 construct encoding a scrambled control miR (CmiR) and GFP (Genecopoeia). To generate doxycycline (DOX)-inducible miR-206 expression, the entire human miR-206 gene was PCR amplified and subcloned in the ClaI/MluI sites of the pTRIPZ vector (GE Dharmacon) and sequence verified (see Suppl. Table 1 for primer sequences). The empty pTRIPZ (TZ) vector was used to produce control cell lines. Expression of miR-206 and RFP was induced by treating cells with 2 μg/mL DOX for 48 h. To produce lentiviruses, HEK293T cells were transfected with miR-206 or control constructs, as well as pCMVΔR8.91 packaging plasmid (15 μg), and pMD.2G envelop plasmid (6 μg) using X-tremeGene HP DNA transfection reagent (Roche). Conditioned media was collected 48 and 72 h after transfection and filtered using 0.45 μm sterile filters. Viral supernatants of similar titers were applied to target cells (MOI ~1), and transduced cell pools were selected using 2 μg/mL puromycin. 2.3. Cell proliferation and cisplatin treatment H1299 TZ-206 and TZ cells were pre-treated with 2 μg/mL DOX for 24 h before being seeded at a density of 2000 cells/well in 96 well plates in DMEM supplemented with 5% FBS and incubated at 37 °C and 5% CO2. The IncuCyte Zoom imaging system (Essen Bioscience) was used to capture 3 images per well at 10× magnification every 2 h over an 84-h period. Images were analyzed to quantify cell proliferation using the IncuCyte Zoom software. To evaluate the effects of miR-206 on H1299 sensitivity to cisplatin, cells were seeded as described above, and treated with increasing concentrations of cisplatin (0.5–20 μg/mL) (CIS; Kingston General Hospital Pharmacy). Cytotoxicity was quantified using the Cytotox Green reagent (Essen Bioscience) at a concentration of 125 nM. The IncuCyte Zoom imaging system was used to capture 3 images per well at 10× magnification every 2 h over a 72-h period. Images were analyzed to quantify cell death using the IncuCyte Zoom software. IC50 values were calculated by dose-response nonlinear regression using GraphPad Prism software. 2.4. Tumor xenograft and tail vein injection assays For subcutaneous tumor xenograft assays, H1299 miR-206 or CmiR cells (106 cells/100 μL in DMEM with 20% Matrigel™) were subcutaneously injected into the hind flank of 6–8-week old male Rag2−/−:IL-2Rγc−/− mice in three independent experiments. After 26

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Table 1 Differentially expressed cancer progression genes in H1299 tumor xenografts with ectopic expression of miR-206. Gene

TGFB1 DAG1 PLAUR BMPR2 VEGFC RPS6KB1 VIM AKAP2 AKT3 FBN2 NME1 ADD1 TYMP TP53 MMP17 RRAS ID1 GDF6 ZEB2

Annotation

Predicted miR-206 binding site

– – – – – – – – – – – – – – – – – Targetscan, miRanda –

Transforming growth factor beta-1 Dystroglycan Urokinase plasminogen activator surface receptor Bone morphogenetic protein receptor type-2 Vascular endothelial growth factor C Ribosomal protein S6 kinase beta-1 Vimentin A-kinase anchor protein 2 RAC-gamma serine/threonine-protein kinase Fibrillin-2 Nucleoside diphosphate kinase A Alpha-adducin Thymidine phosphorylase Cellular tumor antigen p53 Matrix metalloproteinase-17 Ras-related protein R-Ras DNA-binding protein inhibitor ID-1 Growth/differentiation factor 6 Zinc finger E-box-binding homeobox 2

miR-206 vs. CmiR Fold change

p-Value

−1.64 −1.35 −1.74 −1.53 −2.06 1.51 −1.28 −1.88 −1.41 −1.46 1.38 −1.17 −2.65 −1.35 −1.61 −1.55 −2.99 −3.73 1.22

0.0006 0.0007 0.0008 0.0010 0.0011 0.0014 0.0014 0.0015 0.0017 0.0023 0.0029 0.0029 0.0030 0.0037 0.0037 0.0056 0.0072 0.0074 0.0100

from tumor xenografts was hybridized to specific capture and barcoded reporter probes before being immobilized on a cartridge. Each target RNA molecule was quantified by a nCounter Digital Analyzer via the fluorescent barcoded probes. The barcoded images captured by the automated fluorescent microscope were pre-processed for quality control metrics based on field of view registration (FOV) and binding density. All samples had FOV registration > 75%, and binding densities within a 0.05–2.25 range, falling within standard quality specifications. The resulting raw gene expression data from the 8 tumor xenografts was processed and normalized in three steps. First, normalization factors derived from the spiked-in positive controls (Fig. S1) were calculated for each sample by dividing the geometric means of the positive controls across all samples, by the geometric mean of each given sample. Next, a conservative background subtraction was performed using the mean plus two standard deviations of the 8 negative controls across all samples. Finally, sample content normalization factors were calculated in the same manner as for the positive controls, using the geometric mean of the housekeeping genes for the 8 tumor samples. Of the housekeeping genes, 7 were computationally predicted to contain miR206 binding sites and were treated as endogenous genes, leaving 23 genes appropriate for normalization (Fig. S2). All normalization factors fell between 0.75 and 1.35, within the accepted range of 0.3–3. A total of five methods of normalization were compared; four based on subsets of the housekeeping genes filtered into stringency categories based on coefficient of variation, as well as one that used the geometric mean of the 75 most highly expressed genes to generate normalization factors. All methods yielded similar results, with the 10 most significantly differentially expressed genes being largely conserved across normalization methods (Fig. S3). Statistically significant differentially expressed genes comparing H1299 CmiR and miR-206 tumor xenografts were identified using the Welch t-test assuming unequal sample variances. Thresholds of p = 0.01 and p = 0.05 were selected to provide a list of high-confidence genes (see Table 1 and Suppl. Table 3). Volcano and scatter plots of differentially expressed genes were generated using R version 3.3.1, in the RStudio interface. Of the genes included in the PanCancer Progression Panel, those involved in the TGF-β pathway were identified using the KEGG database, online tools GeneMANIA and STRING [31, 32], and a list of genes altered by TGF-β treatment in A549 cells (GSE17708; Ref (33)). Heat maps visualizing TGF-β pathway genes was generated using the HeatMapViewer module on the GenePattern gene expression analysis platform [34].

35–40 days, the mice were sacrificed and primary tumors and lung tissues were harvested. For experimental lung seeding assays, H1299 miR-206 or CmiR cells (106 cells/100 μL in DMEM) were injected into the tail vein of both male and female Rag2−/−:IL-2Rγc−/− mice in two independent experiments. Mice were sacrificed after 14 days and lung tissues were harvested. Fresh lung tissues containing metastases and seeded tumors from both types of experiment were imaged using a fluorescent microscope, and tumor burden was quantified via expression of the GFP reporter using Image-Pro software (Media Cybernetics Inc.). All experiments were approved by the Queen's University Animal Care Committee in accordance with the Canadian Council for Animal Care regulations. 2.5. Small RNA sequencing Total RNA was isolated from H1299 miR-206-expressing and control cell lines and tumor xenografts using TRIzol Reagent (Invitrogen), and small RNA sequencing (RNAseq) was carried out as previously described [29]. Briefly, total RNA (100 ng) was ligated to barcoded 3′ oligonucleotide adaptors. Small RNA molecules with 3′adaptors were then specifically selected by size-fractionation on a denaturing polyacrylamide gel. Samples were then pooled and a second ligation of a 5′ oligonucleotide adaptor was then performed, before reverse-transcription and PCR amplification. The resulting cDNA library was sequenced on an Illumina HiSeq 2500 platform in the Genomics Resource Center at The Rockefeller University. Sequence files were processed and assigned annotations as previously described [30]. A list of differentially expressed miRNAs was generated by comparing mean values for replicate tumor samples. Any miRNA with differences greater than two standard deviations between replicates were discarded. The remaining miRNAs with a fold change greater than two between miR-206 and CmiR tumors are shown in Supplemental Table 2. 2.6. NanoString nCounter gene expression analysis RNA was extracted from H1299 cell lines (stable or inducible miR206 expression), and xenograft tumors (CmiR or miR-206), and hybridized to the human PanCancer Progression codeset containing specific probes to 740 endogenous and 30 housekeeping genes (NanoString Technologies). The panel also contained 6 spiked-in positive controls with concentrations between 128 fM and 0.125 fM, as well as 8 synthetic negative control sequences. Briefly, 100 ng of total RNA isolated 27

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luciferase activities according to the manufacturer's instructions (DualLuciferase Reporter Assay System, Promega). Firefly luciferase luminescence was normalized to that of Renilla luciferase.

2.7. TGF-β treatments, quantitative RT-PCR, and immunoblotting A549 and H1299 cells with stable and inducible miR-206 expression (pre-treated with DOX for 48 h) were plated at a density of 300,000 cells/well in 6-well plates. After an overnight serum-starvation, 10 ng/ mL of TGF-β1 (PeproTech) was added to each well. Cells were harvested at time points between 24 and 72 h to prepare lysates or RNA. For quantitative RT-PCR, total RNA from cultured cells, or xenograft tumors was converted to cDNA by reverse-transcription using the iScript Select cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was then performed using the iQ SYBR Green Supermix Kit (Bio-Rad). Relative gene expression was calculated using the comparative CT method, using GAPDH as a reference housekeeping gene (primer sequences are provided in Suppl. Table 1). For immunoblotting, cells or tumor tissues were lysed in RIPA lysis buffer (50 mM Tris; 5 mM EDTA; 150 mM NaCl; 1% NP-40; 0.5% Sodium Deoxycholate; 0.1% SDS; 10 μg/mL aprotinin; 10 μg/mL leupeptin; 1 mM Na3VO4; 100 μM phenylmethylsulfonyl fluoride; 50 mM NaF). Primary antibodies were used at the following concentrations: N-Cadherin (1:1000; Cell Signalling Tech (CST)); ECadherin (1:1000; CST); Smad3 (1:500; Santa Cruz Biotech (SCBT)); βActin (1:1000; SCBT); Gapdh (1:1000; GeneTex); α-Actinin (1:1000; SCBT). Horseradish peroxidase-conjugated sheep anti-mouse or antirabbit immunoglobulin (1:5000) and ECL Western Blotting Substrate (Thermo Scientific) were used for detection. Densitometry analysis was carried out using ImageJ software (RSB).

2.11. Statistical analysis All statistical analyses were carried out in R or GraphPad Prism software, unless otherwise specified. Bar graphs and scatter plots indicate the mean with error bars representing the standard deviation (SD) or (SEM), where appropriate. Unless otherwise stated, statistical significance was determined by Student's t-test, or by two-way ANOVA. 3. Results 3.1. Ectopic expression of miR-206 causes decreased lung adenocarcinoma cell growth Numerous studies have reported miR-206 downregulation in cancer, with a link to high tumor grade, increased risk of metastasis, and poor prognosis [11–13, 17, 35, 36]. However, few studies have provided insights into the gene networks controlled by miR-206 in these cancers. To investigate these gene networks in lung adenocarcinoma, H1299 cells were transduced with lentiviral vectors allowing stable ectopic expression of precursor miR-206 or a scrambled nontargeting control miRNA (CmiR). For an inducible system, H1299 cells were transduced with a TRIPZ (TZ)-based lentivirus that allows for doxycycline (DOX)-inducible expression of primary miR-206 transcripts (TZ-206). Cells were also transduced with the empty TZ virus to serve as a control for DOX treatments. To measure levels of miR-206 expression achieved in these cells, we performed quantitative barcoded small RNAseq (sequencing statistics provided in Supplementary File 1). We showed that both stable and DOX-inducible expression systems were highly effective in increasing the expression of miR-206 from negligible levels, to between 5 and 6% of the total miRNA reads. As a point of reference, these levels were still well below that of miR-21 (20–30%; Fig. 1A), an endogenous miRNA that is frequently overexpressed in cancers [37]. Importantly, expression of other endogenous gene clusters or sequence family members, which were expressed at very low or undetectable levels in H1299 cells, were not altered in our model (Suppl. Table 4). After confirming the efficacy of our expression systems, we then tested the effects of miR-206 on H1299 cell growth. Over an 84-h period, we found that miR-206 expression led to a significant reduction in cell growth compared to control cells (Fig. 1B). In addition, we tested the effects of miR-206 on the response of H1299 cells to the platinumbased chemotherapy drug cisplatin. Cells with ectopic miR-206 expression were more sensitive to cisplatin-induced killing, with an IC50 of 1.12 μg/mL (95% CI 0.83–1.54 μg/mL) compared to an IC50 of 2.41 μg/mL (95% CI 1.59–5.02 μg/mL) for the control cells (Fig. 1C). These results demonstrate that the levels of miR-206 expression achieved produce a growth suppressive effect, and increased sensitivity to killing by a chemotherapy drug for NSCLC.

2.8. Cell invasion assay H1299 cells with inducible miR-206 expression were seeded in 0.2% FBS DMEM with 2 μg/mL DOX at a density of 20,000 cells/well in 96well Image-Lock plates (Essen Bioscience). Cells were pre-treated with 10 ng/mL TGF-β1 for 24 h before a wound was induced using the WoundMaker tool (Essen Bioscience). The wounds were then overlaid with 0.2% FBS DMEM containing 10% Matrigel™, with and without 10 ng/mL TGF-β. Invasion of cells into the wound area was then monitored over 24 h using the IncuCyte Zoom imaging system capturing images at 10× magnification every 2 h. Images were analyzed to quantify the increase in wound confluence using the IncuCyte Zoom software. 2.9. Thrombospondin-1 protein analysis Thrombospondin-1 protein was detected in homogenates from H1299 tumor xenografts using the Quantikine Human Thrombospondin-1 solid-phase ELISA Immunoassay (R&D Systems). Briefly, recombinant human Thrombospondin-1 protein standard, and H1299 (CmiR or miR-206) tumor xenograft homogenates (n = 4) were added (50 μL) to each well of a microplate pre-coated with a monoclonal antibody specific for human Thrombospondin-1. Subsequent steps for detection were according to the manufacturer's instructions and a microplate spectrometer. 2.10. Luciferase reporter assay

3.2. Ectopic miR-206 expression limits H1299 xenograft tumor growth and metastasis

For Smad2/3-induced luciferase reporter assays, a Caga(12)-Luc construct containing 12 repeated Smad transcriptional response elements (TRE) upstream of Firefly luciferase was used (kindly provided Dr. JJ LeBrun, McGill University). A549 cells (miR-206 or CmiR) were seeded at a density of 750,000 cells/well in 6 well plates. Cells were transfected with both Caga(12)-Luc (2.25 μg) and an empty Renilla luciferase vector control, pLightSwitch_3UTR (0.25 μg), using XtremeGENE HP DNA transfection reagent (Roche). After 24 h, media was replaced with serum-free media for an additional 24 h. After 48 h, media was replaced with either serum-free (unstimulated), or serumfree supplemented with 10 ng/mL TGF-ß1 (PeproTech) for 10 h. Cells were lysed and luciferase activity assayed for Firefly and Renilla

To study the effects of miR-206 on lung adenocarcinoma tumors in vivo, H1299 CmiR and miR-206 cells were injected in the hind flank of Rag2−/−:IL2Rγc−/− mice that lack NK, B and T cells. Subcutaneous tumor growth was monitored until the humane endpoints were reached at 35–40 days for mice harboring H1299 CmiR tumors, with results pooled from three independent experiments. Compared to CmiR tumors, we noted a significant reduction in tumor mass with ectopic miR206 expression (Fig. 2A). This is consistent with suppression of cell growth by miR-206 in vitro, although the magnitude of the effect of miR-206 was greater in vivo. To quantify levels of miR-206 expression 28

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Fig. 1. Ectopic expression of miR-206 causes decreased lung adenocarcinoma cell growth. A) Small RNA sequencing of H1299 cell lines with stable and DOXinducible miR-206 expression was performed. Breakdown of miR-206 levels shown with miR-21 as reference of endogenous miRNA overexpression. B) Proliferation of H1299 TZ and TZ-206 cells treated with 2 μg/mL DOX was measured over 84 h. Significant differences indicated between untreated TZ and TZ-206 cells (n = 3, ** p < 0.01). C) H1299 TZ and TZ-206 cells were treated with 2 μg/mL DOX and increasing concentrations of cisplatin (CIS) (0.5–20 μg/mL). Cell death was measured at 72 h using Cytotox Green fluorescent dye

these experimental metastasis assays for H1299 CmiR cohort, but with a similar reduction in lung seeding for H1299 miR-206 cohort (Fig. 2E and F). These results are consistent with miR-206 functioning as a both a tumor suppressor gene and metastasis suppressor gene in lung adenocarcinoma.

within xenograft tumors, we extracted total RNA and performed quantitative barcoded small RNAseq. This revealed that ectopic miR206 expression was maintained in vivo, with a > 100-fold increase in mature miR-206 levels, from around 0.01% of total mature miRNA sequence reads in the H1299 CmiR tumors, to > 1.5% in the miR-206 group (Fig. 2B). The increased expression of miR-206 expression in xenograft tumors relative to CmiR tumors was also validated by quantitative RT-PCR (Fig. S4). Although ectopic miR-206 expression did not alter expression of the endogenous sequence family (Suppl. Table 5), we did detect differential expression of a number of other miRNAs in the H1299 miR-206 tumors (Suppl. Table 2). This likely reflected effects of miR-206 on transcription factor networks that control expression of these miRNAs. Notably, the most highly differentially expressed miRNA between the CmiR and miR-206 tumors was miR-29b, which was increased by 3.2-fold in tumors with miR-206 expression. Next, we examined the effects of ectopic miR-206 expression on metastasis in H1299 xenograft tumor models. As we showed previously, subcutaneous H1299 tumors primarily metastasize to the lungs in these mice [38, 39]. Indeed, the lung tissues isolated from the CmiR xenograft cohorts described above had numerous spontaneously arising micrometastases (marked by a GFP reporter), with significantly fewer micrometastases observed upon ectopic expression of miR-206 (Fig. 2C and D). These results validate miR-206 as a metastasis suppressor gene in lung cancer. To test the effects of miR-206 on metastasis using an experimental model of lung metastasis, we performed tail vein injections with H1299 CmiR and miR-206 cells to measure lung seeding and colonization efficiencies. As expected, the lung metastases were more extensive in

3.3. TGF-β signalling is suppressed in H1299 xenograft tumors with ectopic miR-206 expression Although several miR-206 target genes with relevance to NSCLC (e.g. MET, KRAS, and EGFR) have been identified [14–16, 21], we interrogated the effects of miR-206 expression on a focused panel of tumor progression genes and pathways in vivo. RNA was isolated from H1299 CmiR and miR-206 xenograft tumors and hybridized to the PanCancer Progression gene codeset (NanoString). Of the genes queried, 421 were expressed above background levels, and 53 genes showed statistically significant differential expression between miR-206 and CmiR tumors (p < 0.05), including several genes with predicted miR-206 binding sites (Suppl. Table 3). Nineteen of these differentially expressed genes met a higher threshold for statistical significance (p < 0.01) (Table 1). Interestingly, this included the TGFB1 transcript, with expression reduced by > 1.5-fold in the miR-206 tumors. To determine the extent of defective TGF-β signalling in the miR-206 tumors, all of the profiled genes were referenced against the KEGG pathway database, GeneMANIA, STRING [31, 32, 40], and a microarray study of TGF-β-regulated genes in A549 cells (GSE17708) [33]. These findings were visualized in a volcano plot, with genes involved in TGF-β signalling highlighted in red (Fig. 3A). Although not all TGF-β-related 29

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Fig. 2. miR-206 limits H1299 xenograft tumor growth and metastasis. A) Graph depicts the mass of xenograft tumors harvested 35–40 days after subcutaneous injection of H1299 miR-206 (n = 12) or CmiR (n = 8) cells in the hind flank of male Rag2−/−IL2Rγc−/− mice (** p < 0.01). B) Small RNAseq was performed on RNA from two tumors from each group (miR-206 and CmiR). Plot depicts the levels of miR-206 as a percent of the total miRNA reads for each sample. C) Spontaneously arising GFP-positive lung micrometastases from mice with subcutaneous tumors were imaged by fluorescence microscopy (CmiR n = 8, miR-206 n = 12; 40× magnification). D) Graph depicts the relative lung area marked by metastases. Percent GFP-positive lung area was quantified using Image-Pro software (** p < 0.01). E) Lung tumor seeding ability of H1299 CmiR and miR-206 cells was assessed following tail vein injections in male and female Rag2−/−IL2Rγc−/− mice. Lung tissues were harvested and tumor burden visualized 14 days after injection. Representative micrographs shown for mice from CmiR and miR-206 groups (n = 8/group). F) Percent GFP-positive lung area was quantified as in B, (* p < 0.05).

may therefore limit the activation of latent TGF-β in the tumor stroma. A heatmap was prepared to visualize the effects of miR-206 on expression of TGF-β pathway-related genes in individual H1299 miR-206 and CmiR tumor samples. Interestingly, 16 of 23 TGF-β pathway genes that were downregulated in the miR-206 tumors (Fig. 3D), were previously shown to be upregulated by TGF-β in A549 cells in vitro [33]. Of four TGF-β-related genes that were significantly upregulated in the miR-206 tumors (Fig. 3D, Table 1, Suppl. Table 3), PLS1 and MYO5C were previously shown to be suppressed by TGF-β [33]. Together, these results provide novel evidence for TGF-β pathway suppression by miR206 in lung adenocarcinoma tumors, with links to autocrine TGF-β production (tumor-derived) and stromal activation of TGF-β via THBS1.

genes were altered in miR-206 tumors, they dominated the list of differentially expressed genes meeting statistical significance. Many of these genes were downregulated in miR-206 tumors, based on the shift to the left along the x-axis (Fig. 3A). In addition to canonical TGF-β signalling-related genes like TGFB1 and PLAUR, other TGF-β family pathway genes were also altered, such as the Type II BMP receptor (BMPR2). Several candidate genes responsive to TGF-β1 signalling (TGFB1, PLAUR, NME1, THBS1) [41–43], were selected for further validation by other methods in xenograft tumors. Reduced expression of TGFB1, PLAUR and THBS1 transcripts, along with increased expression of NME1, were validated in miR-206 tumors compared to CmiR using quantitative RT-PCR (Fig. 3B). Interestingly, NME1 is a known metastasis-suppressor gene that is frequently downregulated in NSCLC [44]. Since Thrombospondin-1 is responsible for stromal release of active TGF-β [33, 45], we performed an ELISA assay on tumor homogenates to test Thrombospondin-1 protein levels. While Thrombospondin-1 was detected in CmiR tumors, the levels were significantly reduced in miR206 tumors (Fig. 3C). Loss of Thrombospondin-1 in miR-206 tumors

3.4. Ectopic miR-206 expression suppresses the EMT response to TGF-β signalling in lung adenocarcinoma cells The ability of TGF-β to promote EMT, including cadherin switching, is linked to cancer progression and metastasis. To interrogate the potential effects of miR-206 on TGF-β-driven EMT and cadherin 30

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Fig. 3. miR-206 suppresses TGF-β signalling in H1299 xenograft tumors. A) Gene expression profiling was performed on H1299 miR-206 and CmiR tumor xenografts using the NanoString PanCancer Progression panel (n = 4/group). All genes expressed above background after normalization are shown in a volcano plot illustrating the log2 fold change and statistical significance (−log10 of the p-value, Welch's t-test) between miR-206 and CmiR tumors. Vertical dotted red lines indicate a fold change of +/− 2, and the horizontal dotted blue lines indicate p-values of 0.05 and 0.01, with statistical significance increasing vertically. Points filled in red indicate genes that are known to be involved in, or responsive to TGF-β signalling. B) Quantative RT-PCR validation of differentially expressed mRNA transcripts using specific primers and RNA from the same tumor xenografts used in NanoString profiling (n = 4). Relative transcript expression normalized to GAPDH, (*p < 0.05, ** p < 0.01). C) THBS1 protein levels detected by ELISA in H1299 tumor xenograft homogenates (n = 4) (** p < 0.01). D) Heat map visualizing the TGF-β pathway-related genes identified out of those significantly differentially expressed between tumor xenograft groups. +/− indicate whether expression of the gene is known to be induced or suppressed after TGF-β stimulation of A549 cells [47]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Ectopic miR-206 expression dampens TGF-β signalling in lung adenocarcinoma cells. A and B) Expression of CDH1, CDH2, and THBS1, in H1299 (TZ and TZ206) and A549 (miR-206 and CmiR) cells was measured by quantitative RT-PCR after a 48 h stimulation with 10 ng/mL TGF-β. Graph depicts the relative foldchange, normalized to GAPDH. Significance between Control + TGF-β and miR-206 + TGF-β treatment groups indicated on both graphs (n = 3, * p < 0.05). Error bars indicate SEM. C and D) A549 miR-206 and CmiR cells were treated with 10 ng/mL TGF-β for up to 72 h and subjected to immunoblot analysis with antibodies to E- and N-Cadherin, with β-actin and α-actinin as loading controls. E and F) Relative fold change for E-cadherin and N-cadherin immunoblots determined by densitometry analysis (n = 3). G) Invasion of H1299 TZ and TZ-206 cells treated with DOX and TGF-β (10 ng/mL) into a wound area covered in Matrigel, and measured over 24 h (n = 4, * p < 0.05; ns, not significant).

(Fig. 4A). However, the cadherin switching effect of TGF-β was impaired in miR-206 expressing cells, with no significant changes in CDH1, CDH2 or THBS1 expression upon treatment (Fig. 4A). To test whether autocrine production of TGF-β in H1299 cells had any impact on these results, we repeated these assays following transient transfection with siRNA targeting TGFB1 (or GFP as a control). With similar levels of TGFB1 transcript silencing in H1299 TZ and TZ-206 cells, we observed modest effects of autocrine TGF-β on expression of EMT genes

switching, we treated miR-206-expressing H1299 and A549 lung adenocarcinoma cell lines, or their controls, with or without TGF-β1 (10 ng/ml) for 48 h. RNA was isolated and subjected to quantitative RTPCR to measure changes in several genes that are either induced (CDH2, THBS1), or repressed (CDH1) by TGF-β. In H1299 TZ control cells, treatment with recombinant TGF-β led to expected changes in gene expression, with suppression of CDH1 (encoding E-cadherin), and increased CDH2 (encoding N-Cadherin) and THBS1 expression 32

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ultimate effects of miR-206 on Smad2/3-driven transcription, we used a firefly luciferase reporter controlled by 12 tandemly repeated Smad transcriptional response elements (TRE) (Fig. 5C). This construct was co-transfected with a constitutively expressed Renilla Luc construct in A549 CmiR or miR-206 cells. After 48 h, cells were treated with or without TGF-β1 (10 ng/ml) for 10 h, and the relative Smad-induced luciferase activity was measured. In CmiR cells, TGF-β treatment caused a ≈ 41-fold increase in Smad-driven luciferase expression, compared to a ≈ 25-fold increase in miR-206 cells based on repeated experiments (Fig. 5D). These results are consistent with reduced Smad2/3-driven transcription of target genes in cells with miR-206 expression. In light of the reduced Smad3 expression and Smad2/3-driven transcription in cells with miR-206 expression, we compared the differentially expressed genes from the H1299 CmiR and miR-206 xenograft tumors with a publicly available database sequential chromatin immunoprecipitation dataset for promoter regions bound by Smad3 following TGF-β1 stimulation (GSE28346) [43]. These Smad3 target genes from TGF-β1 treated cells were also enriched within the differentially expressed genes from the miR-206-expressing xenograft tumors, and included three of the most highly significant genes: TGFB1, PLAUR, and NME1 (Fig. S6). Together, these findings suggest that the suppression of TGF-β signalling by miR-206 in lung adenocarcinoma may be due to silencing of Smad3 expression.

(Fig. S5). SMAD3 levels were significantly reduced in TZ control relative to siGFP controls, consistent with positive regulation by autocrine TGF-β, and disruption of this positive feedback loop in TZ-206 cells (Fig. S5). As expected, levels of THBS1 were lowest in TZ-206 cells, and significant when compared to siGFP controls (Fig. S5). In A549 cells, the suppressive effect of miR-206 on THBS1 induction was validated, and a similar trend was observed for CDH2, however, no statistically significant effects were observed for CDH1 and CDH2 transcripts after 48 h of treatment with TGF-β (Fig. 4B). To extend on these findings, we performed a time course of treatments with recombinant TGF-β using A549 CmiR and miR-206 cell lines. The A549 cells were selected for these studies since they exhibit a partial EMT phenotype that is driven to completion by TGF-β treatment [46]. Cells were stimulated with TGF-β for up to 72 h and lysates were subjected to immunoblot to assess levels of E-cadherin and N-cadherin proteins, along with loading controls (β-actin, α-actinin). In CmiR control cells, TGF-β treatment led to > 50% reduction in E-cadherin levels and 300% increase in N-Cadherin within 48 h (Fig. 4C and D). These treatment effects were greatly reduced in A549 cells with ectopic miR-206 expression (Fig. 4C–F). To further test the consequence of the partial EMT defect with ectopic miR-206 expression in lung adenocarcinoma cells, we performed cell invasion assays. In H1299 cells with inducible miR-206 expression, the effects of media supplementation with recombinant TGF-β failed to significantly potentiate H1299 cell invasion through Matrigel compared to TZ control cells (Fig. 4G). Together, these results demonstrate a suppressive effect of miR-206 on cadherin switching and induction of the EMT response in lung adenocarcinoma cells responding to TGF-β1.

4. Discussion In recent years, miRNAs have emerged as key tumor suppressor and/or metastasis suppressor genes [7, 47]. Several studies have identified miR-206 as a putative tumor and metastasis suppressor gene when overexpressed in cancer cells [10–13, 17, 21, 48, 49]. Many of these studies have validated a limited number of cancer-related targets of miR-206, however, the broader impact of miR-206 on tumor progression gene networks remains poorly defined. In this study, we engineered lung adenocarcinoma cell lines with stable or inducible ectopic expression of miR-206, and performed tumor xenograft assays to identify genes and pathways regulated by miR-206 that are implicated in tumor progression and metastasis. We found that tumor growth and metastasis were considerably reduced by miR-206 expression, validating its utility as a tumor and metastasis suppressor in lung adenocarcinomas. We detected differential expression of numerous TGF-β pathway genes linked to cancer progression and metastasis within

3.5. Smad3 expression is suppressed in the presence of ectopic miR-206 in lung adenocarcinoma cells Next, to address how miR-206 suppresses TGF-β signalling we measured effects on the canonical pathway, including SMAD2 and SMAD3. First, we measured the levels of SMAD2 and SMAD3 transcripts in A549 and H1299 cells with CmiR or miR-206 expression by quantitative RT-PCR. SMAD3 transcript levels were significantly lower with miR-206 expression in A549 cells, and trended lower in H1299 cells (Fig. 5A, data not shown). In addition, Smad3 protein levels were reduced by 20–60% in A549 and H1299 cell models using both stable and inducible miR-206 expression systems (Fig. 5B). To measure the

Fig. 5. Smad3 expression is suppressed in the presence of ectopic miR-206 in lung adenocarcinoma cells. A) SMAD3 transcript levels were measured in H1299 (miR-206 and CmiR, as well as TZ and TZ206) and A549 (miR-206 and CmiR) cells using quantitative RT-PCR. Graph depicts the relative foldchange, normalized to GAPDH (n = 3). Error bars indicate SEM (* p < 0.05; ns, not significant). B) A549 (miR-206 and CmiR) and H1299 (miR-206 and CmiR, as well as TZ and TZ-206) cells were subjected to immunoblot analysis with antibodies to Smad3, with β-actin as a loading control. Relative fold changes determined by densitometry analysis. C) Schematic of the luciferase construct expressing firefly luciferase (Luc) under the control of tandemly repeated Smad transcriptional response elements (TRE) bound by p-Smad2/3. D) The TRE-Luc reporter and constitutively expressed renilla Luc constructs were transfected into A549 CmiR and miR206 cells. After 48 h, wells were treated with or without TGF-β (10 ng/ml) for an additional 10 h. Bar graph shows firefly luminescence normalized to renilla, and relative to levels in untreated control cells (n = 3, ** p < 0.01).

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feedback loop that drives further expression of THBS1 as a result of increased TGF-β signalling [45]. Loss of expression of these activating factors in the extracellular matrix may lead to reduced signalling from stromal-derived TGF-β, resulting in the widespread disruption of the pathway observed in the tumor xenografts. In melanoma cells, treatment with exogenous TGF-β led to increased autocrine TGF-β1 production [58], with similar results found in A549 cells [33]. Although TGFB1 does not appear to be a direct target of miR-206, the loss of key activators of latent TGF-β in the ECM, like Thrombospondin-1, may disrupt this positive feedback by stromal TGF-β on the autocrine production of TGFB1 within the tumors. Due to these findings, and the observation that miR-206 expression reduced both spontaneous and experimental metastasis, we propose that regulation of TGF-β signalling by miR-206 spans both autocrine production and stromal activation of the ligand. However, further studies using a syngeneic model of lung adenocarcinoma will be needed to more fully define the role of miR-206 in regulating TGF-β signalling in the tumor microenvironment in the presence of an intact immune system. Increased expression of TGF-β1 has been associated with poor survival, enhanced metastasis, increased EMT, and resistance to chemotherapy in lung adenocarcinoma [25–28]. In this study, we have confirmed that ectopic expression of miR-206 causes defects in lung adenocarcinoma tumor growth and metastasis, and suppresses the TGFβ signalling pathway in vivo. Given the known role of TGF-β in modulating the immune response [59], these findings also suggest that blockade of TGF-β signalling following the diagnosis of lung adenocarcinomas is a therapeutic avenue worth exploring. This may limit immune suppression, and enhance the response rate for immune checkpoint inhibitor treatment regimes, which have improved outcomes in lung adenocarcinoma [3]. These findings also highlight the utility of our method of in vivo tumor xenograft profiling as a tool for elucidating miRNA-gene network relationships. The widespread effects of miR-206 on the TGF-β pathway were not as apparent when gene expression profiling was conducted in cells grown in 2D culture. By profiling tumors grown in vivo, the role of miR-206 in regulating factors that influence the tumor microenvironment were revealed. This represents a critical limitation of miRNA targeting studies carried out in 2D cell culture that often focus on single targets, and is a shortfall that should be addressed in future studies. In light of the nature of miRNA-mediated regulation of gene expression, this study demonstrates the ability of a miRNA to regulate large networks of genes, and highlights the advantages of using in vivo methods to identify critical pathways driving tumor progression and metastasis. These findings also demonstrate the potential value of miRNA-based therapeutics as adjuvant therapies to broadly disrupt these driver pathways. Such therapeutics could provide robust effects, helping to bypass issues of genetic heterogeneity and single-target drug resistance that often render current treatment options ineffective for many cancer patients. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellsig.2018.06.008.

xenograft tumors with enforced miR-206 expression. This included transcriptional targets of Smad3 including TGFB1, and a number of proteins involved in stromal TGF-β activation, including Thrombospondin-1. In vitro, we confirmed that miR-206 suppresses TGF-β signalling and expression of several downstream target genes, potentially due to the suppression of Smad3 expression that we observed in multiple lung adenocarcinoma cell lines. Together, these findings suggest that miR-206 is able to suppress TGF-β signalling through multiple mechanisms involving both the suppression of autocrine production in lung tumors, and downregulation of secreted factors responsible for stromal activation of TGF-β. The link between miR-206 and TGF-β was first established in murine myogenic progenitor cells, where it was found that TGF-β treatment inhibited expression of the mature forms of miR-206 and miR-29, which could in turn reciprocally inhibit TGF-β and prevent TGF-β-induced expression of the epigenetic regulator HDAC4 [50]. A more recent study using A549 cells also showed that the human SMAD3 transcript is directly targeted by miR-206, leading to negative feedback inhibition between miR-206 and TGF-β1 [19]. Here, we have validated the suppressive effect of miR-206 on Smad3 protein levels, however a statistically significant reduction of SMAD3 mRNA transcripts was not observed in all cell lines tested, or tumor xenografts with miR-206 expression. Since direct targeting of SMAD3 by miR-206 has been demonstrated, this less consistent reduction in transcript level could be accounted for by the mechanism of miRNA-mediated gene silencing whereby translation is repressed in the absence of transcript degradation [51]. Despite the subtle effect on miR-206 on SMAD3 transcript levels, the significant decrease in Smad2/3-driven transcription with ectopic miR-206 expression can be explained, at least in part, by the overall reduction in Smad3 protein levels. In addition, since TGFB1 is a direct transcriptional target of Smad3 [43], the significantly reduced expression of autocrine TGFB1 we observed in our miR-206-expressing cell lines and tumors is likely due to the reduction in Smad3 protein expression. Interestingly, although suppression of SMAD3 by miR-206 has been shown to be direct in humans [19], an indirect mechanism was proposed in murine cells [50]. This supports the notion that alternate mechanisms of Smad3 and TGF-β signalling repression by miR206 also exist. These additional mechanisms may include the disruption of feedback loops regulating autocrine TGF-β production, like the increase in miR-29b expression we observed in the H1299 miR-206 tumors (Suppl. Table 2), which is also known to be reciprocally inhibited by TGF-β [50]. Another relevant feedback mechanism involves BMPR2, which was suppressed in the miR-206-expressing tumors, and whose ligand BMP2 (a member of the TGF-β superfamily) is known to block miR-206 maturation [52]. Considering our findings of reduced tumor-derived human TGFB1 transcripts in the H1299 miR-206 tumor xenografts, a reduction in autocrine signalling due to decreased Smad3 and disrupted feedback mechanisms provides a partial explanation for the suppression of the TGF-β pathway by miR-206. In fact, a recent study in estrogen receptor positive breast cancer cells also demonstrated that miR-206 inhibited autocrine production of TGF-β, limiting EMT and cell migration and invasion [53]. However, many TGF-β-signalling-related genes are not targets of Smad3. TGF-β can also be produced by stromal cancer-associated fibroblasts in lung adenocarcinoma [54], and the extent of the expression changes in TGF-β pathway genes in the tumor xenografts with miR-206 expression would suggest the significance of stromal TGF-β in vivo. A number of the genes downregulated in the H1299 miR206 tumor xenografts (THBS1, PLAUR, FBN1 and FBN2) are directly involved in latent TGF-β activation and signalling in the ECM [55]. One of the most highly downregulated genes, PLAUR, is vital in the activation of plasmin, which is able to activate latent TGF-β [56]. FBN1 and FBN2 assemble into microfibrils that also play a role in regulating the localization and activation of TGF-β in the ECM [57]. Thrombospondin1 is responsible for activation of latent TGF-β by inducing conformational changes in the latency associated peptide (LAP), contributing to a

Acknowledgments The authors kindly thank Stephanie Young and Colleen Schick for technical assistance with tumor xenograft studies. This work was supported by grants from Cancer Research Society (20142) and Canadian Institutes for Health Research (MOP 119562) to AWC. Salary support was provided by Ontario Graduate Scholarship award to KW, and the Terry Fox Transdisciplinary Training Program in Cancer Research at Queen's University to KW and DN. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. 34

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