CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells

Accepted Manuscript CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells Eun Jung Sohn, Deok-Beom Jung, HyoJung Lee, In Han, Jihyun Lee, Hyemin Lee, Sung-Hoon Kim PII:

S0304-3835(17)30621-3

DOI:

10.1016/j.canlet.2017.09.052

Reference:

CAN 13540

To appear in:

Cancer Letters

Received Date: 2 September 2017 Revised Date:

27 September 2017

Accepted Date: 28 September 2017

Please cite this article as: E.J. Sohn, D.-B. Jung, H. Lee, I. Han, J. Lee, H. Lee, S.-H. Kim, CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells, Cancer Letters (2017), doi: 10.1016/j.canlet.2017.09.052. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells

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Eun Jung Sohn, Deok-Beom Jung, HyoJung Lee, In Han, Jihyun Lee, Hyemin Lee, SungHoon Kim*

Cancer Molecular Targeted Herbal Research Center, College of Korean Medicine, Kyung

*

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Corresponding Author

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Hee University, Seoul, South Korea.

Sung-Hoon Kim, K.M.D., Ph.D., Cancer Molecular Targeted Herbal Research Center, College of Korean Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul

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131-701, South Korea.

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Tel: 82-2-961-9233; Fax: 82-2-964-1064; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abbreviations: CCR4-Not complex; carbon catabolite repressed 4-negative on TATA-less complex EGFR; epidermal growth factor receptor HER2; human epidermal growth factor receptor 2 TNBC;

modified Eagle's medium

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triple negative breast cancers VEGF; vascular endothelial growth factor DMEM; Dulbecco's FBS; fetal bovine serum ChIP; Chromatin immunoprecipitation

HUVECs; Human Umbilical Vein Endothelial Cells CAM; Chick Chorioallantoic Membrane PPAR γ; peroxisome proliferator-

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HIPK2; Homeodomain Interacting Protein Kinase2

activated receptor γ C/EBPα; enhancer-binding protein α GSK3α/β; Glycogen synthase

Real-time polymerase chain reaction

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kinase 3 α/β ATG5; autophagy-related gene 5 PBS; Phosphate-buffered saline RT-qPCR; HRP; Horseradish peroxidase TBST; Tris buffered

saline containing 0.1 % Tween 20 DMSO; dimethyl-sulfoxide OD; Optical density SD; standard deviation;ECL; enhanced chemoluminescence SDS; sodium dodecyl sulfate SDS-

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PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis RIPA buffer; Radioimmunoprecipitation assay PSG5; Pregnancy Specific Beta-1-Glycoprotein 5 TGF-ߚ;

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Transforming growth factor- ߚ

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ACCEPTED MANUSCRIPT Abstract Here the underlying role of CNOT2, a subunit of CCR4-NOT complex, was elucidated in cancer progression. CNOT2 was overexpressed in HIT-T15, ASPC-1, BXPC-3, PC-3, LNCaP,

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MCF-7 and MDA-MB-231 cell lines, which was confirmed by Tissue array in various human tumor tissues. Also, CNOT2 depletion suppressed proliferation and colony formation of MDA-MB-231 cells. Of note, microarray revealed decreased expression of CNOT2, VEGFA, HIF2 alpha (<0.5 fold) and increased expression of UMOD1, LOC727847, MMP4, hCG

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and other genes (> 2.0 fold) in CNOT2 depleted MDA-MB-231 cells compared to untreated

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control. Consistently, downregulation of VEGF, CNOT2 and HIF2 alpha was verified in CNOT2 depleted MDA-MB-231 cells by RT-qPCR. Additionally, CNOT2 depletion inhibited VEGF induced tube formation in HUVECs and reduced neovascularization in CAM assay. Furthermore, the growth of CNOT2 depleted MDA-MB-231 cells was significantly reduced in Balb/c nude mice along with decreased expression of VEGF and PCNA by

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immunohistochemistry compared to untreated control group. Overall, our findings provide evidences that CNOT2 promotes proliferation and angiogenesis via VEGF signaling in

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MDA-MB-231 breast cancer cells as a potent molecular target for breast cancer treatment.

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Keywords:CNOT2, Breast cancer, Tissue array, CAM, VEGF

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Introduction Breast cancer is well known the most common type of cancer in women worldwide[1,2]. Hence many target therapies have been developed for prevention or treatememt of breast

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cancer. Though abouit75% breast cancer is associated with estrogen or progesterone, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are overexpressed in approximately 40% and 25% of breast cancers, respectively, in

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hormone insensitive triple negative breast cancers (TNBC) with poor prognosis[1,3,4,5]. Also, it is well known that angiogenesis is critically involved in the progression and metastasis of

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breast cancers[6,7,8]. Thus, the level of angiogenic factors including VEGF is important in reflection of aggressiveness of braest cancers.

CR4-NOT (CNOT) complex, a large (>2 MDa) multisubunit, is composed of nine core subunit (CNOT1, CNOT2, CNOT3, CNOT6, CNOT6L, CNOT7, CNOT8, CNOT9/RQCD1,

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and CNOT10) [9,10]. CNOT complex plays a critical role in multiple cellular functions in regulating mRNA stability [11], translation [12], RNA polymerase I and II transcriptions. In yeast, CCR4-NOT complex regulates the gene expression, cell growth and glucose

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metabolism [13]. Furthermore, CNOT2 regulates the deadenylase activity and structural integrity of the CCR4–NOT complex as well as embryonic development in C. elegans and D.

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melanogaster [14,15]. Accumulating evidences reveal that CNOT2 depletion induces apoptosis in a caspase-dependent manner in HeLa [16] and interacts with cyclin- dependent kinase 11 which was cleaved by caspases [17]. In addition, CNOT2 knockdown is known to reduce the expression level of HIPK2 protein which plays a pivotal role as a signal integrator of DNA damage, hypoxia, and reactive oxygen intermediates [18]. Notably, our group reported that CNOT2 promotes the differentiation of 3T3-L1 preadipocytes via upregulation 4

ACCEPTED MANUSCRIPT of PPAR

and CEBPα and inhibition of GSK3α/β and β-catenin signaling [19] and also

suggested the critical role of CNOT2 as a negative regulator in ATG5 dependent autophagy

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[20]. Neverthelss, the underlying oncogenic functions of CNOT2 still remain unclear to date. Thus, in the present study, the critical role of CNOT2 was elucidated in proliferation and angiogenesis of TNBC MDA-MB-231 breast cancer cells by using proliferation assay, colony

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formation assay, wound healing assay, tube formation assay, CAM assay, tissue array,

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microarray, ChIP assay, RT-qPCR and Western blotting and also the growth of CNOT2 depleted MDA-MB-231 cells was monitored in BALB/c athymic nude mice along with

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immunohistochemistry.

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Materials and methods

Cell culture

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Human breast cancer cell lines (MDA-MB-231, MCF-7), prostate cancer cell lines (PC-3, LNCap), pancreas cancer cell lines (ASPC-1, BXPC-3), and normal cell lines (HIT-T15, RWPE-1, MCF-10A) were obtained from the American Type Culture Collection (ATCC;

with

10%

fetal

bovine

serum

(FBS),

2

µM

L-glutamine

and

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supplemented

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Manassas; VA), and cultured in RPMI1640 medium (Welgene, Daegu, South Korea)

penicillin/streptomycin at 37°C and 5% CO2. MCF-10A cells were maintained in Dulbecco's modified Eagle's medium-F12 (DMEM/F12) (Invitrogen, Carlsbad, CA,USA) supplemented with 5% horse serum, 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA,USA), 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin (Sigma, St. St. Louis, MO, USA),

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and 20 ng/ml recombinant human EGF (Pepro Tec Inc, MD,USA).

Plasmid constructs and transfection

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H1299 cells were transfected with siRNA oligoribonucleotides targeted against human

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CNOT2 or a RNA interference negative control. Each well was incubated for 48 h with 1 nM of siRNA using INTERFERIN siRNA transfection reagent (Polyplus, 409-50) according to the manufacturer’s protocol MDA-MB-231 cells at 40%-50% confluence were transfected with siRNA oligoribonucleotides targeted against human CNOT2 or pSG5-CNOT2 (Origene, Rockville, MD, USA) and a RNA interference negative control using Lipofetamine Transfection Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. 6

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Cytotoxicity assay

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The cytotoxicity of MDA-MB213 cells (1 104 cells/well) transfected with CNOT2 shRNA or control vector was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, MDA-MB213 cells (1 104 cells/well) transfected with

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CNOT2 shRNA or control vector were seeded onto 96-well culture plate and cultured for 24

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h. The cells were incubated with MTT (1 mg/mL) (Sigma Chemical) for 2 h and then treated with MTT lysis solution overnight. Optical density (OD) was measured using a microplate reader (Molecular Devices Co., USA) at 570 nm. Cell viability was calculated as a percentage

Proliferation assay

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of viable cells of CNOT2 depleted MDA-MB213 cells versus untreated control.

Cell proliferation was continuosly monitored in MDA-MB-231 transfected with CNOT2

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siRNA plasmid using INTERFERin® reagent (Polyplus, IIIkirch, France) for 3 days with xCELLigence RTCA (Real-Time Cell Analyzer) DP (Dual plate) instrument (Roche Applied

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Science, Mannheim, Germany). Background impedance was measured in 100 µl cell culture medium per well. The final volume was adjusted to 200 µl cell culture medium, including 5 × 103 cells/well. After plating, impedance was recorded in 15 min intervals. All experiments were performed in triplicates. Also, to confirm the proliferation of CNOT2 depleted MDAMB-231 cells by using RTCA DP instrument, MTT assay was also conducted in MDA-MB231 cells for 7 days. The cells were treated with MTT lysis solution overnight. Optical 7

ACCEPTED MANUSCRIPT density (OD) was measured using a microplate reader (Molecular Devices Co., USA) at 570 nm. Cell viability was calculated as a percentage of viable cells in drug treated groups

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versus untreated control.

Colony formation assay

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MDA-MB-231 cells transfected with CNOT2 shRNA or control plasmid were suspended in the complete medium containing 0.3%-0.4% noble agar in 6 well plates with bottom layer.

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Cells were cultured for 14 days and stained with 0.1% crystal violet. The number of cell colonies was photographed and counted under an inverted microscope.

Wound healing assay

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For siRNA transfection, MDA-MB-231 cells were tranfected using Inteferin siRNA transfection reagent with CNOT2 siRNA plasmid. Two days later, when the cells reached 90%

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confluency, a single wound was created by a sterile plastic pipette tip. Then, the cells were incubated for 24 h. To visualize how the cells migrate into the wounded area or protruded

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from the border of the wound, the cells were fixed and stained with 1% crystal violet. Wound areas were visualized and photographed under an inverted microscope. Each experiment was performed at least three times independently

Real-time quantitative RT-PCR (RT-qPCR)

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ACCEPTED MANUSCRIPT Total RNA was isolated from MDA-MB-231 cells with QIAzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out with a reverse transcription kit (Promega, Madison, WI,USA). RT-qPCR was performed with the LightCycler TM instrument (Roche

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Applied Sciences, Indianapolis, IN,USA) according to the manufacture’s protocol. The mRNA level of each target gene was normalized to that of GAPDH. The primers used to be as follows: GAPDH forward : 5′-CCA CTC CTC CAC CTT TGA C-3′, reverse : 5′-ACC

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CTG TTG CTG TAG CCA-3′. HIF-2 forward:5’-GCGCTAGACTCGAGAACAT-3’reverse 5’-TGGCCACTTACTACCTGACCCTT-3’

VEGF

forward:

5-

hCNOT2

forward:

GGTAACCCAACTCCATTAATAAACC

hCNOT2

reverse:

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TGCTGGTTTTGTTACCATTCC.

Western blot analysis

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CCAGCAGAAAGAGGAAAGAGGTAG, Reverse-5’CCCCAAAAGCAGGTCACTCAC3’.

MDA-MB-231 cells transfected with CNOT2 shRNA or control plasmid were lysed in RIPA

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buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholic acid-Na, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF and protease inhibitors cocktail). Protein samples were

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quantified by using a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA), separated by electrophoresis on 8 to 15% SDS-PAGE gel and electro transferred onto a Hybond ECL transfer membrane (Amersham Pharmacia, Piscataway, NJ). After blocking with 3-5% nonfat skim milk, the membrane was probed with antibodies for CNOT2, VEGF, Cyclin D1 (Santa Cruz Biotech, USA), Akt, JNK,phospho Akt,phospoho JNK, c-myc, β-actin (Cell signaling, Danvers, MA) and exposed to horseradish peroxidase (HRP)-conjugated secondary anti9

ACCEPTED MANUSCRIPT mouse or rabbit antibodies. Protein expression was measured by using enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ). Chromatin immunoprecipitation (CHIP) assay

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Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Briefly, to crosslink protein to DNA, 1% final concentration of formaldehyde

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was added to MDA-MB-231 cells for 10 min at room temperature, harvested, and then

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incubated on ice for 10 min in lysis buffer. Pellet nuclei were digested by micrococcal nuclease, sonicated and centrifuged. Sheared chromatin was incubated with anti-CNOT2 (Santa Cruz, USA) or IgG overnight at 4°C. After adding protein G bead, the chromatin was incubated for 2 h. Following elution of antibody-bound protein/DNA complexes, PCR analysis was carried out. The primers to amplify the human VEGF promoter were as follows: forward-CCCCAGTCACTCCAGCCTG,

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P1

P1

backward-

GCCGTCTGCACACCCCGGCTC, P2 forward- GAGCCGGGGTGTGCAGACGGC P2

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backward- GCCTGAGAGCCGTTCCCTCT.

Luciferase assay

VEGF promoter construct was transfected into CNOT2 siRNA transfected MDA-MB-231 cells along with Renilla luciferase reporter plasmid. Also, c-myc promoter construct was transfected into CNOT2 depleted MDA-MB-231 stable cells along with Renilla luciferase reporter plasmid. Two days after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). 10

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Tissue-array analysis

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The tissue array chips were purchased from Super Bio Chips Laboratories (Seoul, Korea). The tissue sections from tumor and adjacent non-cancerous tissues were immune-stained in a regular way using anti-CNOT2 antibody. Immunohistochemistry data were photographed and

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analyzed with the help of pathologist.

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Microarray analysis

Commercial two CNOT2 siRNAs that specifically target different regions of CNOT2 were purchased from the Thermo scientific company. CNOT2 siRNA sequence 25: (sense sequence-G.U.U.G.G.A.C.C.U.U.U.C.A.G.A.U.U.U.U.U.

anti-sense

sequence-

A.A.A.U.C.U.G.A.A.A.G.G.U.C.C.A.A.U.C.U.U) (Dharmacon, Thermo Scientific Thermo

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scientific, USA). Briefly, MDA-MB-231 cells at 40%-50% confluence were transfected with nonspecific or 80 nM siRNA using Lipofetamine Transfection Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Two days later, the cells were

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collected for the Gene expression array.Total RNAs from CNOT2 siRNA treated MDA-MB231 cells were extracted with QIAzol (Invitrogen, Carlsbad, CA, USA) according to the

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manufacturer's protocol (Nippon Gene). For each RNA, the synthesis of target cRNA probes and hybridization were performed using Agilent’s LowInput QuickAmp Labeling Kit (Agilent Technologies, USA) according to the manufacturer’s instructions. The gene expression data were analyzed using GeneSpringGX 7.3.1 software (Agilent). A hierarchically clustered heat map was generated using MeV v. 4.9.0 software.

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ACCEPTED MANUSCRIPT Tube formation assay Matrigel (BD Biosciences, Bedford, MA,USA) was dissolved at 4°C overnight. After coating with 150 µl Matrigel in each 48 well plates and the plates were incubated at 37°C for 30min. HUVEC cells (1 × 10

5

cells/well) were seeded on the layer of

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After gel formation,

polymerized Matrigel in cultured media from CNOT2 depeleted or untreated MDA-MB-231 cells. After 8 h incubation, the cells were fixed with 4% formaldehyde and randomly chosen

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fields were photographed under an Axiovert S 100 light microscope (Carl Zeiss, USA) at

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100× magnification. Tube network was quantified using NIH Scion image program.

CAM (Chick chorioallantonic membrane) assay

To check in vivo angiogenic activity of CNOT2, CAM assay was used as previously described [32]. Briefly, cultured media from CNOT2 depeleted or untreated MDA-MB-231

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cells were loaded onto a 1/4 piece of thermonox disc (Nunc, Naperville, IL,USA). The disc was applied to the CAM of a 9-day-old embryo and incubated for 48 h. Then fat emulsion was injected under the CAM and the number of newly formed blood vessels was

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group).

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photographed and counted. The experiment was repeated twice in two groups (15 eggs per

Measurement of VEGF production by ELISA.

To determine the production of VEGF, ELISA was performed by using a Human Vascular Endothelial Growth Factor ELISA kit (Biosource International, Camarillo, CA, USA).

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ACCEPTED MANUSCRIPT Establishment of CNOT2 shRNA stably transfected cells. To establish stable MDA-MB-231 cell line with CNOT2 depletion by shRNA, the MISSION short-hairpin RNA (shRNA) plasmid CNOT2 (Sigma-Aldrich, St.Louis, MO, U.S.A) or the

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shRNA:shRNA#1;TRCN0000015129;CCGGCGGGTTACTAACATTCCTCAACTCGAGT TGAGGAATGTTAGTAACCCGTTTTT,shRNA#2;TRCN0000234898;CCGGATGAATGG AGGAGACGTATTACTCGAGTAATACGTCTCCTCCATTCATTTTTTG)and

control

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(shControl; pLKO.1puro; SHC002, Sigma-Aldrich, USA) were transfected into MDA-MB231 cells using the Lipofetamine reagent (Thermo Fisher Scientific, USA). Lentivectors

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containing the shCNOT2 and shControl constructs were transduced into the MDA-MB-231 and MCF-7 cells. Puromycin (1µg/mL) was used to select shRNA knockdown cells. The efficacy of CNOT2 knockdown was evaluated by Western blotting.

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Xenograft mouse model

All procedures of animal study were conducted under guidelines approved by the Institutional Animal Care and Use Committee, Kyung Hee University (KHUASP(SE)-14-033). MDA-

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MB-231 cells transfected with Control or CNOT2 shRNA plasmid were harvested and washed twice with ice cold PBS.

The cells (2 × 105 cells) were suspended in 200 µL of

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matrigel (5 mg/mL) in PBS and 200µL of the cells was subcutaneously injected into the right flank of 6-week-old male BALB/c athymic nude mice (Central Lab. Animal, Inc. Seoul, Korea). Four weeks later when tumors were approximately 10–15 mm at their largest diameter, tumors were removed, photographed, weighed and used for immunohistochemistry analysis.

Immunohistochemistry 13

ACCEPTED MANUSCRIPT Slides with paraffin sections were deparaffinized and dehydrated. To block endogenous peroxidase activity, 0.3% hydrogen peroxide for 15 min was incubated. For antigen retrieval, slides were boiled in 10 mM citrate buffer (pH 6.0) for 10 min in a microwave. To block

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nonspecific binding, 10% normal rabbit serum for 30 min was incubated. The slides were incubated with anti-CNOT2, VEGF and PCNA antibodies (Santa Cruz, USA) overnight at

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4 °C.

Statistical analysis

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The results were expressed as means ± SD from at least three independent experiments.

Statistical analyses were conducted by Student s t-test using SigmaPlot version 12 (Systat

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Software Inc., San Jose, CA, USA). P value < 0.05 was considered statistically significant.

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ACCEPTED MANUSCRIPT Results

CNOT2 is overexpressed in a variety of human tumor tissues and cancer cell lines.

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To investigate how CNOT2 is expressed in human cancer cell lines, CNOT2 expression was comparatively evaluated in the normal cells and several cancer cell lines such as pancreas, prostate or breast cancer cell lines. As shown in Figure 1A, Western blotting revealed that CNOT2 was overexpressed in pancreas cancer (ASPC-1, BXPC-3), prostate cancer (PC-3,

Furthermore, to determine whether the

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(HIT-T15, RWPE-1, MCF-10A), respectively.

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LNCap), and breast cancer cell lines (MCF-7, MDA-MB-231) compared to normal cell lines

expression of CNOT2 is correlated with tumor stages in various cancers, tissue array analysis (benign 0, low grade stage 1, intermediate grade stage 2, and high grade stage 3) was performed. As shown in Figure 1B and C, the expression of CNOT2 was relatively correlated with the stage levels of tumor tissues from breast, liver, urinary bladder, ovary and prostate

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cancers compared to normal tissues.

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CNOT2 enhances the proliferation of MDA-MB-231 cells.

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To further examine the cellular function of CNOT2, proliferation of CNOT2 depleted breast cancer cells was monitored at every 1 h intervals for 72 h using the xCELLigence System (Roche). Also, to confirm the proliferative activity of CNOT2 depleted breast cancer cells, MTT assay was conducted in a time course for 7 days. As shown in Figure 2 A, proliferative activity of two CNOT2 siRNAs (CNOT2 si-1, CNOT2 si-2) transfected MDA- MB-231 cells for 72 h compared to untreated control. Furthermore, MDA-MB-231, or MCF-7 breast cancer cell lines were established by transfection of stable CNOT2 shRNA plasmids by lentivirus15

ACCEPTED MANUSCRIPT expressing two shRNAs targeting different regions of CNOT2. Consistent with the results by using xCELLigence System, silencing of CNOT2 by shRNA reduced cell proliferation by 80 to 82.8% in MDA-MB-231 cells 7 days after culture compared to untreated control (301

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x104± 10 x 104) (Figure 2B). However, anti-proliferative potential was not sensitive in MCF7 cells more than in MDA-MD-231 cells (Figure 2B). In addition, colony formation assay revealed that the number of colonies was significantly reduced in CNOT2 shRNA transfected

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MDA-MB-231 cells compared to untreated control, while the colonies were significantly increased in pGFP-CNOT2 overexpression plasmid transfected MDA-MB-231 cells

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compared to untreated control (Figure 2C).

Differentially expressed gene profile in CNOT2 depleted MDA-MB-231 cells. To further examine the effect on the mRNA gene profile by CNOT2 knockdown, microarray

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was conducted in CNOT2 depleted MDA-MB-231 cells. Interestingly, CNOT2 depletion up-regulated 96 genes and down-regulated 58 genes by two fold up or down compared to untreated control. The Gene ontology annotation was obtained from the KEGG database the changes of major 15 biological processes such as angiogenesis (1.1%), aging

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showed

(1.1 %), cell proliferation (0.9%), cell differentiation (0.9%), apoptosis (0.8%), cell cycle

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(0.3%), and others (Figure 3A).

Most significant downregulated or upregulated 10 genes

between control and CNOT2 depleted groups were listed in Figure 3B and Supplementary Table 1. The expression level of VEGF and HIF-2 was attenuated in two CNOT2 depleted MDA-MB-231 cells by RT-qPCR (Figure 3C). Similarly, mRNA expression of VEGF was abrogated in CNOT2 shRNA depleted MDA-MB-231 cells (Figure 3D), while overexpression of CNOT2 by pSG5-CNOT2 plasmid transfection increased the mRNA 16

ACCEPTED MANUSCRIPT expression of VEGF expression in MDA-MB-231 cells (Figure 3E). Also, the production of VEGF was significantly reduced in CNOT2 silenced MDA-MB-231 cells compared to untreated control by ELISA (Figure 4F). Consistently, Western blotting revealed that CNOT2

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depeletion attenuated the expression of VEGF and proliferation related genes such as c-myc, β-catenin and cyclin D1 in MDA-MB-231 cells (Figure 3G and H).

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CNOT2 transcriptionally regulates VEGF and c-myc in MDA-MB-231 cells.

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To check whether CNOT2 activates the VEGF promoter, a full-length VEGF-luciferase reporter constructs were used. As shown in Figure 4A, VEGF promoter activity was inhibited in CNOT2 depleted MDA-MB-231 cells compared to untreated controll. Furthermore, to determine whether CNOT2 binds to human VEGF promoter, ChIP assay was conducted using

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the anti-CNOT2 antibody. Cross-linked chromatin fragments were immunoprecipitated with CNOT2 or the control IgG and the two different regions (P1: from -602 to - 837; P2: from 319 to-602) of the VEGF promoter was amplified from the immunoprecipitates using the

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CNOT2 antibody or IgG (Figure 4B). Our results show that strong amplification of the P2 region in the VEGF promoter was induced compared to IgG control by immunoprecipitation

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with anti-CNOT2 antibody. Furthermore, c-myc promoter activity was significantly reduced in CNOT2 shRNA depleted MDA-MB-231 cells compared to shRNA vetcror control (Figure 4C).

CNOT2 has angiogenic potential in HUVECs and MDA-MB-231 cells.

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ACCEPTED MANUSCRIPT To examine the angiogenic potency of CNOT2 in HUVECs, tube formation assay was conducted in VEGF treated HUVECs. Tube formation was significantly reduced in CNOT2 depleted HUVECs compared to VEGF control (Figure 5A). Furthermore, to evaluate

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angiogenic property of CNOT2 in vivo, Chick embryo chorioallantoic membrane (CAM) assay was performed. Here CNOT2 depletion by using shRNA transfection significantly abrogated the number of neoangiogenic vessels compared to untreated control by CAM assay

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(Figure 5B). Next, the effect of CNOT2 was evaluted on the migratory activity of MDA-MB231 cells by wound-healing assay. CNOT2 knockdown by shRNA in MDA-MB-231 cells

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significantly suppressed the migration of MDA-MB 231 cells(Figure 5C), while overexpression of CNOT2 by transfection with pSG5-CNOT2 plasmids increased the migratory activity of MDA-MB-231 cells (Figure 5D).

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Silencing of CNOT2 suppresses in vivo growth of MDA-MB-231 cells in Balb/c nude mice. To examine the in vivo antitumor activity by CNOT2 depeletion, CNOT 2 shRNA or control

mice.

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plasmids transfected MDA-MB-231 cells were injected on the right flanks of Balb/c nude As shown in Figure 6A. the growth of MDA-MB-231 cells was significantly retarded

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in mice group bearing MDA-MB-231 cells compared to untreated control group. However, body wieght was not different between two groups with no statistical significance (Figure 6B). Also, the tumor sizes and weight were remarkabley reduced in CNOT2 depleted group compared to untreated control group (Figure 6 C and D). Of note, Immunohistochemistry revealed that the expression of VEGF as an angiogenesis marker and PCNA as a prolifetation biomarker was significantly attenuated in CNOT2 depleted group compared to untreated 18

ACCEPTED MANUSCRIPT control group. Interestingly, the expression of VEGF and PCNA was in parallel with that of

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CNOT2 between two groups by Immunohistochemistry (Figure 6 E and F).

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Discussion Though CNOT2, a component of the CCR4-NOT cytoplasmic deadnylase complex, is known to have transcriptional regulation in nucleus, and chromatin modifying activity[9,21], its

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function in cancer progression is still not fully understood. Thus, in the cureent study, the oncogenic role of CNOT2 was eludicadted in assoaciation with angiogenesis and proliferation. Here tissues array revealed that CNOT2 was overexpressed in a variety of

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cancer cells and tissues (prostate, liver, urinary baldder, pancreas, breast and ovary cancers) compared to non-tumorigenic cells, implying oncogenic potential of CNOT2. Futhremore,

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the expression level of CNOT2 was closely associated with the stages of tumors. Among tested cancers, CNOT2 was most overexpressed in breast cancers including MDA-MB-231 cells. Thus, next experiment was conducted mainly in aggressive MDA-MB-231 cells. Previous studies demonstrated that CNOT2 was identified as a tumor specific amplicon in

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salivary duct carcinoma through genome-wide SNP analysis [22]. Also, deficiency of CNOT2 induced apoptosis and enhanced CHOP mRNA level in Hela cells [16]. Likewise, knockdown

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of CNOT3, CNOT6, CNOT7 or CNOT8 resulted in a decrease of cell growth and proliferation [23,24,25]. Herein CNOT2 depletion suppressed proliferation of MDA-MB-231

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and MCF 7 breast cells in a time course. Also, CNOT2 depeletion reduced the numbers of colonies and motility of MDA-MB-231 cells by using siRNA transfection method, indicating antiproliferative and antimigratory effects of CNOT2 depletion. Interstingly, microarray that has been usefully applied to detect gene expression signature in several cells [26]. Our microarray revealed decreased expression of CNOT2, VEGF-A, HIF2 alpha (<0.5 fold) and increased expression of UMOD1, LOC727847, MMP4, hCG and other genes (> 2.0 fold) in CNOT2 depleted MDA-MB-231 cells compared to untreated control. 20

ACCEPTED MANUSCRIPT Also, the KEGG database showed

the changes of major 15 biological processes such as

angiogenesis (1.1%), aging (1.1%), cell proliferation (0.9%), cell differentiation (0.9%), apoptosis (0.8%), cell cycle (0.3%) in order. Consistently, downregulation of VEGF, CNOT2

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and HIF2 alpha was verified in CNOT2 depleted MDA-MB-231 cells by RT-qPCR, indicating the important role of angiogenesis related genes in CNOT2 depletion induced antitumor effect in MDA-MB-231 cells.

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There are accumulating evidences that VEGF-A acts as a proliferative factor in breast cancer cells [21,27] and a key regulator of angiogenesis [28]. Also, VEGF-A exerts autocrine and

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paracrine effects in cancer cells and surrounding endothelial cells [28,29]. Furthermore, VEGF is well known as a potent angiogenic growth factor and a prognostic factor in breast cancer cells [30,31,32]. Notably, CNOT2 depletion inhibited the motility of MDA-MB-231 cells, VEGF induced tube formation in human HUVECs and redcued neovascularization in

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CAM assay. Additionally, CNOT2 transcriptionally targets VEGF by promoter assay and ChIP assay and also CNOT2 depeletion attenuated the expression of VEGF and proliferation related genes such as c-myc, β-catenin and cyclin D1 in MDA-MB-231 cells, demonstrating

cancer cells.

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that CNOT2 is as a key regulator in the proliferation and angiogenesis of MDA-MB-231

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However, considering the discrepancy of our data that expression of CNOT2 in MCF-7 cell is higher than MDAM-MB-231 cells, while the inhibitory effect of CNOT2 depletion on MCF7 is less than that found in MDA-MB-231 cells, other signalings such as c-Myc, β-catenin, VEGF or other molecules may be working together with CNOT2 in proliferation of breast cancers, which should be confirmed in further study. Consistent with in vitro data, the in vivo growth of CNOT2 depleted MDA-MB-231 cells was 21

ACCEPTED MANUSCRIPT significantly reduced along with decreased expression of VEGF and PCNA by immunohistochemistry compared to untreated control group in Balb/c nude mice, demonstrating antitumor effect of CNOT2 depletion via antiproliferative and antiangiogenic

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activity. However, Faraji et al reported that CNOT2 overexpression suppressed the growth of 6DT1 primary tumor mass by 22% with no significance (p=0.02) but significantly reduced pulmonary metstasis by 46% (p=0.006) [33], which looks sort of contradictory to our results.

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Given that TGF-ߚ has dual roles as an oncogene or tumor suppressor, the underlying mechanism of CNOT2 should be further investigated in vitro and in vivo in the future, though

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the previous evidences on the effect of CNOT2 on apoptosis or metastasis are not surprising under specific cellular or microenvironmental conditions.

Collectively, our study demonstrate that CNOT2 was overexpressed in breast cancer cells and tumor tissues. Furthermore, inhibition of CNOT2 suppresses proliferation, migration and

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colony formation in MDA-MB-231 cells. Also, inhibition of CNOT2 reduced VEGF induced tube formation of HUVEC and attenuated neovascularization by CAM assay ex vivo. Furthermore, CNOT2 targets VEGF by promoter and ChIP assays. Consistently, CNOT2

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depletion suppressed the growth of MDA-MB-231 cells implanted in Xenograft mice models with reduced expression of VEGF and PCNA. Overall, these findings provide evidences that

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CNOT2 is critically involved in the proliferation and angiogenesis of breast cancers via VEGF signaling.

Acknowledgements

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ACCEPTED MANUSCRIPT EJ Sohn, DB Jung and Sung-Hoon Kim designed the experiment and wrote MS, HJ Lee, I Han, J Lee and H Lee performed in vitro and in vivo experiments.

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Funding This work was supported by the National Research Foundation of Korea (NRF) Grant funded

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by the Korea Government (MEST) (no. 2017R1A2A1A17069297).

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Conflict of interest

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All authors declare no competing financial interests.

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Figure legends Figure 1. Expression levels of CNOT2 in various normal and cancer cells and tissues. (A) Expression levels of CNOT2 in various normal and cancer cells. Protein samples from

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normal pancreas epithelial HIT-T15 cells, normal prostate epithelial RWPE-1 cells, normal breast epithelial MCF-10A cells, pancreatic cancer (ASPC-1, BXPC-3), prostate cancer (PC3, LANCap), breast cancer (MCF-7, MDA-MB-231) cell lines were isolated and subjected to

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Western blotting with antibodies of CNOT2 and β-actin. (B) Corelation of CNOT2 expression and tumor stages in several cancer tissues. Human cancer tissue array was

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conducted in breast, liver, urinary bladder, ovary, pancreas and prostate cancer tissues. Normal, low grade, high grade and metastasis tissues were stained with anti-CNOT2 antibody. Data represent means ± SD. * p<0.05, ** p<0.01, *** p<0.001 vs untreated control. (C) Representive pictures for CNOT2 overexpression in breast, liver, urinary baldder, ovary,

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pancreas and prostate tumor tissues stained with anti-CNOT2 antibody (Magnification 40x). Figure 2. Depletion of CNOT2 suppresses the proliferation in MDA-MD-231 cells. (A)

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Inhibitory effect of CNOT2 knockdown on the proliferation of MDA-MB-231 cells for 3 days. MDA-MB-231 cells were transfected with control or CNOT2 siRNA plasmids (CNOT2

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siRNA-1, siRNA-2), respectively, and proliferation of MDA-MD-231 cells was monitored for 3 days by using xCELLigence RTCA DP instrument (Roche Applied Science, Mannheim, Germany) (Left panel). Western blot represents the efficiency of CNOT2 knockdown (Right panel). (B) Effect of CNOT2 depletion using CNOT2 and control shRNA plasmids on the proliferation of MDA-MB-231 (Left panel) or MCF-7 cells (Right panel) for 7 days. MDAMB-231 and MCF-7 cells were transfected by CNOT2 and control shRNA plasmids and proliferation was determined every day for 7 days by using MTT assay. (C) Effect of CNOT2 29

ACCEPTED MANUSCRIPT depletion on colony forming ability of MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with CNOT2 or control shRNA plasmid and colony formation was monitored in MDA-MB-231 cells. Colonies formed after 14 days culture were fixed in methanol and then

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stained with 0.1% crystal violet in PBS. The number of cell colonies was photographed and counted under an inverted microscope.

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Figure 3. Differentially expressed gene profile in CNOT2 depleted MDA-MB-231 cells (A) Diagram of hierarchical clustering of gene expression profile in CNOT2 depleted MDA-

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MB-231 cells. (B) Selected down-regulated genes in CNOT2 depleted MDA-MB-231 cells (< 0.5 fold compared to untreated control). (C) Real-time PCR analysis of selected genes involved in angiogenesis and proliferation. Effect of CNOT2 on HIF-2 and VEGF expression in CNOT2 depleted MDA-MB-231 cells. Data represent means ± SD. * p<0.05, ** p<0.01,

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*** p<0.001 vs untreated control. (D) mRNA level of VEGF and CNOT2 in CNOT2 depleted MDA-MB-231 cells. Data represent means ± SD. ** p<0.01 vs untreated control. (E) mRNA level of VEGF and CNOT2 in pSG5 or pSG5-CNOT2 plasmid transfected MDA-MB-231

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cells. * p<0.05, ** p<0.01, vs untreated control. (F) VEGF production in CNOT2 depleted MDA-MB2131 cells by ELISA.

*** p<0.001 vs untreated control. (G) Effect of CNOT2

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depletion on c-Myc, β-catenin, VEGF and cyclin D1 in CNOT2 depleted MDA-MB2131 cells by Western blotting.

Figure 4. Silencing of CNOT2 reduced the activity of VEGF and c-Myc promoter. (A) pGL3-VEGF promoter constructs were co-transfected with renilla luciferase in control or CNOT2 depleted MDA-MB-231 cells and their luciferase activity was measured. Data 30

ACCEPTED MANUSCRIPT represent means ± SD. *** p<0.001 vs untreated control. (B) The binding of CNOT2 to VEGF by ChIP assay. Chromatin from MDA-MB-231 cells immunoprecipitated with either rabbit IgG or CNOT2 was amplified by PCR with the indicated primers. (C) pGL-3 basic and

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c-Myc promoter constructs were co-transfected with renilla luciferase in control or CNOT2 siRNA transfected MDA-MB-231 cells. * p<0.05 vs untreated control.

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Figure 5. Effect of CNOT2 depletion on angiogenic property in vitro and ex vivo

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(A) Effect of conditioned medium from CNOT2 depleted MDA-MA-231 cells on tube formation in HUVECs. Bar graphs represent quantification of tube formation. Data represent means ± SD. ** p<0.01, *** p<0.001 vs untreated control (B) Effect of conditioned medium from CNOT2 depleted MDA-MA-231 cells on neovascularization of CAM compared to

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VEGF or untreated control by CAM assay. Representative images of an in vivo CAM assay. CAMs of 9-day-old chicken embryos were injected with conditoned medium from control shRNA or CNOT shRNA transfected

MDA-MB-231 cells.

One week after inoculation,

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angiogenesis in yolk sac was monitored and representative images were photographed. Data represent means ± SD. ** p<0.01 (C) CNOT2 siRNA inhibits cell migration of MDA-MB-

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231 cells by wound healing assay. MDA-MB-231 cells were treated with CNOT2 siRNA (si1, si-2). After 48 h transfection, a wound was created with a plastic tip in control or CNOT2 siRNAs treated cells. After 24 h of incubation, the migration of cells was measured. Bar graphs represent quantification of migrated cells. ** p<0.01, *** p<0.001

(D)

Overexpression of CNOT2 enhanced the migration of MDA-MB-231 cells. Cells were transfected with pGFP-CNOT2 or pGFP plasmid. Bar graphs represent quantification of 31

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Figure 6. Silencing of CNOT2 retarded the growth of MDA-MB-231 cells.

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(A) Effect of CNOT2 silencing in MDA-MB-231 cells on subcutaneous tumour growth. CNOT2 depletion significantly decreased tumor growth compared to untreated control by two-tailed Student t-test. P <0.01 (n=8 mice in each group). ** p<0.01 (B) Body weight. (C)

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Photograph of the dissected tumor from control or CNOT2 shRNA mediated group. A decrease of tumor size was observed in mice injected by CNOT2 silenced MDA-MB-231

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cells compared to untreated control group. (D)Tumor weight of sacrificed mice. Silencing of CNOT2 resulted in reduction of tumor growth in MDA-MB 231 cells by two-tailed Student ttest. ** p<0.01. n=8 mice per group. (E) Effect of CNOT2 depletion on VEGF, PCNA expression by immunohistochemistry. Immunohistochemistry was carried out in tumor

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sections with antibodies of PCNA for proliferation, and VEGF for angiogenesis. (F) Bar graph represents quantification of immunohistochemistry data. Data represent means ± SD. *

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p<0.05, ** p<0.01, *** p<0.001

Supplementary Table 1. Upregulated and downregulated gene profiles in CNOT2

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depleted MDA-MB-231 cells.

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Highlights •

CNOT2 was overexpressed in HIT-T15, ASPC-1, BXPC-3, PC-3, LNCaP, MCF-7

and MDA-MB-231 cell lines compared to normal cells. CNOT2 depletion suppressed proliferation and colony formation of MDA-MB-231

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•

cells.

CNOT2 depletion inhibited VEGF induced tube formation in HUVECs and reduced neovascularization in CAM assay.

CNOT2 depletion suppressed the growth of MDA-MB-231 cells implanted in Balb/c nude mice along with

decreased

expression

of VEGF and

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•

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PCNA by

immunohistochemistry compared to untreated control.

Overall, CNOT2 promotes proliferation and angiogenesis via VEGF signaling in

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treatment

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MDA-MB-231 breast cancer cells as a potent molecular target for breast cancer

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•