Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelial-mesenchymal transition and promotes stemness in mammary epithelial cells

Accepted Manuscript Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelial-mesenchymal transition and promotes stemness in mammary epithelial cells

Eunhye Oh, Ji Young Kim, Daeil Sung, Youngkwan Cho, Nahyun Lee, Hyunsook An, Yoon-Jae Kim, Tae-Min Cho, Jae Hong Seo PII: DOI: Reference:

S0898-6568(17)30138-9 doi: 10.1016/j.cellsig.2017.05.009 CLS 8921

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

9 January 2017 8 May 2017 9 May 2017

Please cite this article as: Eunhye Oh, Ji Young Kim, Daeil Sung, Youngkwan Cho, Nahyun Lee, Hyunsook An, Yoon-Jae Kim, Tae-Min Cho, Jae Hong Seo , Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelial-mesenchymal transition and promotes stemness in mammary epithelial cells, Cellular Signalling (2016), doi: 10.1016/ j.cellsig.2017.05.009

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Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelialmesenchymal transition and promotes stemness in mammary epithelial cells Eunhye Oha,b,1, Ji Young Kima,b,1, Daeil Sunga,b, Youngkwan Choa,b, Nahyun Leea,b, Hyunsook Ana,b , Yoon-Jae Kima,c, Tae-Min Choa,b and Jae Hong Seoa,b,* a

These two authors contributed equally to this work.

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Division of Medical Oncology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 152-703, Republic of Korea. b Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea University, Seoul 152-703, Republic of Korea. c Department of Biomedical Research Center, Korea University Guro Hospital, Korea University, Seoul 152-703, Republic of Korea.

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Corresponding author. Address: Korea University, Guro Hospital Campus, 97 Gurodong-gil, Guro-gu, Seoul 152703, Republic of Korea. Tel: +82-2-2626-3059, Fax: +82-2-862-6453; E-mail address: [email protected] (JH. Seo)

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Disclosure summary: The authors declare no conflict of interest.

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Abstract Ubiquitin-specific protease 34 (USP34) is a deubiquitinating enzyme that regulates Axin stability and plays a critical role in Wnt/β-catenin signaling. We sought to investigate the role of USP34 on epithelial-mesenchymal (EMT) induction and its effects on mammary

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epithelial stem cells. USP34 expression levels were relatively lower in MDA-MB-231 and 4T1 mesenchymal-like cells when compared to epithelial-like cells. Inhibition of USP34 in

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NMuMG cells induced EMT, as evidenced by the upregulation of EMT markers including N-

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cadherin, phospho-Smad3, Snail and active-β-catenin, as well as the downregulation of Axin 1 and E-cadherin. USP34 knockdown (KD) in these cells also resulted in the acquisition of

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invasive behavior, and promoted stemness as indicated by enhanced mammosphere-forming

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ability, concomitant with the upregulation of Nanog, Oct4 and Sox2 mRNA expression. Endogenous USP34 expression was observed to be at low levels in virgin mouse mammary

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glands in vivo. When USP34-KD cells were transplanted into the cleared mammary fat pads

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(CFP) of mice, these cells reconstituted the mammary gland with ductal tree development within 3 months. Our findings suggest a previously unknown role for USP34 in mammary

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gland development.

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Keywords: USP34, EMT, stemness, mammary gland development

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1. Introduction Over the last few decades, evidence for the existence of stem cells in mammary glands has accumulated. Mammary stem cells (MaSCs) possess specific biological properties including self-renewal capacity and the ability to differentiate into more specialized cell types

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[1, 2]. Under serum-free suspension in vitro, MaSCs give rise to mammospheres, which are

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cell masses highly enriched in stem/progenitor cells capable of reconstituting entire

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mammary ductal trees upon transplantation into the cleared mammary fat pads of mice [3, 4]. Recent studies have highlighted a significant relationship between MaSC

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characteristics and epithelial-mesenchymal transition (EMT) [3, 5]. During mammary gland development, the EMT program orchestrates the formation of special structures and

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functional properties of the glands, thereby contributing to ductal morphogenesis [6, 7]. EMT is a critical process for many aspects of normal development including wound healing and

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mesoderm formation, and arises from the loss of cell-cell adhesion and cell polarity, after

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which cells are more likely to migrate and invade surrounding tissue [8, 9]. For this reason, its dysregulation has been implicated in the development and metastasis of various cancers,

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as it can allow circulating tumor cells to exit the bloodstream and form peripheral tumors [10]. Although EMT is known to be tightly regulated, the process overall remains poorly

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understood due to the considerable number of factors and the complexity of the events involved. The process during mammary gland development is known to be regulated by multiple signaling factors including TGF-β/Smad, Wnt/β-catenin and Notch, as well as stromal microenvironment constituents including hormones, receptor tyrosine kinases, growth factors and matrix metalloproteinases (MMPs) [11, 12]. Canonical Wnt/β-catenin pathway activity is pivotal for the maintenance and dynamics of normal mammary stem cells

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and accelerates formation of mammary placodes, suggesting that this pathway is involved in the initiation of mammary gland morphogenesis [13, 14]. The ubiquitin proteasome system (UPS) has been implicated in the regulation of numerous biological processes including cell survival, apoptosis, autophagy, EMT, cell

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motility and cell cycle control, as well as embryonic development [15-17]. Deubiquitination is the reverse of ubiquitination and is mediated by a family of cysteine proteases known as

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deubiquitinating enzymes (DUBs) [18, 19]. Ubiquitin-specific proteases (USP) are a subclass

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of DUBs [19]. Recent studies have found that USP34 is a component of the Axin complex, and directly regulates the β-catenin destruction complex by mediating Axin stability [20].

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USP34 activation is involved in the regulation of Wnt/β-catenin and TGF-β/BMP signaling,

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and also serves as a DNA damage checkpoint control and contributor to DNA repair [20-22]. However, the existence of a role for USP34 in EMT regulation and mammary epithelial cell

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stemness has not previously been elucidated. In the present study, we sought to investigate

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the involvement of USP34 during EMT induction and its associated effects on mammary

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epithelial stem cells and mammary gland outgrowth.

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2. Materials and Methods 2.1. Reagents and antibodies Triton X-100, propidium iodide (PI), phosphate buffered saline (PBS) tablets and cycloheximide were obtained from Sigma-Aldrich (St Louis, MO). Transforming growth

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factor beta 1 (TGF-β1) was supplied by R&D systems (Minneapolis, MN). Phosphatase

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inhibitor and protease inhibitor cocktail tablets were purchased from Roche Applied Sciences (Penzberg, Germany). The following primary antibodies were used: phospho-Smad3

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(Ser423/425), vimentin, Nanog, Oct4 and Sox2 (Cell Signaling, Beverly, CA); USP34, Snail,

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Cytokeratin 8/18 (Abcam, Cambridge, MA); Smad4 (Santa Cruz Biotechnology Inc, CA); Ecadherin, N-cadherin (BD Biosciences, Franklin Lakes, NJ); active-β-catenin (Millipore,

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Billerica, MA); Axin 1 (Thermo Scientific Inc., Rockford, IL); and β-actin (Sigma-Aldrich, St Louis, MO). The secondary antibodies used included horseradish peroxidase (HRP)-

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conjugated anti-rabbit and mouse IgG (Bio-Rad Laboratories, Hercules, CA); Alexa Fluor-

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488 or -594 goat anti-mouse and rabbit IgG, and Texas Red-X Phalloidin (Invitrogen,

2.2. Cell culture

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Carlsbad, CA).

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The human breast cancer cell lines MCF7 and MDA-MB-231, the mouse mammary epithelial cell line NMuMG (American Type Culture Collection, Manassas, VA, USA), and the mouse mammary carcinoma cell line 4T1 (Japanese Collection of Research Bioresources Cell Bank) were cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum (FBS), streptomycin-penicillin (100 U/ml), Fungizone (0.625 μg/ml) and insulin (10 μg/ml). Cells were incubated at 37℃ in an atmosphere of 5% CO2.

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2.3. Si-RNA for USP34 transfection Cells were seeded into 6-well plates 24 h prior to transfection. NMuMG cells were transfected with si-USP34 (Origene, Rockville, USA) or control si-RNA (30 nM) using Lipofectamine 2000 (Invitrogen) for 24 h according to the manufacturer’s instructions. The

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USP34 si-RNA sequences were as follows; 5’-CGGAUAGACAAAUUCGAAUGAGATT-3’

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2.4. Western blot analysis

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The procedures were performed as previously described [23]. The primary antibody dilutions were: [USP34 (1:2000), Axin 1 (1:2000), active-β-catenin (1:2000), E-cadherin

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(1:2000), N-cadherin (1:2000), vimentin (1:2000), phospho-Smad3 (1:1000), Smad4 (1:2000),

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Snail (1:2000), Nanog (1:1000), Oct4 (1:1000), Sox2 (1:1000) and β-actin (1:20000)], followed by incubation with horseradish peroxidase (HRP)-conjugated rabbit or mouse IgG

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(1:3000–1:10,000). Signal intensity was detected using an Enhanced Chemiluminescence Kit

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(Thermo Scientific Inc., Rockford, IL, USA) and x-ray film (Agfa Healthcare, Mortsel, Belgium) and quantitated using AlphaEaseFC software (Alpha Innotech, San Leandro, CA,

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USA).

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2.5. RT-PCR analysis

Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Amplification of transcripts was achieved by reverse transcriptase-polymerase chain reaction (RT-PCR) using 1 μg/μl total RNA, Molony Murine Leukemia Virus reverse transcriptase (MMLV; Gibco/BRL, Gaithersburg, MD, USA), and oligo-d(T)15 primers (Roche Applied Sciences). PCR amplification was achieved using a Takara PCR Thermal Cycler (Thermo Scientific Inc., Rockford, IL) with the following primers: Nanog, forward 5’-AGGACAGGTTTCAGAAGCAGAAGT-3’, reverse

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5’-TCAGACCATTGCTAGTCTTCAACC-3’; Oct4, forward 5’-TGGCTTCAGACTTCGC CTTC-3, reverse 5’-GGAGGTTCCCTCTGAGTTGC-3’; Sox2, forward 5’-TGCCTCTTTA AGACTAGGGCT-3’, reverse 5’-CGAGTTGTGCATCTTGGGGT-3’; Actin, forward 5’GCCAACCGCGAGAAGATGAC-3’, reverse 5’-GAAGGTAGTTTCGTGGATGC-3’. The

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PCR products were separated on 1.2% agarose gels and visualized using a Gel Doc™ XR+

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System (Bio-Rad Laboratories).

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2.6. Immunocytochemistry

Cells on 8-well chamber slides (BD Biosciences, Franklin Lakes, NJ) were fixed

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with 4% paraformaldehyde, washed with PBS, and incubated with 0.2% Triton X-100 for 10

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min. Primary antibodies in antibody diluent (Dako, Glostrup, Denmark) were incubated overnight at 4oC and then incubated with fluorescence-conjugated secondary antibodies

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(Alexa Fluor®-488 or -594). Cells were mounted with ProLong Gold Antifade Reagent with

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DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired using a Carl Zeiss confocal microscope (Weimar, Germany). Intensity of fluorescence was analyzed using

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fluorescence profiling, as previously described [24].

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2.7. Invasion assay

Invasion chambers were coated with matrigel matrix (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s recommendations. Cells (1.5×105) were trypsinized, washed and suspended onto the upper chamber with serum-free media. Conditioned media was added to the lower chambers. The chambers were incubated for 48 h at 37℃ in a humidified atmosphere of 5% CO2. Invaded cells were fixed and stained with Diff-Quik (Sysmex, Kobe, Japan) and quantified under a BX51 microscope (Olympus, Tokyo, Japan).

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2.8. MMP-2 and TGF-β ELISA assay Mouse MMP-2 and TGF-β expression levels were measured using ELISA kits (R&D systems, Minneapolis, MN), according to the manufacturer’s instructions. The quantity of MMP-2 or TGF-β was determined by measuring absorbance at 450 nm using a Spectramax

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Plus384 microplate analyzer (Molecular Devices, Sunnyvale, CA, USA).

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2.9. Mammosphere formation assay

Mammosphere-forming ability was analyzed as previously described [25]. Cells were

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plated in ultralow attachment dishes (Corning, NY) and cultured in HuMEC basal serum-free medium (Gibco), supplemented with B27 (1:50, Invitrogen, Carlsbad, CA), 20 ng/mL basic

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fibroblast growth factor (bFGF, Sigma-Aldrich), 20 ng/mL mouse epidermal growth factor (mEGF, Sigma-Aldrich), 4 μg/ml heparin, 1% antibiotic-antimycotic, and 15 μg/mL

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gentamycin at 37°C in an atmosphere of 5% CO2.

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2.10. Mammary gland transplantation and whole mounts

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All animal procedures were carried out in accordance with animal care guidelines approved by the Korea University Institutional Animal Care and Use Committee (IACUC). The fourth inguinal mammary glands of 3 week-old female Balb/c mice were surgically removed from the nipple region up to the lymph node by cauterization. NMuMG (5×104 /10 μl) or USP34-KD cells in PBS were implanted with a Hamilton syringe (Hamilton, Reno, NV) attached to a 30-gauge needle into the left and right cleared fourth inguinal mammary fat pads, respectively. After 3 or 10 months, the animals were sacrificed and the fourth mammary glands dissected and removed (n=5 per time point). Whole mammary glands were

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fixed in Carnoy’s solution (ethanol: chloroform: glacial acetic acid=6:3:1) and rinsed in 70% ethanol for 15 min and washed in water for 5 min. Tissues were stained overnight with carmine alum solution including carmine and aluminum potassium sulfate (Sigma-Aldrich). Tissues were dehydrated through a graded alcohol series to water, cleared in xylene and

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mounted with Permount (Fisher Scientific, Pittsburg, PA). Mammary gland images were

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captured using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan).

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2. 11. Immunohistochemistry

Mammary gland sections from at least 7 mice were used to investigate the expression

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of USP34 by immunohistochemical analysis. At sacrifice, tissue samples from the fourth

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mammary gland were removed, fixed in 10% neutral-buffered formalin, and paraffin embedded. Tissue sections of 5-μm thickness were mounted on positively-charged glass

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slides and then deparaffinized with xylene and dehydrated through a graded alcohol series to

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water. For antigen retrieval, sections were boiled in citric acid buffer (pH 6.0) for immunofluorescence analysis. Tissue sections with primary antibodies (USP34; 1:100 and Cytokeratin-8/18; 1:100) in antibody-diluent (Dako, Glostrup, Denmark) were incubated

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overnight at 4oC. For secondary antibody reactions, the sections were incubated with the

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Alexa Fluor®-488 or -594 conjugated secondary antibody (Invitrogen, Carlsbad, CA) at RT for 2 hours, and then incubated with ProLong gold antifade reagent with DAPI.

2.12. Statistical analysis All data were analyzed using GraphPad Prism 5.0 statistical software (San Diego, CA). The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by student’s t-test and one- and two-way ANOVA, as appropriate. A

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two-way ANOVA was used to assess the effects and interactions of two variables and multiple comparisons were achieved using Bonferroni’s post hoc test. Statistical significance

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was defined at p< 0.05.

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3. Results 3.1. USP34 downregulation facilitates EMT in mammary epithelial cells Epithelial and mesenchymal cells are distinguished by their unique phenotypes, cell morphology, expression patterns and the subcellular distribution of EMT markers such as

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vimentin, E-cadherin, N-cadherin and Smad/Snail [26]. MCF7 and NMuMG cells exhibit

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typical features of epithelial-like phenotype, while MDA-MB-231 and 4T1 cells exhibit

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mesenchymal-like phenotype with elongated spindles (Fig. 1A). MDA-MB-231 and 4T1 cells were found to express a higher level of vimentin and active-β-catenin and a lower level of E-

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cadherin when compared to the MCF7 and NMuMG cells (Fig. 1B). The reverse was found for E-cadherin and vimentin expression between MDA-MB-231 and MCF7 cells, as observed

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by double-immunofluorescence analysis (Fig. 1C). Interestingly, we found that endogenous level of USP34 was relatively lower in mesenchymal-like phenotype cells compared to

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epithelial-like phenotype (Fig. 1B). The addition of TGF-β induced morphological changes,

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characterized by elongated spindles and the re-arrangement of cytoskeletal F-actin in NMuMG cells (Fig. 1D). TGF-β-induced EMT was associated with the downregulation of E-

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cadherin and the upregulation of N-cadherin, phospho-Smad3, Snail and active-β-catenin (* p<0.05, Fig 1E). Analysis of USP34 protein levels revealed a significant difference between

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the control and TGF-β treatment group. Based on these observations, we postulated that USP34 may be involved in the regulation of EMT in mammary gland epithelial cell development.

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3.2. Inhibition of USP34 induces EMT and increases invasive behavior Knockdown of USP34 was found to reduce Axin 1 and E-cadherin expression, and increase N-cadherin and the expression of EMT-related transcription factors such as phospho-Smad3,

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Snail,

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active-β-catenin

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p<0.05,

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2A).

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Immunofluorescence analysis further showed a significant downregulation of cytoplasmic E-

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cadherin expression (*** p<0.001, Fig. 2B) and considerable upregulation of nuclear phospho-Smad3 during USP34 knockdown (Fig. 2C). Evidence suggests that EMT promotes

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invasive behavior in cells [27, 28] and we next investigated whether the absence of USP34

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facilitates cell invasion. USP34 knockdown caused NMuMG cells to extensively invade through a matrigel assay (*** p<0.001, Fig. 2D). Furthermore, the USP34 knockdown

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resulted in significantly increased levels of MMP-2 secretion (* p<0.05, Fig. 2E) and TGF-β (*** p<0.001, Fig. 2F). Increased TGF-β levels may therefore contribute to the induction of

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EMT allowing for activation of Smad3/4 and Snail upregulation. We further examined

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whether TGF-β could enhance these responses. Low concentrations of 1 ng/ml TGF-β upregulated protein expression of N-cadherin, p-Smad3, Smad4, Snail, while downregulating

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E-cadherin and β-catenin activation in NMuMG cells in the presence of USP34 knockdown (* p<0.05, Fig. 3A). This response was also associated with a significant increase in

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invasiveness (** p<0.01 and ## p< 0.01, Fig. 3B) and MMP-2 secretion levels (** p<0.01 and ## p< 0.01, Fig. 3C).

3.3. Inhibition of USP34 promotes mammosphere formation and increases Nanog, Oct4 and Sox2 mRNA expression As mammospheres are highly enriched in stem/progenitor cells, sphere-forming assays can be a valuable tool for investigating stemness [29]. Adherent NMuMG cells under

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a 2D culture system exhibit limited expression of embryonic stem cell markers including Nanog, Oct4 and Sox2, while mammospheres in a 3D culture system express relatively higher levels. USP34 was considerably downregulated in mammospheres when directly compared to adherent NMuMG cells (Fig. 4A). Both ectopic expression of mesenchymal

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factors such as Snail and Twist, and TGF-β treatment is known to generate self-renewal in epithelial cells that have a high CD44high/CD24low subpopulation and are capable of producing

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mammospheres [3]. To examine whether USP34 has an impact on stemness, mammosphere-

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forming ability was determined in the presence or absence of USP34. We observed that inhibition of USP34 significantly increased the number and volume of mammospheres

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formed (** p<0.01, Fig. 4B and C). This effect was accompanied by significant increases in

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mRNA expression of Nanog, Oct4 and Sox2 (* p<0.05, Fig. 4D). TGF-β treatment enhanced

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the expression of all transcription factors during USP34 knockdown (Fig. 4E).

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3.4. USP34 inhibition reconstitutes mammary glands To examine the importance of our in vitro observations, CTL-KD or USP34-KD

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cells were transplanted into the cleared mammary fat pads of 3 week-old female Balb/c mice,

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before mammary gland outgrowth was observed for up to 10 months (Fig. 5A). Reconstituted mammary glands with striking ductal trees, large bulbous terminal end buds (TEBs) and furcating TEBs were observed at 3 months (Fig. 5B). At 10 months, USP34-KD cells were observed to have further developed reconstituted mammary glands with ductal trees and alveolar buds. High magnification images of ductal structures arising from USP34-KD transplantation revealed that the secondary- and tertiary-side branches were formed along the trailing duct. In contrast, CTL-KD cells failed to reconstitute mammary glands to any significant extent. Normal 10 month-old female mice exhibited complete ductal

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morphogenesis and mammary glands with side branches (Fig. 5B). TEBs normally disappear from the mammary gland after ducts elongate and proliferate throughout the entire fat pad [14]. Both the USP34-KD transplanted mice and the normal 10 month-old mice showed no

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evidence of TEBs at the tips of the ducts.

3.5. Endogenous USP34 expression is at low levels in virgin mouse mammary glands in

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vivo.

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To assess USP34 expression during mammary gland development, gland tissues

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from virgin female mice (1-week to 10-months old) and lactating mice were obtained for a double-label immunohistochemical analysis with USP34 and cytokeratin 8/18 (CK8/18). The

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luminal epithelial marker CK8/18 was used, which is specifically present in the luminal cells of all gland tissues at different stages of mammary gland development. The endogenous

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levels of USP34 were low in mammary glands of 1-, 2-, 3-week and 3-, 10-month-old virgin

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mice, while strong USP34 expression was predominantly visible in the cytoplasm and nucleus of the vacuolated alveolar cells in lactating tissues, as evidenced by intense red

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fluorescent signal (Fig. 6A). This observation was further supported by USP34 expression intensity profiling in mammary glands, showing that USP34 is expressed at low levels in both

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immature and mature virgin mice (Fig. 6B). Our findings suggest that USP34 may be an important determinant of mammary gland development.

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4. Discussion

EMT has been implicated in embryonic mammary morphogenesis and development as well as breast cancer progression, tumor cell invasion and metastasis [30, 31]. EMT in

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mammary epithelial cells is governed by specific transcription factors including Snail, Smad3/4, Twist and β-catenin. TGF-β/Smad-induced EMT is known to be regulated by the

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hetero-trimeric Smad family, which include two of the receptor-regulated Smads (Smad2/3)

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and a co-Smad (Smad4). The trimeric Smads translocate to the nucleus and form a complex with Snail, allowing for the inhibition of E-cadherin expression [26, 32]. Wnt signaling-

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induced EMT is mediated by the oncoprotein β-catenin via inhibition of the destruction

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complex consisting of Axin and adenomatous polyposis coli (APC) [26, 33]. Growing evidence indicates that crosstalk between TGF-β/Smad and Wnt/β-catenin signaling

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transcription factors [34, 35].

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facilitates the interaction between the activated Smad3/4 complex and Wnt-activating

In the present study, we found that USP34 expression levels were relatively lower in mesenchymal-like cells when compared to epithelial-like cells. USP34 knockdown-induced

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EMT was associated with TGF-β and Wnt signaling, as evidenced by significant increases in

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levels of phospho-Smad3, Smad4 and Snail, as well as active-β-catenin upregulation. Under normal physiological conditions, β-catenin interacts with E-cadherin and the actin cytoskeleton at the adherens junction and this complex is indispensable for the adhesion and integrity of the epithelial cell layer [36]. We observed that inhibition of USP34 caused the loss of E-cadherin from cell-cell contacts, an effect mirrored by TGF-β challenge. Disruption of the complex could also conceivably affect β-catenin activation, thereby promoting EMT. This response was associated with the acquisition of invasive behavior in NMuMG cells.

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USP34 deubiquitinates and destabilizes Axin 1, which in turn regulates Wnt/βcatenin signaling in colon cancer [20]. Overexpression of Axin 1, a negative regulator of the Wnt signaling pathway, abrogates β-catenin expression and its nuclear accumulation [37, 38]. Furthermore, increased Axin stability following treatment with a tankyrase inhibitor

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attenuates TGF-β-stimulated EMT leading to the upregulation of Snail, Twist and α-smooth muscle actin (α-SMA), through the inhibition of β-catenin activation in a lung fibrosis murine

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model [39]. Based on these findings, we speculated that Axin 1 instability could be involved

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in USP34 downregulation-mediated EMT, thereby allowing for Smad/Snail activation and active-β-catenin upregulation. Our results show that USP34 knockdown results in a

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significant downregulation of Axin 1, indicating that Axin stability could eventually be

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governed by USP34. Furthermore, a cycloheximide pulse-chase assay revealed that USP34 knockdown caused significant degradation of Axin 1 (Supplementary Fig. S1), implying that

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USP34 may be a negative regulator of the EMT process.

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The pluripotent transcription factors Nanog, Oct4 and Sox2 have been identified as embryonic stem cell (ESC) markers that maintain pluripotency. A recent study has reported that Sox2 regulates mammary stem cell self-renewal activity and mammary regeneration,

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leading to ductal outgrowth formation via G protein-coupled receptor (Lgr4)/Wnt signaling

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[40]. Nanog and Oct3/4 are predominantly expressed in the nucleus of cells in both the mesenchyme and epithelium in mouse embryonic mammary placodes, with a likely role in the initiation and regulation of embryonic mammary gland development [41]. We observed that levels of all three factors were markedly higher in mammospheres derived from NMuMG cells compared to their adherent counterparts, an observation in agreement with previous findings in primary human breast epithelial mammospheres [42, 43]. USP34 was also expressed at a relatively lower level in mammospheres than adherent cells and its

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knockdown significantly augmented mammosphere-forming ability, coinciding with significant increases in Nanog, Oct4 and Sox2 levels. We found that USP34 was kept at very low level in mammary glands until 10 months of age in virgin mice. In contrast, lactating mice exhibit a high level of USP34 in mammary

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alveoli in vivo. We next examined the effect of USP34 on ductal outgrowth by transplanting USP34-KD cells into the cleared mammary fat pads of virgin mice. It was observed that

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USP34-KD cells reconstituted ductal trees with bulbous-shaped TEBs at the tips of ducts, and

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bifurcating TEBs could be observed at 3 months. By 10 months, mammary glands with ductal trees including secondary-side branches were observed in USP34-KD cell-transplanted fat

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pads, suggesting a role for USP34 in the regulation of mammary epithelial self-renewal and

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mammary gland formation.

Mammary cell invasion is an essential step that occurs during mammary gland ductal

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elongation and breast cancer metastatic progression [1, 44]. MMP-2 activation is essential for

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cell invasion and metastasis through ECM degradation in mammary fat pads. MMP-2 resides in an active form in the stromal environment of mammary ducts and fosters ductal penetration and elongation at the initial event during gland morphogenesis [45, 46]. Of note,

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MMP-2 knockout mice exhibit subdued invasion of the mammary duct into the stromal fat

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pad [45]. We found that USP34 knockdown significantly increases MMP-2 secretion levels and this response was enhanced by TGF-β treatment. Following USP34-KD cell transplantation, increased MMP-2 secretion likely contributes to mammary ductal invasion. In the present study, we have demonstrated for the first time that: (i) USP34 is downregulated in mesenchymal-like phenotype cells; (ii) Inhibition of USP34 induces EMT and increases invasive properties; (iii) USP34 knockdown promotes stemness, as evidenced by increased mammosphere-forming capacity and upregulation of Nanog, Oct4 and Sox2 in mammary epithelial cells in vitro; (iv) TGF-β enhances these responses in the absence of

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USP34; (v) USP34 is endogenously expressed at low levels in the mammary glands of virgin female mice in vivo; and (vi) USP34-KD mice exhibit outgrowth of mammary glands with ductal trees and alveolar buds in vivo. Taken together, these findings suggest that USP34

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plays an important role in EMT and stemness during the development of mammary glands.

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Figure Legends Fig. 1. USP34 is downregulated in mesenchymal-like cells. (A) Phase contrast images of MCF7 and NMuMG with epithelial-like phenotypes and the MDA-MB-231 and 4T1 cells with mesenchymal-like phenotypes. (B) Differential expression

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patterns of E-cadherin, vimentin, active-β-catenin and USP34 between epithelial- and

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mesenchymal-like cell lines, as determined by Western blot analysis. (C) Expression and

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subcellular localization of E-cadherin (green) and vimentin (red) in MCF7 and MDA-MB231 cells, determined by double-label immunofluorescence analysis. Cells were

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immunostained for E-cadherin (1:100) and vimentin (1:100) with DAPI nuclear stain (blue) and images were acquired using a Carl Zeiss confocal microscope (Weimar, Germany). (D)

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NMuMG cells were treated with TGF-β (0, 1, 5 ng/ml, 24 h) and changes in cell morphology were observed by phase-contrast microscopy (top panel). Re-arrangement of cytoskeletal F-

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actin with elongated spindles following TGF-β challenge (bottom panel). Cells were stained

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with fluorescent phalloidin (F-actin, red) and nuclei were stained with DAPI (blue). (E) Effect of TGF-β (0, 1, 5 ng/ml, 24 h) on protein expression of USP34 and EMT-related

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markers. Actin was used as a loading control. The star (*) indicates a non-specific band. Quantitative graphs of USP34 and E-cadherin, N-cadherin, p-Smad3 Snail and active-β-

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catenin levels are shown in the right panels. Control solvent (1 mg/ml bovine serum albumin (BSA)/4 mM HCl) was used as a control. The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test, (* p<0.05 compared with control).

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Fig. 2. Knockdown of USP34 induces EMT in NMuMG cells. (A-F) Effect of USP34 knockdown on EMT, cell invasion and secretion levels of MMP-2 and TGF-β. NMuMG cells were transiently transfected with si-USP34 or si-CTL (30 nM) for 48 h. (A) USP34 knockdown significantly increases N-cadherin, Smad4, Snail and active-β-

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catenin and reduces Axin 1 and E-cadherin expression. Quantitative graphs of Axin 1 and

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EMT-related markers are shown in the right panel (* P<0.05). (B) Inhibition of USP34 causes a loss of cytoplasmic E-cadherin (green) as determined by immunofluorescence

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analysis. The E-cadherin fluorescence intensity was analyzed using a histogram tool in the

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Carl Zeiss software package. Data was analyzed by unpaired Student's t-test (*** p<0.001 compared with si-CTL). (C) Nuclear accumulation of phospho-Smad3 following knockdown

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of USP34. Cells were immunostained for phospho-Smad3 (green) with DAPI. Nuclear phospho-Smad3 intensity profile is represented in arbitrary units defined by the software and

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scaled on the y-axis. Green fluorescence indicates nuclear phospho-Smad3 expression

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through the central nuclear regions of cells. (D) USP34 knockdown confers invasive ability of NMuMG cells. Images were captured with an inverted microscope (×400 magnification)

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and selected areas enlarged at high magnification (×800). The total number of invading cells in the fields (×400) were quantified and are shown in the right panel (*** p<0.001). (E)

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Effect of MMP-2 in NMuMG cells following USP34 knockdown. The MMP-2 levels in supernatant extracts from either si-CTL or si-USP34 (30 nM) transfected cells were determined by MMP-2 ELISA assay. (F) Measurement of TGF-β secretion levels from culture supernatants in si-CTL- and si-USP34-transfected cells. Fresh culture medium was used as a negative control. The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by student’s t-test (* p<0.05 compared with siCTL).

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Fig. 3. TGF-β enhances EMT and invasiveness following USP34 knockdown. (A) Effect of TGF-β on expression levels of EMT-related markers in the presence or absence of USP34. Quantitative graphs of E-cadherin, N-cadherin, phospho-Smad3, Smad4, Snail and

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active-β-catenin expression are shown in the right panel (* p<0.05). NMuMG cells were

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transiently transfected with si-USP34 or si-CTL (30 nM, 24 h) and then treated with TGF-β

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(1 ng/ml, 24 h). (B) TGF-β enhances USP34 knockdown-induced NMuMG cell invasion. Images were captured with an inverted microscope (×200 magnification). The total number

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of invading cells in the fields was counted and is shown in the right panel. (C) MMP-2 secretion levels (ng/ml) were determined by MMP-2 ELISA assay. The results are presented

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as mean ± SEM of at least three independent experiments. Data were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (** p<0.01, si-CTL vs si-USP34; ## p< 0.01,

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si-USP34 alone vs TGF-β treatment in the absence of si-USP34).

Fig. 4. USP34 downregulation promotes self-renewal capability.

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(A) Comparison of expression levels of USP34 and embryonic stem cell (ESC) markers

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between adherent cells and mammospheres derived from NMuMG cells. Phase contrast images of NMuMG cells and mammospheres (top panel) and Western blot images of USP34, Nanog, Oct4 and Sox2 expression (bottom panel). NMuMG cells were cultured in ultralow attachment plates for 3 days and mammosphere images were captured with an optical microscope. (B) Effect of USP34 knockdown on mammosphere formation. si-CTL and siUSP34-transfected cells were cultured in ultralow attachment plates for 3 days and mammosphere-forming ability was observed. Selected areas are shown at high magnification (bottom panels, scale bars; 100 µm). (C) The number (per 105 cells) and volume (mm3) of

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mammospheres were quantified (** p<0.01). (D) Inhibition of USP34 significantly increases mRNA transcript levels of embryonic stem cell markers. Quantitative graphs of Nanog, Oct4 and Sox2 mRNA abundance is shown in the right panel (* p<0.05). The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by student’s t-

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test. (E) Effect of TGF-β on Nanog, Oct4 and Sox2 mRNA levels in the presence or absence of USP34. NMuMG cells were transiently transfected with si-USP34 or si-CTL (30 nM, 24 h)

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and then treated with TGF-β (1 ng/ml, 24 h).

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Fig. 5. Inhibition of USP34 reconstitutes mammary outgrowth. (A) Experimental scheme. 5×104 CTL-KD or USP34-KD cells were transplanted into the left

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and right cleared fourth inguinal mammary fat pads of 3 week-old female Balb/c mice, respectively. (B) Representative whole-mount images of mammary glands from CTL-KD- or

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USP34-KD cell-transplanted mammary fat pads at 3 and 10 months after cell transplantation.

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Selected area indicates the bifurcating terminal end buds (TEBs). 10 month-old female virgin mice were used as a normal control. Whole mammary glands were stained with Carmine-

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Alum solution and images were captured with an inverted microscope (×25 magnification).

Fig. 6. Expression of USP34 during mammary gland development. (A) Hematoxylin and eosin (H&E) staining of mammary gland features at different stages. The fourth mammary glands from 1-, 2- and 3-week old, 3- and 10-month old Balb/c virgin female mice were removed. Lactating tissues were obtained from mice sacrificed 1 day after giving birth. Tissues were immunostained with cytokeratin 8/18 (CK 8/18, 1:100, green) and USP34 (1:100, red) antibodies with DAPI (nuclei, blue). The selected areas (yellow box) in images are shown at high magnification (×1000) and white arrows indicate peak

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concentrations of USP34 in the cytoplasm and nuclei of vacuolated alveolar cells during lactation. (B) USP34 expression intensity profile. Red fluorescence indicates USP34 expression through the nuclear and cytoplasmic regions of cells, with nuclei stained blue (DAPI). USP34 expression intensity (y-axis) in mammary glands at different developmental

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stages is represented in arbitrary units as defined by the software. The horizontal line (yellow dotted line) indicates 100 intensity units (y-axis on the left, a range scale 0-250 unit). White

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arrows indicate high USP34 expression through red fluorescence. Normal mouse IgG was

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used as a negative control (CTL) and adjacent sections of 2-week old glands were used for

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negative IHC staining.

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Author Contributions Conceived and designed the experiments: JYK EHO and JHS. Performed the experiments: EHO JYK DIS YKC NHL HSA YJK and TMC. Analyzed the data: EHO and JYK.

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Contributed reagents/materials/analysis tools: EHO JYK DIS YKC NHL HSA YJK TMC and JHS.

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Wrote the paper: EHO and JYK.

Conflict of interest

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The authors declare no conflicts of interest.

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Final approval of the manuscript: EHO JYK DIS YKC NHL HSA YJK TMC and JHS.

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Acknowledgements

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We thank Lee Farrand for editing the manuscript. This research was supported by the Basic Science Research Program through a National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0023605) and the Ministry of

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Science, ICT & Future Planning (MSIP, grant number: 2015R1C1A2A01053747), a grant

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from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI12C1852), and the Brain Korea (BK) 21 Plus Program.

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Highlights  USP34 knockdown increases self-renewal ability in mammary epithelial cells.  Inhibition of USP34 promotes epithelial-mesenchymal transition.

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 Inhibition of USP34 promotes invasive behavior and reconstitutes mammary gl ands.