TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1

Accepted Manuscript TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1

Helena J. Janse van Rensburg, Dulcie Lai, Taha Azad, Yawei Hao, Xiaolong Yang PII: DOI: Reference:

S0898-6568(18)30185-2 doi:10.1016/j.cellsig.2018.08.012 CLS 9165

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

29 May 2018 16 August 2018 17 August 2018

Please cite this article as: Helena J. Janse van Rensburg, Dulcie Lai, Taha Azad, Yawei Hao, Xiaolong Yang , TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1. Cls (2018), doi:10.1016/j.cellsig.2018.08.012

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ACCEPTED MANUSCRIPT TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1 Helena J. Janse van Rensburg1, Dulcie Lai1,2, Taha Azad1,2, Yawei Hao1, Xiaolong Yang1* 1.

Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario

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K7L 3N6, Canada

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2. Equal Contribution

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Financial support: Canadian Institute of Health Research (#119325, 148629) (X Yang), Canadian Breast Cancer Foundation/Canadian Cancer Society (X Yang)

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*Correspondence: Dr. Xiaolong Yang

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Richardson Laboratories, 88 Stuart Street, Kingston, Ontario K7L 3N6, Canada 613-533-6000 x75998

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[email protected]

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Declarations of interest: none

ACCEPTED MANUSCRIPT ABSTRACT WW domain-containing transcriptional regulator 1 (TAZ) is a transcriptional co-activator and effector of the Hippo signaling pathway. In certain breast cancer subtypes, Hippo signaling is dysregulated leading to activation of TAZ and altered expression of TAZ transcriptional

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targets. Over the past decade, we and others have found that TAZ transcriptionally regulates

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genes that affect multiple aspects of breast cancer cell behaviour. However, while cancer cell-

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intrinsic oncogenic functions of TAZ have emerged, less is known about whether TAZ might also contribute to tumourigenesis by sensitizing tumour cells to factors present in the tumour

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microenvironment or in systemic circulation. Here, we show that TAZ directly regulates the

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expression of insulin receptor substrate 1 (IRS1) in breast cancer cells. TAZ or IRS1 overexpression induces a similar proliferative transformation phenotype in MCF10A mammary

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epithelial cells. TAZ enhances IRS1 mRNA, protein levels and downstream signaling in

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MCF10A. Mechanistically, TAZ interacts with the IRS1 promoter through the TEAD family of transcription factors and enhances its activity. Critically, TAZ-induced IRS1 upregulation

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contributes to the proliferation of TAZ-overexpressing MCF10A in 3-dimensional (3D) Matrigel

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culture. Therefore, we offer compelling evidence that TAZ regulates signaling through the insulin pathway in breast cancer cells. These findings highlight an additional mechanism by

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which TAZ promotes breast cancer tumourigenesis and progression by modulating cancer cell responses to exogenously produced factors.

KEYWORDS: TAZ, IRS1, NT157, Hippo pathway, insulin signaling, spheroid formation

ACCEPTED MANUSCRIPT 1. INTRODUCTION The Hippo pathway is an evolutionarily conserved regulator of tissue growth and homeostasis that is frequently dysregulated in human cancers [1,2]. In the mammalian Hippo pathway, MST1/2 serine/threonine (S/T) kinases phosphorylate and activate LATS1/2 S/T

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kinases which subsequently phosphorylate two transcriptional co-activators, TAZ and its paralog

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YAP [3–7]. Phosphorylation of TAZ or YAP by LATS1/2 leads to their sequestration by 14-3-3

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proteins in the cytoplasm, degradation and functional inactivation. The Hippo pathway effector TAZ is an oncogene that is highly expressed in breast cancer [8]. Over the past decade, mounting

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evidence has emerged implicating TAZ in breast cancer tumourigenesis and progression. We and

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others have shown that TAZ directly regulates gene targets that are involved in various aspects of breast cancer cell behaviour (e.g. anchorage-independent growth, epithelial-to-mesenchymal

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transition, cell migration/invasion, chemotherapy resistance and stem cell phenotypes) [8–13].

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Therefore, we have concluded that TAZ has evident cancer cell-intrinsic oncogenic functions. However, relatively less is known about whether TAZ can contribute to breast tumourigenesis by

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sensitizing transformed cells to factors that might be present in the tumour microenvironment or

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in systemic circulation. Indeed, although several growth factors (e.g. CTGF, AREG) have been found to be directly regulated by TAZ, whether TAZ regulates genes that confer breast cancer

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cells with a selective advantage in the presence of certain exogenously-produced growth signals is not completely understood [12,14]. IRS1 is an adaptor protein and a component of the insulin signaling pathway [15,16]. In the presence of insulin or IGF-I/II, IRS1 binds to the cytoplasmic domains of the insulin receptor (IR) or IGF-IR and facilitates signal transduction downstream to the PI3K/AKT and MAPK signaling pathways thereby inducing metabolic and mitogenic responses in target cells [17,18].

ACCEPTED MANUSCRIPT Like Hippo signaling, insulin signaling is dysregulated in breast cancer [19,20]. Hyperinsulinemia or elevated plasma IGF-I levels have been linked to increased breast cancer incidence while approximately half of all breast cancers show expression and activation of IR or IGF-IR [21–24]. Interestingly, IRS1 acts as an oncogene in breast cancer despite having no

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enzymatic activity. Mechanistically, IRS1 overexpression enhances breast cancer cell sensitivity

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to insulin/IGF-I and is sufficient for increased cell proliferation/suppression of apoptosis in vitro

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as well as for tumourigenesis and metastasis in vivo in transgenic mice [25,26]. While these phenotypes are compelling, the mechanisms underlying endogenous IRS1 upregulation in breast

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cancer have been poorly understood with only a few transcriptional regulators of IRS1 (e.g.

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PKC, estrogen) having been identified to date [27–30].

In an effort to characterize the molecular mechanisms by which TAZ acts as an oncogene

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in breast cancer, we have previously performed a whole human genome microarray screen for

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TAZ-regulated genes in an immortalized breast epithelial cell line, MCF10A, with stable TAZ overexpression [12]. In this screen, we identified CTGF and CYR61 as genuine TAZ

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transcriptional targets and we have since validated several other genes (e.g. BMP4, Np63) in

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subsequent work [11–13]. IRS1 was among the top upregulated candidate TAZ gene targets that were discovered in our initial microarray analysis. Here, we show that IRS1 is transcriptionally

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regulated by TAZ in human breast cancer cells. We further show that upregulation of IRS1 by TAZ contributes to the phenotype of TAZ-overexpressing MCF10A in 3D Matrigel culture. Through this work, we have uncovered an additional mechanism by which TAZ promotes breast cancer cell transformation and, in doing so, have provided evidence that TAZ activation may sensitize breast cancer cells to exogenously-produced growth factors.

ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS 2.1. Cell culture MCF10A, MDA-MB-231, HBE-135 and SK-BR-3 cell lines were purchased from ATCC. Cells were maintained at 37C with 5% CO2 in the following culture media: MCF10A: DMEM/F12-

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Ham, 5% Horse Serum, 1% P/S, 2.5 mM L-glutamine, 10 μg/mL insulin, 0.5 μg/mL

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hydrocortisone, 100 ng/mL cholera toxin and 20 ng/mL hEGF; MDA-MB-231: DMEM, 10%

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FBS, 1% P/S, 1% non-essential amino acids; HBE-135: Keratinocyte-SFM, 2.5 ng/mL EGF, 50

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μg/mL bovine pituitary extract, 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 1% P/S; SK-BR-3: McCoy's 5A, 10% FBS, 1% P/S. 293T cells from ATCC were used for virus production and

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were maintained in DMEM with 10% FBS and 1% P/S. The Doxycyline (Dox)-inducible stable cell lines were treated with 1 μg/mL Dox for 48 hours to induce overexpression of TAZ, YAP or

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IRS1 constructs. To assess AKT and ERK1/2 phosphorylation in MCF10A, cells were cultured

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overnight in complete media without supplemental hEGF. To determine the efficacy of IRS1 inhibition, MCF10A were treated with an IRS1 inhibitor, NT157 (ApexBio), at 500 nM, 1 μM or

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5 μM for 24 hours. All experiments were performed using cells with passage number less than

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40.

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2.2. Plasmid construction, establishment of stable cell lines and site-directed mutagenesis To generate constructs for the Dox-inducible stable cell lines, cDNAs were cloned into a modified pTRIPZ vector (puromycin resistant) using the following primers: AgeI-TAZ-F (5’ ATACCGGTACCATGAATCCGGCCTCGGCG 3’), MluI-HA-TAZ-R (5’ CATACGCGTTT ATGCGTAGTCTGGGACATCGTATGGATACAGCCAGGTTAGAAAGG 3’), AgeI-IRS1-F (5’ ATACCGGTATGGCGAGCCCTCCGGAGAGC 3’), MluI-IRS1-R (5’ ACGACGCGTCTA

ACCEPTED MANUSCRIPT CTGACGGTCCTCTGGCTG 3’), AgeI-YAP-F (5’ ATACCGGTACCATGGATCCCGGGCA GCAGCCG 3’) and MluI-YAP-R (5’ CGACGCGTCTATAACCATGTAAGAAAG 3’). Methods for lentivirus production/infection are as previously described [31]. For transient gene expression, cDNAs were cloned into pCDNA3.1 using the following primers: BamHI-TAZ-F (5’

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CGGGATCCACCATGAATCCGGCCTCGGCG 3’), NotI-TAZ-R (5’ CGCGTTAACTGCGGC

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CGCTTACAGCCAGGTTAGAAAGG 3’), BglII-TEAD1-F (5’ GAAGATCTATGGAAAGGA

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TGAGTGACTC 3’), NotI-TEAD1-R (5’ GTAATCATGCGGCCGCTGTTCAGTCCTTTACA AGCC 3’), BamHI-TEAD2-F (5’ ATGGATCCATGGGGGAACCCCGGGCTGG 3’), XbaI-

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TEAD2-R (5’ ACTCTAGACCTTCAGTCCCTGACCAGGC 3’), BamHI-TEAD3-F (5’

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CGGGATCCATGATAGCGTCCAACAGCTGGAACGCC 3’), NotI-TEAD3-R (5’ GTAATCATGCGGCCGCCTAGTCTTTGACGAGCTTGTAGAC 3’), BamHI-TEAD4-F (5’

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CGGGATCCATGTTGGAGGGCACGGCCGGCAC 3’), NotI-TEAD4-R (5’ GTAATCATG

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CGGCCGCTCATTCTTTCACCAGCCTG 3’) and NotI-TAZΔ227-R (5’ GTAACAATGCGGC CGCTCAAGTGGTCAGCGCATTGGGCATAC 3’). The RUNX2 plasmid was a generous gift

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from Dr. G. Karsenty. Mutant forms of TAZ, YAP and TEAD4 were constructed using

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overlapping PCR and the following primers: TAZ-S89A-F (5’ CATGTCCGCTCGCACGCGTC GCCCGCGTCCCTG 3’), TAZ-S89A-R (5’ CAGGGACGCGGGCGACGCGTGCGAGCGGA

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CATG 3’), YAP-S127A-F (5’ CATGTTCGAGCTCATGCCTCTCCAGCTTCTCTG 3’), YAPS127A-R (5’ CAGAGAAGCTGGAGAGGCATGAGCTCGAACATG 3’), TAZ-F52/53A-F (5’ GGAAGAAGATCCTGCCGGAGTCTGCCGCTAAGGAGCCTGATTCGGGCTCG 3’), TAZF52/53A-R (5’ CGAGCCCGAATCAGGCTCCTTAGCGGCAGACTCCGGCAGGATCTTCT TCC 3’), TEAD4-Y429H-F (5’ GCTCAGCACCACATCCACAGGCTGGTGAAAGAA 3’) and TEAD4-Y429H-R (5’ TTCTTTCACCAGCCTGTGGATGTGGTGCTGAGC 3’). To construct

ACCEPTED MANUSCRIPT the IRS1 promoter (IRS1-P) reporter, a fragment corresponding to positions -1000 to +82 in the IRS1 promoter was PCR-amplified from HeLa cell genomic DNA using the following primers and was cloned into a pGL3-basic vector: MluI-IRS1-P-F: 5’ TAAACGCGTCTGTGTGTGAA ACAAACATTTCAGCC 3’) and XhoI-IRS1-P-R: 5’ AATCTCGAGCTCGGAGAGTTGCCGA

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GAGCCCCAAC 3’). The TEAD response element mutant IRS1 promoter reporter (IRS1-P-

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TREM) was constructed by overlapping PCR using the following primers: IRS1-TREM-F (5’

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GCGGGGCGGGCTCGGCCCAAAAAGTAGAGACCCGGGCGGGA 3’) and IRS1-TREM-R

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(5’ TCCCGCCCGGGTCTCTACTTTTTGGGCCGAGCCCGCCCCGC 3’).

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2.3. Three-dimensional (3D) Matrigel spheroid formation assay Methods for 3D Matrigel spheroid formation assay are as described [32]. In brief, TAZ-S89A or

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IRS1-overexpressing MCF10A cells were stimulated with 1 μg/mL Dox for 24 hours. The

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following day, 8-well chamber slides (Ibidi) were coated with 50 μL Matrigel (Corning) per well and 1.5 x 103 MCF10A cells were plated in 300 μL media with 2% Matrigel (with or without 1

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μg/mL Dox) on top of the base Matrigel layer. Spheroid formation was monitored each day after

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plating and the upper layer of media with 2% Matrigel was replaced with fresh media (with or without Dox) every fourth day. For some experiments, cells were plated on Matrigel with various

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concentrations of IRS1/2 inhibitor NT157 (500 nM, 1 μM, 5 μM) or an equal volume of DMSO. In another experiment, cells were plated on Matrigel with IR/IGF-IR inhibitors (Linsitinib, NVPAEW541 or GSK1904529A from Selleckchem) at 1 μM or an equal volume of DMSO. Representative images of spheroids were captured at 4X, 10X, 40X and 60X magnification using a Nikon TE-2000U inverted microscope. Relative spheroid sizes were quantified from 4X magnification images using the Particle Analysis tool from ImageJ software. For DAPI staining,

ACCEPTED MANUSCRIPT spheroids were fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.5% Triton X-100 at 4C for 10 minutes and then stained with DAPI (1:300 in PBS) for 30 minutes at room temperature. Confocal images of spheroids were captured at 10X magnification using a Quorum Wave FX spinning disc confocal microscope and were analyzed

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using MetaMorph image analysis software.

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2.4. Western blot and antibodies

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Methods for Western blot are as previously described [3]. Antibodies and their corresponding dilution ratios used were as follows: IRS1 (Cat. No. 2382), 1:500; YAP/TAZ (D24E4), 1:1000;

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phospho-AKT (S473) (D9E), 1:1000; AKTI (2H10), 1:1000; phospho-ERK1/2 (T202/Y204) (D13.14.4E), 1:2000; ERK1/2 (137F5), 1:2000 and PTEN (26H9), 1:1000 from Cell Signaling;

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TAZ (M2-616), 1:1000 from BD Biosciences; TEAD4 (Cat. No. ab58310), 1:1000 from Abcam

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and -actin (A5441), 1:10000 from Sigma.

2.5. Quantitative real-time PCR (qRT-PCR)

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Methods for total RNA collection and qRT-PCR are as previously described [31]. Primers used are as follows: qIRS1-F (5’ TGGACATCACAGCAGAATGAAGAC 3’), qIRS1-R (5’

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ATGGATGCATCGTACCATCTACTG 3’), qrRNA-F (5’ TCCCCATGAACGAGGAATTCC 3’) and qrRNA-R (5’ AACCATCCAATCGGTAGTAGC 3’).

2.6. Transient gene knockdown using siRNA For transient knockdown of TAZ, YAP or TEADs, targeting siRNAs (or scrambled siControl) were transfected into cells using Lipofectamine RNAiMAX reagent (Life Technologies)

ACCEPTED MANUSCRIPT according to the manufacturer’s standard protocol and a final concentration of 50 nM siRNA per reaction. The siRNA sequences used were as follows: siTAZ (5’ GACAUGAGAUCCAUCACU AAUAATA 3’), siYAP (5’ GGUCAGAGAUACUUCUUAAAUCACA 3’) and siTEAD (5’ CACAAGACGUCAAGCCUUU 3’ and 5’ UUGUGGAUGAAGUUGAUCAUU 3’). 48 hours

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after transfection, knockdown efficiency was assessed by Western blot.

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2.7. Dual luciferase assays

Methods for luciferase assays are as previously described [33]. In brief, 100 ng IRS1 promoter

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reporter (IRS1-P) were co-transfected into SK-BR-3 alongside 200 ng of TAZ construct and/or

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200 ng TEAD construct using PolyJet transfection reagent (SignaGen). 10 ng/well of Renilla luciferase pRL-TK plasmid was included as an internal control. Total DNA was adjusted to 510

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ng/well using pCDNA3. 24 hours after transfection, transfection media was replaced with fresh

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media. 48 hours after transfection, luciferase assays were performed using the Dual-Luciferase Reporter Assay kit from Promega. Relative IRS1 promoter activity was calculated as the ratio of

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Firefly luciferase signal to Renilla luciferase signal which was then normalized to the

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Firefly/Renilla ratio of control wells that had been transfected with the promoter reporter alone.

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2.8. Chromatin Immunoprecipitation (ChIP) ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) from Cell Signaling as described [33]. MCF10A-TAZ-S89A were stimulated with 1 μg/mL Dox for 72 hours to induce TAZ-S89A expression. 2 x 107 cells were treated with 1% formaldehyde to crosslink chromatin and associated proteins. Chromatin was digested using 3.75 μL micrococcal nuclease and nuclei were disrupted by gentle sonication. 10 μg of the digested chromatin was

ACCEPTED MANUSCRIPT used for immunoprecipitation with 2 μg TAZ antibody (M2-616 from BD Biosciences) or with 2 μg normal mouse IgG (Santa Cruz) and protein G magnetic beads. After ChIP, a fragment from the IRS1 promoter was PCR-amplified from the eluted DNA using the following primers: IRS1P-(-161)-F (5’ TCGGCGCGGGCGCCGCTGCAGCA 3’) and IRS1-P-(-34)-R (5’

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CTCCGCGCTCGGCAGCCGGGCAG 3’). Total chromatin extract was used as a positive

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(“input”) control for the PCR. The PCR products were visualized by standard agarose gel

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electrophoresis.

2.9. Statistical analysis

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All statistical analysis was performed using a Student’s T-test and/or ANOVA (with post-hoc

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analysis). p-value (p) < 0.05 was defined as the threshold for statistical significance.

ACCEPTED MANUSCRIPT 3. RESULTS 3.1. TAZ increases mammary cell proliferation in 3D Matrigel culture in the presence or absence of supplemental insulin In order to determine whether IRS1 is a TAZ-regulated gene that contributes to the

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oncogenic activity of TAZ, we first compared the transformation phenotypes induced by TAZ or

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IRS1 overexpression in 3D cell culture and assessed whether insulin contributes to these

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phenotypes. We established an MCF10A that overexpresses constitutively active TAZ (MCF10A-TAZ-S89A) in a Dox-inducible manner. When cultured on Matrigel, wild-type

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MCF10A form small and highly organized spheroids or acini (Figure 1A) [32,34]. However,

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under the same culture conditions, Dox-treated MCF10A-TAZ-S89A formed larger spheroids with a more disorganized structure (Figure 1A-C). We next investigated the role of insulin in the

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normal growth of MCF10A on Matrigel. Indeed, MCF10A growth media typically includes 10

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μg/mL of supplemental insulin. When cultured in complete growth media lacking this supplemental insulin, wild-type MCF10A formed smaller spheroids (Figure 1D,E). However,

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Dox-treated MCF10A-TAZ-S89A remained capable of forming larger, disorganized spheroids

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even in the absence of supplemental insulin (Figure 1F,G). The haphazard spheroid structure induced by TAZ-S89A was readily appreciable with higher magnification microscopy (Figure

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1H) and when nuclei were visualized with DAPI (Figure 1I). Therefore, TAZ enhances mammary cell proliferation and disrupts spheroid structure in 3D Matrigel culture in the presence or absence of supplemental insulin.

3.2. IRS1 overexpression enhances mammary cell proliferation in 3D culture We next established a Dox-inducible MCF10A cell line that overexpresses IRS1 (MCF10A-IRS1). Like TAZ-S89A, IRS1 overexpression increased the size of MCF10A

ACCEPTED MANUSCRIPT spheroids grown on Matrigel both in the presence and absence of supplemental insulin (Figure 2A-C). IRS1-overexpressing spheroids also demonstrated irregular structure under higher magnification (Figure 2D) but showed relatively more cellular organization than their TAZS89A-overexpressing counterparts when nuclei were visualized with DAPI (Figure 2E).

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Therefore, IRS1 promotes mammary cell proliferation in 3D Matrigel culture in the presence or

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absence of supplemental insulin and induces a proliferative phenotype in this system that

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resembles that of TAZ-S89A.

3.3. TAZ regulates IRS1 expression and downstream signaling through AKT and ERK1/2

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In order to further validate the relationship between TAZ and IRS1, we measured endogenous IRS1 levels in our MCF10A-TAZ-S89A cell line. TAZ-S89A induced IRS1 protein

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and mRNA upregulation in MCF10A (Figure 3A,B). In contrast, IRS1 protein and mRNA levels

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were unchanged in response to overexpression of constitutively active TAZ paralog YAP-S127A in MCF10A. Thus, IRS1 primarily appears to be regulated by TAZ but not YAP. Consistent with

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this, transient knockdown of TAZ in MDA-MB-231 human breast cancer cells suppressed IRS1

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mRNA expression whereas YAP knockdown had no effect on IRS1 mRNA expression levels in this cell line (Figure 3C).

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As previously described, IRS1 functions downstream of IR and IGF-IR to activate signaling through PI3K/AKT and MAPK pathways [17,18]. Therefore, we further assessed whether TAZ can induce signaling downstream of IRS1 through these pathways. Indeed, TAZS89A enhanced phosphorylation of AKT and ERK1/2 in MCF10A that were cultured in complete growth media with supplemental insulin (but without EGF) (Figure 3D). Although others have reported that YAP increases AKT phosphorylation through miRNA-29-induced

ACCEPTED MANUSCRIPT downregulation of PTEN, we did not observe downregulation of PTEN by TAZ-S89A overexpression in MCF10A (Supplementary Figure S1) [35]. Similar increases in IRS1 expression as well as AKT phosphorylation were observed when TAZ-S89A was overexpressed in a human immortalized lung/bronchus epithelial cell line, HBE135 (Figure 3E).

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Phosphorylation of AKT was also apparent in our inducible IRS1-overexpressing MCF10A cell

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line (Figure 3F). Interestingly, only minor changes in ERK1/2 phosphorylation were observed in

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TAZ-S89A-overexpressing HBE135 and in IRS1-overexpressing MCF10A (Figure 3E,F). Given that we observed increased cell proliferation with TAZ-S89A overexpression in

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the absence of supplemental insulin in our 3D Matrigel culture experiments, we also evaluated

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whether TAZ regulates IRS1 and its downstream signaling in complete culture media without supplemental insulin or EGF. Interestingly, under these culture conditions, TAZ-S89A induced

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IRS1 upregulation and phosphorylation of ERK1/2 but not AKT (Figure 3G). This finding

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suggests that TAZ-induced AKT phosphorylation may be dependent at least in part on the

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presence of supplemental insulin in growth media.

factors

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3.4. TAZ transcriptionally regulates IRS1 through the TEAD family of transcription

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We next set out to elucidate the mechanisms by which TAZ upregulates IRS1. TAZ acts as a transcriptional co-activator and associates with various transcription factor families (e.g. TEADs, RUNX2) to regulate gene expression [36–39]. Therefore, we tested whether interactions with TEADs are necessary for upregulation of IRS1 by TAZ. When overexpressed in MCF10A, a constitutively active, TEAD-binding mutant form of TAZ (MCF10A-TAZ-S89A-F52/53A) could not upregulate IRS1 expression or signaling through AKT (Figure 4A). Similarly,

ACCEPTED MANUSCRIPT transient knockdown of TEADs abolished IRS1 upregulation by TAZ-S89A and phosphorylation of AKT in MCF10A (Figure 4B). Thus, TEAD family transcription factors are essential for TAZ-induced upregulation of IRS1 and its downstream signaling through AKT. We further constructed a luciferase-based reporter for the IRS1 promoter (IRS1-P)

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spanning positions -1000 to +82 relative to the transcriptional start site. We tested the

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responsiveness of this reporter to TAZ and TEADs in SK-BR-3 human breast cancer cells. Co-

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expression of wild-type TAZ and TEADs (TEAD1-4) enhanced IRS1 promoter activity (Figure 4C). Moreover, constitutively active TAZ-S89A and TEAD4 co-expression caused a further

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increase in IRS1 promoter activity while TAZ without its transcriptional co-activation domain

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(TAZ227), TAZ-binding mutant TEAD4 (TEAD4-Y429H) and RUNX2 all could not increase IRS1 promoter activity (Figure 4D). Within the IRS1 promoter, we recognized a sequence that

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could correspond to a putative TEAD response element (TRE; GGAAT; positions -94 to-90). We

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mutated this sequence to generate a TRE mutant IRS1 promoter reporter (IRS1-P-TREM)

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(Figure 4E). This construct showed dramatically reduced activation by TAZ and TEAD4 compared to the wild-type construct indicating that these five nucleotides are likely to interact

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with TAZ/TEAD4 during their activation of the IRS1 promoter (Figure 4F). Finally, we evaluated whether TAZ physically interacts with the IRS1 promoter by ChIP.

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We used our TAZ-S89A-overexpressing MCF10A cell line to immunoprecipitate TAZ alongside its associated chromatin. Indeed, a fragment from the IRS1 promoter (positions -161 to -34) was detectable by PCR after TAZ immunoprecipitation (Figure 4G). Collectively these data suggest that TAZ physically interacts with the IRS1 promoter through the TEAD family of transcription factors to enhance promoter activity and upregulate IRS1 expression.

ACCEPTED MANUSCRIPT 3.5. Pharmacological inhibition of IRS1 suppresses TAZ-induced mammary cell proliferation in 3D culture We finally set out to determine whether upregulation of IRS1 by TAZ contributes to the TAZ overexpression phenotypes that we had observed in 3D Matrigel culture. We

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pharmacologically inhibited IRS1 using a compound (NT157) that causes IRS1/2 degradation

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[40]. Indeed, treatment of MCF10A-TAZ-S89A with NT157 suppressed TAZ-induced IRS1

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upregulation in a dose-dependent manner (Figure 5A). IRS1 inhibition with NT157 also reduced the proliferation of MCF10A-TAZ-S89A on Matrigel in the presence or absence of supplemental

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insulin (Figure 5B,C). We further examined whether inhibition of the insulin pathway receptors

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(IR/IGF-IR) upstream of IRS1 might also affect TAZ-S89A-stimulated mammary cell proliferation in 3D culture. Indeed, TAZ-S89A-induced cell proliferation on Matrigel was

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suppressed by various IR/IGF-IR-targeting agents (Linsitinib, NVP-AEW541 or GSK1904529A)

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(Figure 5D,E). Therefore, we conclude that TAZ directly upregulates IRS1 expression through TEADs leading to activation of the insulin signaling pathway as well as enhanced mammary cell

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proliferation in 3D culture (Figure 5F).

ACCEPTED MANUSCRIPT 4. DISCUSSION The Hippo pathway and its effectors are critical proteins in cancer biology. Dysregulated signaling through MST1/2, LATS1/2, TAZ and YAP alters the expression of TAZ/YAP transcriptional targets and causes cancer cell-intrinsic changes consistent with neoplastic

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transformation [41]. Screens for transcriptional targets of TAZ have provided important insights

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into the molecular mechanisms by which Hippo pathway dysregulation affects cancer cell

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behaviour. In this study, we validated IRS1 as a novel transcriptional target of TAZ. We further showed that IRS1 participates in TAZ-induced mammary cell proliferation in 3D culture. In

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doing so, we provided evidence that TAZ regulates gene targets involved in determining cancer

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cell sensitivity to growth factors and uncovered a novel mechanism by which IRS1 is upregulated in breast cancer cells.

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Our study adds to the growing body of literature demonstrating crosstalk between the

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Hippo and insulin pathways in physiology and pathology. Indeed, several previous publications have illustrated bidirectional interactions between these two pathways. First, the Hippo pathway

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has been found to act as an effector of insulin signaling in the cellular response to insulin/IGF-I.

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In 2010, Kim et al. observed that IGF-I-induced AKT activation can suppress the pro-apoptotic and growth-suppressive activities of MST2 in breast cancer cells [42]. Consistent with this,

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knockdown of TAZ/YAP in endometrial cancer cell lines has been shown to reduce insulin- and IGF-I-induced cell proliferation while similar observations have been reported to occur with the YAP homolog in Drosophila, Yorkie [43,44]. Additional evidence implicates the Hippo pathway in regulating insulin signaling. YAP and Yorkie can upregulate the upstream receptors of insulin signaling during development [43,45]. Moreover, a correlation between TAZ/YAP and IRS1 expression has been proposed to occur in endometrial cancer [44]. Very recently, TAZ/YAP

ACCEPTED MANUSCRIPT were reported to regulate IRS2 expression during hepatic steatosis and liver cancer [46]. In our study, we showed that TAZ directly regulates the expression of IRS1 and that IRS1 contributes to TAZ-induced cell proliferation in mammary epithelial cells. Evidently, Hippo and insulin signaling are heavily interconnected and likely function within a complex network mediating

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insulin sensitivity and cellular responses.

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Insulin and IGF-I are essential factors in normal physiology that orchestrate numerous

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responses in target tissues [47]. In cancer cells, upregulation of insulin signaling pathway components enhances cell sensitivity to insulin or IGF-I present in circulation and allows cancer

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cells to benefit from the metabolic and mitogenic effects of these proteins [48]. Increased

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signaling through the insulin pathway is a frequently observed event in breast cancer and is associated with poor survival [21]. Given this, there is avid interest in exploiting this pathway for

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breast cancer treatment [49]. Despite promising pre-clinical data, efforts to target insulin

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signaling in breast cancer patients have yielded disappointing results in clinical trials [50]. It is possible that a greater understanding of the molecular mechanisms regulating insulin signaling

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pathway activity may facilitate efforts to efficaciously and predictably modify this pathway or to

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stratify patients for treatment. Furthermore, since aberrant signaling through the insulin pathway is observed across neoplastic and non-neoplastic disease processes, our observation that TAZ

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directly regulates a central component of the insulin signaling pathway in human cells may also provide a rationale for future investigations into functions for the relationship between Hippo and insulin signaling in breast cancer and other insulin-related pathologies. IRS1 has no intrinsic enzymatic activity rather functions as an oncogene by increasing cancer cell sensitivity to insulin pathway ligands [25,26]. Therefore, our finding that TAZ or IRS1 overexpression enhances proliferation of MCF10A in 3D culture in the absence of

ACCEPTED MANUSCRIPT supplemental insulin warrants further discussion. Indeed, given that these experiments were performed by culturing spheroids in growth media with horse serum, our results obtained in complete growth media without supplemental insulin should not be interpreted to represent a culture condition where insulin/IGF-I are absent. Rather, we propose that IRS1 overexpression

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(or upregulation by TAZ) increases MCF10A proliferation in the absence of supplemental

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insulin by increasing their sensitivity to low levels of insulin and/or IGF-I that are provided by

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the horse serum. This interpretation is supported by our observation that inhibition of IR/IGF-IR suppressed TAZ-induced proliferation under these experimental condition and is also consistent

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with work performed by Dearth et al. who showed that IRS1 overexpression cannot increase

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AKT or ERK1/2 phosphorylation or cell proliferation in MCF10A that are cultured in serum-free media [25]. However, since horse serum may also contain other factors that signal through IRS1

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(e.g. cytokines), it remains possible that IRS1 overexpression or upregulation by TAZ enhances

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mammary cell proliferation by acting downstream of insulin/IGF-I as well as other ligands [51,52]. While future work will be necessary to clarify the relative contributions of each of these

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ligands to IRS1 overexpression phenotypes, this possibility has intriguing implications as it

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suggests that upregulation of a single gene target, IRS1, by TAZ could sensitize breast cancer cells to multiple exogenously-produced factors.

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In our overexpression experiments, we observed that both TAZ and IRS1 enhanced mammary cell proliferation in 3D culture in the presence or absence of supplemental insulin. This observation provided a rationale for investigating IRS1 as a transcriptional target of TAZ that might participate in TAZ-induced cell proliferation under these conditions. Consistent with this, pharmacological inhibition of IRS1 suppressed TAZ-induced proliferation of MCF10A in 3D culture. From these experiments, we conclude that IRS1 upregulation by TAZ contributes to

ACCEPTED MANUSCRIPT this aspect of the TAZ overexpression phenotype. However, it should be noted that other aspects of the TAZ overexpression phenotype were not entirely recapitulated in IRS1-overexpressing cells. TAZ-S89A-overexpressing MCF10A spheroids were highly disorganized and demonstrated greater invasive potential than their IRS1-overexpressing counterparts (Figure

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1H,I; Figure 2D,E). In fact, in our assays, we observed that TAZ overexpression frequently

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caused MCF10A spheroids to invade through the base Matrigel layer and form 2-dimensional

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structures along the bottom of the tissue culture vessel. While this invasive phenotype occurred with TAZ-S89A overexpression in both the presence or absence of supplemental insulin, it was

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only rarely observed with IRS1 overexpression. We therefore suspect that other transcriptional

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targets of TAZ contribute to TAZ-induced cell invasion. Future studies should aim to elucidate the identity of these TAZ gene targets and fully characterize their relationships to Hippo

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signaling.

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Finally, it should also be noted that our use of NT157 as an IRS1 inhibitor has potential limitations. This agent was originally discovered as an allosteric inhibitor of IR/IGF-IR that

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destabilizes interactions between these receptors and IRS1/2 thereby causing degradation of the

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insulin receptor substrates [40]. This compound has subsequently been found to modulate STAT3 function independent of its effects on IRS1/2 [53]. Given these nuances, future studies

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should aim to determine the extent to which each of these proteins contributes to TAZ overexpression phenotypes in vitro and in vivo. In summary, in this study we have validated IRS1 as a transcriptional target of TAZ that participates in TAZ-induced cell proliferation in mammary epithelial cells. These findings not only contribute to our understanding of TAZ as an oncogene in breast cancer but may provide insights into potential methods for targeting TAZ-high breast cancers therapeutically.

ACCEPTED MANUSCRIPT 5. CONCLUSIONS In conclusion, in this study we have characterized IRS1 as a transcriptional target of TAZ that contributes to TAZ-induced cell proliferation in human mammary epithelial cells. This finding offers new insights into the molecular mechanisms by which TAZ acts as an oncogene in

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breast cancer and provides evidence that TAZ-induced transcriptional changes can regulate

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breast cancer cell sensitivity to growth factors.

AUTHOR CONTRIBUTIONS

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HJJVR, DL, TA and XY designed the study and individual experiments. HJJVR, DL, TA and

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YH performed experiments with supervision from XY. XY provided resources/equipment and

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secured funding. HJJVR and XY wrote the manuscript.

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ACKNOWLEDGMENTS

We would like to thank Canadian Institute of Health Research (CIHR#119325, 148629) and

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Canadian Breast Cancer Foundation (CBCF)/Canadian Cancer Society (CCS) for their financial

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support. HJJVR is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology. We would like to thank Matt Gordon for providing technical expertise with

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confocal imaging and Dr. G. Karsenty for providing us with the RUNX2 plasmid. We have no competing interests to declare.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. TAZ overexpression enhances mammary cell proliferation and disrupts spheroid formation in 3D Matrigel culture in the presence or absence of supplemental insulin. (A-C) TAZ-S89A overexpression in MCF10A enhances cell proliferation in Matrigel. Representative

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images are shown in (A) (scale bar denotes 250 μm). Relative spheroid size is quantified at Day

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4 in (B) and at Day 8 in (C) (Mean + SEM, n ≥ 130 spheroids, ***p < 0.001, control refers to

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relative spheroid size in -Dox condition). (D,E) MCF10A form smaller spheroids when grown in media without 10 g/mL supplemental insulin. Representative images of spheroids are shown in

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(D) and higher magnification images are shown in (E) (scale bar denotes 250 μm and 100 μm,

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respectively). (F,G) TAZ-S89A overexpression in MCF10A increases cell proliferation in Matrigel in the presence or absence of 10 g/mL supplemental insulin. Representative images

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are shown in (F) (scale bar denotes 250 μm). Relative spheroid size is quantified in (G) (Mean +

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SEM, n ≥ 80 spheroids, ***p < 0.001, control refers to relative spheroid size in -Dox condition). (H) TAZ-S89A-overexpressing MCF10A spheroids demonstrate irregular morphology in the

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presence or absence of 10 g/mL supplemental insulin (scale bar denotes 100 μm). (I) TAZ-

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S89A overexpression disrupts normal MCF10A spheroid formation in the presence or absence of

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10 g/mL supplemental insulin. Spheroids were fixed with paraformaldehyde, permeabilized and nuclei were stained with DAPI (scale bar denotes 100 μm).

Figure 2. IRS1 overexpression enhances mammary cell proliferation in 3D Matrigel culture in the presence or absence of supplemental insulin. (A-C) IRS1 overexpression in MCF10A increases cell proliferation in Matrigel in the presence or absence of 10 g/mL supplemental insulin. Representative images are shown in (A) (scale bar denotes 250 m). Relative spheroid

ACCEPTED MANUSCRIPT size is quantified at Day 4 in (B) and at Day 8 in (C) (Mean + SEM, n ≥ 60 spheroids, ***p < 0.001, control refers to relative spheroid size in -Dox condition). (D,E) IRS1-overexpressing MCF10A spheroids demonstrate irregular morphology in the presence or absence of 10 g/mL supplemental insulin (scale bar denotes 100 m). Phase contrast images are shown in (D) and

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spheroids were fixed, permeabilized and nuclei were stained with DAPI in (E) (scale bar denotes

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100 m).

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Figure 3. TAZ enhances IRS1 expression and downstream signaling through AKT and

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ERK1/2. (A,B) IRS1 protein (A) and IRS1 mRNA (B) levels are upregulated by TAZ-S89A overexpression but not YAP-S127A overexpression in MCF10A (Mean + SEM, n= 2, **p <

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0.01, ns, not significant, control refers to IRS1 mRNA levels in -Dox condition for each cell

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line). (C) Transient knockdown of TAZ but not YAP reduces IRS1 mRNA expression in MDAMB-231 (Mean + SEM, n = 2, *p < 0.05, control refers to IRS1 mRNA levels in siControl

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condition). (D) TAZ-S89A overexpression increases IRS1 expression and phosphorylation of

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AKT and ERK1/2 in MCF10A cultured with 10 g/mL insulin but without hEGF. (E) TAZS89A overexpression increases IRS1 expression and phosphorylation of AKT in HBE135

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cultured with 5 g/mL insulin and 2.5 ng/mL hEGF. (F) IRS1 overexpression increases phosphorylation of AKT in MCF10A cultured with 10 g/mL insulin but without hEGF. (G) TAZ-S89A overexpression increases IRS1 expression and phosphorylation of ERK1/2 but not AKT in MCF10A cultured without supplemental insulin or hEGF.

ACCEPTED MANUSCRIPT Figure 4. TAZ transcriptionally regulates IRS1 expression by enhancing IRS1 promoter activity through the TEAD family of transcription factors. (A) TAZ-S89A increases IRS1 expression and signaling through AKT in MCF10A in the presence of 10 g/mL insulin whereas overexpression of TEAD-binding mutant TAZ (TAZ-S89A-F52/53A) does not enhance IRS1

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expression or AKT phosphorylation. (B) Transient knockdown of TEAD transcription factors

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suppresses IRS1 upregulation by TAZ-S89A and AKT phosphorylation in MCF10A cultured

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with 10 g/mL insulin. (C) TAZ and TEAD transcription factor family members (TEAD1-4)

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increase the activity of an IRS1 promoter reporter (IRS1-P; nucleotides -1000 to +82) in SK-BR3 (Mean + SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, control refers to basal IRS1-P

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activity). (D) Wild-type (WT) TAZ, WT TEAD4 and/or TAZ-S89A increase IRS1 promoter reporter activity in SK-BR-3 while TAZ without its transcriptional co-activation domain

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(TAZΔ227), TAZ-binding mutant TEAD (TEAD-Y429H) and RUNX2 cannot activate the IRS1

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promoter reporter (Mean + SEM; n= 3, **p < 0.01, ***p < 0.001, control refers to basal IRS1-P

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activity). (E) The IRS1 promoter contains a putative TEAD response element (TRE; GGAAT) spanning positions -94 to -90. A mutant IRS1 promoter reporter (IRS1-P-TREM) was

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constructed with the putative TRE mutated to AAAAA. (F) The mutant IRS1-P-TREM shows

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diminished activation by TAZ and TEAD4 compared to the wild-type IRS1-P (Mean + SEM, n = 3, ***p < 0.001, control refers to basal IRS1-P activity). (G) TAZ binds to the IRS1 promoter in MCF10A-TAZ-S89A. Chromatin and associated proteins were crosslinked and chromatin associated with TAZ was precipitated using a mouse monoclonal antibody (ChIP). Normal mouse IgG was used as a negative control (IgG). A region from the IRS1 promoter (nucleotides 161 to -34) was PCR-amplified and visualized on an agarose gel. Total chromatin extract was used as a positive control for PCR (“input”).

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Figure 5. Pharmacological inhibition of IRS1 disrupts TAZ-induced mammary cell proliferation in 3D Matrigel culture in the presence or absence of supplemental insulin. (A) TAZ-S89A-induced IRS1 upregulation in MCF10A is suppressed by NT157 in a dose-dependent

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manner (24 hours treatment). (B,C) TAZ-S89A overexpression in MCF10A increases cell

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proliferation in Matrigel in the presence or absence of 10 g/mL supplemental insulin while pharmacological inhibition of IRS1 using various concentrations of NT157 suppresses this

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effect. Representative images are shown in (B) (scale bar denotes 250 m). Relative spheroid size is quantified in (C) (Mean + SEM, n ≥ 90 spheroids, **p < 0.01, ***p < 0.001, control refers

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to relative spheroid size in -Dox DMSO with 10 g/mL supplemental insulin condition). (D,E)

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TAZ-S89A overexpression in MCF10A increases cell proliferation in Matrigel in the presence or

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absence of 10 g/mL supplemental insulin while pharmacological inhibition of IR/IGF-IR using 1 M of Linsitinib, NVP-AEW541 or GSK1904529A suppresses this effect. Representative

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images are shown in (D) (scale bar denotes 100 m). Relative spheroid size is quantified in (E)

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(Mean + SEM, n ≥ 50 spheroids, *p < 0.05, control refers to relative spheroid size in -Dox DMSO with 10 g/mL supplemental insulin condition). (F) Model for how TAZ increases

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mammary cell proliferation in Matrigel through the transcriptional regulation of IRS1. TAZ acts downstream of the Hippo pathway (MST1/2, LATS1/2) to regulate IRS1 promoter activity through TEAD transcription factor family members. TAZ-induced IRS1 expression subsequently leads to signaling through AKT and ERK1/2 and increased cell proliferation.

ACCEPTED MANUSCRIPT HIGHLIGHTS  The Hippo pathway effector TAZ regulates IRS1 in mammary epithelial cells 

TAZ activates the IRS1 promoter through TEAD transcription factors

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TAZ and IRS1 both enhance proliferation of mammary epithelial cells in 3D culture in

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TAZ increases proliferation of mammary epithelial cells in 3D culture through IRS1

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upregulation

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the presence or absence of supplemental insulin

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