Neuromedin U is upregulated by Snail at early stages of EMT in HT29 colon cancer cells

    Neuromedin U is upregulated by SNAIL at early stages of EMT in HT29 colon cancer cells Patrycja Przygodzka, Izabela Papiewska-Pajak, Helena Bogusz, Jakub Kryczka, Katarzyna Sobierajska, M. Anna Kowalska, Joanna Boncela PII: DOI: Reference:

S0304-4165(16)30259-8 doi: 10.1016/j.bbagen.2016.07.012 BBAGEN 28552

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

5 May 2016 7 July 2016 15 July 2016

Please cite this article as: Patrycja Przygodzka, Izabela Papiewska-Pajak, Helena Bogusz, Jakub Kryczka, Katarzyna Sobierajska, M. Anna Kowalska, Joanna Boncela, Neuromedin U is upregulated by SNAIL at early stages of EMT in HT29 colon cancer cells, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.07.012

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ACCEPTED MANUSCRIPT Neuromedin U is upregulated by SNAIL at early stages of EMT in HT29 colon cancer cells Patrycja Przygodzka1*, Izabela Papiewska-Pajak1, Helena Bogusz1, Jakub Kryczka1, Katarzyna Sobierajska2, M. Anna Kowalska1,3, Joanna Boncela1*

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Author details Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland

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Department of Molecular Cell Mechanisms, Medical University, 6/8 Mazowiecka Street, 92215 Lodz,

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Poland

Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA

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*Corresponding authors: Patrycja Przygodzka, [email protected] and Joanna Boncela, [email protected]

E-mail addresses: [email protected] (I. Papiewska-Pajak), [email protected] (H.

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Bogusz), [email protected] (J. Kryczka), [email protected] (K. Sobierajska), [email protected] (M. A. Kowalska)

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Highlights

Snail regulates phenotype conversion in HT29 up to an intermediate epithelial state

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Neuromedin U (NMU) is upregulated in colon cancer cells with induced EMT signals

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NMU upregulation is Snail dependent

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NMU protein and mRNA is released from HT29-Snail cells

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Abbreviations CM: conditioned medium; CRC: colorectal cancers; CIMP: CpG island methylation phenotype; CIN: chromosomal instability; DEGs: differentially expressed genes; EGF: epidermal growth factor; EMT: epithelial-mesenchymal transition; IPA: Ingenuity Pathway Analysis; NMU: neuromedin U; OSCC: squamous cell carcinoma; TGF-β: transforming growth factor-β

ACCEPTED MANUSCRIPT ABSTRACT Background: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a

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key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown

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and new predictive markers of the process are awaited.

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Methods: Genes expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU

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detection was performed by ELISA.

Results: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell

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motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media.

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Conclusions: These results show that Snail in HT29 cells regulates early phenotype conversion towards

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an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells.

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General Significance: We described the global, early transcriptomic changes induced through Snail in

Keywords: Snail

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HT29 colon cancer cells and suggested NMU involvement in this process.

Epithelial-mesenchymal transition Metastasis Colon cancer Neuromedin U

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ACCEPTED MANUSCRIPT 1. Introduction Colorectal cancer (CRC) is the third most common cancer worldwide with 1.36 million new cases described in 2012 [1]. Despite the progress in diagnosis and therapy, metastasis and tumor recurrence are two critical processes influencing the survival rate for CRC that is only 67% within a 5-year period [2]. In most cases CRC cells metastasize to the liver, lung and peritoneum but metastasis to other sites such as

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bone, spleen, brain and distant lymph nodes, has also been reported [3]. Molecular knowledge of the

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metastatic process is limited but systematic studies comparing gene expression in primary tumors and their metastases revealed the cellular and biochemical cascades activated during tumor spread [4,5].

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One of the first steps of local dissemination from solid tumors and the subsequent evolution of metastasis is the epithelial-mesenchymal transition program (EMT), which contributes to the phenotypic transformation of epithelial carcinoma into mesenchymal-like cells. Under physiological conditions, EMT

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represents a fundamental mechanism involved in wound healing and organism development [6]. EMT was also found to be associated with carcinoma progression [7–9]. It was shown that cancer cells at the invasive front of a primary tumor lose epithelial features, exhibit decreased polarity and intercellular

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adhesion, undergo cytoskeletal reorganization and become more motile [10,11]. Many in vitro studies have demonstrated that the cells undergoing EMT have enhanced stemness, reduced anoikis and increased drug resistance. Also gene expression profiling provided clinical evidence that EMT is

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important in primary human carcinogenesis [12–14]. Previous studies have attempted to characterize

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EMT in in vivo mouse models of breast [15] and pancreatic cancer progression [10]. Improving the knowledge of the early phase of EMT is important, as increasing evidence suggests that the dissemination and seeding of epithelial cells precede the actual detection of a primary tumor [10].

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Transcription factor Snail (Snai1) is an early core regulator of the EMT that functions through the repression of E-cadherin and other epithelial markers but also through the upregulation of mesenchymal, pro-invasive genes [16–18]. Snail is regulated through various signals from the tumor microenvironment,

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including extracellular matrix components and soluble factors, such as transforming growth factor-β (TGF-β) and other cytokines (reviewed in [19]). Notably, the TGF-β pathway is considered a primary EMT inducer through Snail upregulation in a SMAD-dependent manner [20]. Enhanced Snail expression has been observed in colon cancer cells and wide range of other carcinomas (breast, ovarian, pancreas, prostate), and was associated with more aggressive phenotypes, poorer clinical outcomes and frequent distant metastases [21–24]. However, the molecular basis of Snail involvement in the EMT transition, independently of other transcription factors implicated in the regulation of the EMT, such as ZEB and Twist families [16], has not been fully clarified. The ability of Snail to induce phenotype conversion in epithelial cells has been previously examined [17,24], but the status of colon cancer cells in the EMT spectrum and the global transcriptomic changes induced by elevated Snail levels has not been reported.

Extensive studies of CRC have led to the discovery of many putative colorectal cancer biomarkers for the prediction of long-term prognosis and the response to therapy; however, only a few markers show value as prognostic factors (EGFR, BRAF, MSI phenotype, 18q AI expression, p53, and KRAS) [25,26]. Also full-length mRNA molecules have been previously detected in cancer cell culture medium [27] and in 3

ACCEPTED MANUSCRIPT cancer patient serum, and these molecules have been proposed as markers for breast [28] and colon cancer [29,30] diagnostics.

In the present study, the transcriptomic observations of Snail overexpressing HT29 CRC cell line let us identify new pathways and genes not associated previously with early stages of metastasis.

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One of them was upregulation, both on transcriptional and protein level, of neuromedin U (NMU).

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Neuromedin U is a secreted neuropeptide with widespread distribution throughout the body. This protein is synthetized as a 174 amino acid precursor and cleaved to an active 25-amino acid peptide through as-

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yet-unknown proteases [31]. Two G-coupled receptors for NMU have been identified, NMU-R1 and NMU-R2, which are distributed in various tissues, including the gastrointestinal tract [32]. NMU has been far associated with a myriad of different functions, such as feeding behavior, stress, pain, smooth muscle

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contractions, immune responses, hormone release, and cancer cachexia (reviewed in [33]). Limited evidence exists connecting NMU with cancer. So far, the reported findings have been contradictory and dependent on cancer type. Fairly sparse studies have shown an association between NMU abundance and

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the progression of ovarian [34], bladder [35], renal [36], pancreatic [37] breast [38], lung [39], endometrial cancers [40] and myeloid leukemia [41]. In contrast, preliminary investigations of oral [42] and esophageal [43] cancers suggested the suppressive activities of NMU. Interestingly, NMU

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overexpression increased tumor formation and metastasis only in vivo, indicating that the impact of NMU

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is highly related to tumor microenvironment, consistent with the emerging paradigm of cancer progression modulation through a tumor niche [44]. Here we found that NMU and NMUR2 are coexpressed in colon cancer cell line with Snail overexpression. Our data for the first time associate NMU

2. Methods

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expression and release with Snail transcription factor activity and colon cancer progression.

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2.1. Cell culture and reagents

The HT29 colon cancer cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in McCoy’s 5A medium (LifeTechnologies, Waltham, MA, USA), supplemented with 10% FBS (LifeTechnologies, Waltham, MA, USA) and antibiotics - streptomycin and penicillin (SigmaAldrich, St. Louis, MO, USA), primocin (Invivogen, San Diego, CA, USA) in a 90-95% humidified atmosphere of 5% CO2. The cells were periodically tested for mycoplasma every 4 weeks using the PlasmoTest (Invivogen, San Diego, CA, USA). 2.2. HT29 nucleofection and stable clone generation The pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) and pcDNA3.1 vector expressing Snail was obtained from Prof. Muh-Hwa Yang (Institute of Clinical Medicine, National Yang-Ming University Taipei, Taiwan) [45]. HT29 cells were grown to 85% confluence and transfected with 5 μg DNA/106 cells using the Amaxa® 4D nucleofector

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X Unit (Lonza, Basel, Switzerland). Subsequently, the cells were

cultured in medium supplemented with 200 μg/mL G418/Geneticin (Gibco/LifeTechnologies, Waltham, MA, USA). The selection medium was refreshed every 48 h. After 2 weeks in culture, well-separated colonies were isolated. Snail expression was verified through real-time PCR and Western blot analysis. 4

ACCEPTED MANUSCRIPT Snail-siRNA and non-targeting control siRNA, 60 pmoli/106 cells (Dharmacon, Lafayette, CO, USA) was delivered to HT29-Snail clones using Amaxa® 4D nucleofector (as above). 72h post-nucleofection cell lysates were collected and Snail mRNA expression and protein level were verified. 2.3. Western Immunoblotting Proteins from cells were extracted with NP-40 lysis buffer (50 mM TRIS, pH 8.0 containing 1% Nonidet-

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Igepal, 150 mM NaCl, 5 mM EDTA) with the Halt protease inhibitor cocktail (Thermo Scientific,

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Waltham, MA, USA) and the soluble protein fraction was collected through centrifugation. The equal amount of protein extracts (concentration measured BCA method (Pierce/Thermo Scientific, Waltham,

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MA, USA) were subjected to SDS-PAGE analysis, transferred onto PVDF membranes (BioRad, Hercules, CA, USA) and blotted with mouse anti- Snail antibodies (Cell Signaling Tech, Danvers, MA, USA). The control antibody, rabbit anti-β-actin was obtained from Abcam (Cambridge, UK). Secondary

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HRP-conjugated antibody was from Santa Cruz Biotechnology, Dallas, TX, USA, enhanced chemiluminescence kit from Thermo Scientific, Waltham, MA, USA, and Kodak BioMax Light Film from Eastman Kodak, Rochester, NY, USA.

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2.4. Confocal imaging

The cells were grown on coverslips (Nunc/Thermo Scientific, Waltham, MA, USA) until 60-70% confluency and incubated with Hoechst33342 (Molecular Probes/ LifeTechnologies, Waltham, MA,

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USA) for 15 min at 37oC. After washing the cells were fixed in cold acetone, blocked and incubated with

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anti-E-cadherin (1: 200) or anti-vimentin (1: 50) antibodies (both from Santa Cruz Biotechnology, Dallas, TX, USA) following by incubation with secondary antibodies conjugated with Alexa Fluor-488 (LifeTechnologies, Waltham, MA, USA). The slides were washed with PBS, mounted with Mowiol

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(Sigma-Aldrich, St. Louis, MO, USA) and the cells were visualized under a confocal microscope (Nikon D-Eclipse C1; Nikon, Tokyo, Japan) with a 40x objective and analysed with EZ-C1 version 3.6 software. 2.5. Motility assay with the use of time-lapse microscopy

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Cells were seeded (105 cells/well) onto 12-well plates pre-coated with poly-L-lysine, fibronectin, vitronectin (Sigma-Aldrich, St. Louis, MO, USA) or type I collagen (Merck, Darmstadt, Germany). After 8 h cells movement was examined using a Leica AF7000 Live Imaging System (Leica, Wetzlar, Germany) containing Inverted Leica DMI6000 microscope and High Speed Camera Andor iXon DU-885. The plates were incubated under 5% (v/v) CO2 in air at 37°C , and differential interference contrast (DIC) images were captured and recorded for 14 h with 5-min time intervals. Individual cell trajectories were tracked manually using the MetaMorph software. Track Points function to denote the coordinates of the center of each cell in the DIC image at the pixel level and tracking function to measure distance for cells moving in almost 170 consecutive frames were applied. Based on obtained data average speed of cell movement (μm/h), i.e., total length of cell trajectory divided by time of recording and total length of cell displacement (μm), i.e., the direct distance from the starting point to the final position of the cell was calculated. 2.6. Transcriptomic analysis of HT29 clones Transcriptomic analysis was done in triplicate on mRNA samples isolated from HT29-pcDNA, HT29Snail 3 and HT29-Snail 8 clones. Microarray expression analysis was performed using GeneChip human 5

ACCEPTED MANUSCRIPT genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA) and the Affymetrix GeneChip system according to the manufacturer's instructions. The Pearson's analysis was applied to estimate the correlation between linear variables. The CEL files were imported into the Partek Genomic Suite v 6.6 software using GCRMA (GC Robust Multiarray Averaging). The qualitative, Principal Component Analysis and the quality assessment using Analysis of Variance (ANOVA) were performed (with the

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cutoff values: p-value with FDR < 0.05 and -2 > Fold Change > 2). The selected lists were subjected to

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cluster analysis to identify genes and samples with similar profiles using Hierarchical Clustering algorithm. The results were interposed onto the database of Ingenuity using Ingenuity Pathway Analysis

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software (Ingenuity®Systems, Redwood City, CA, USA; http://www.ingenuity.com) containing information about the gene functions. The microarray experiments and data analysis were performed with the collaboration of the Corelab Laboratory for Microarray Analysis (Institute of Biochemistry and

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Biophysics, Polish Academy of Sciences, Warsaw, Poland).

2.7. RNA isolation from cells and real-time PCR Analysis

Total RNA was isolated from cells using miRCURYTM RNA Isolation Kit (Exiqon, Denmark) according

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to manufacturer’s instructions, with additional RNAse-free DNAse I (Ambion/ LifeTechnologies, Waltham, MA, USA) treatment. The quality control of isolated RNA was performed using the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions.

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Briefly, 1 μg of the isolated total RNA from cells (RIN ≥ 8) were reverse transcribed using the High

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Capacity cDNA Reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. TaqMan Gene Expression Assays for Snail (HS00195591_m1), neuromedin (Hs00183624_m1),

(Hs01023894_m1),

NMUR1

vimentin

(Hs00173804_m1),

(Hs00185584_m1),

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U

NMUR2 claudin-1

(Hs00173930_m1), (Hs00221623_m1),

E-cadherin β-actin

(Hs01060665_g1), GAPDH (Hs99999905_m1) were further used for transcripts quantification through real-time quantitative PCR using the TaqMan Universal PCR master mix and the ABI Prism7900-HT

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detection system (both from Applied Biosystems, Foster City, CA, USA). A standard PCR cycle with incubation at 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min was used. GADPH and β-actin mRNA transcripts were used as internal control genes. The amount of target mRNA in the various samples was estimated using the 2-ΔΔCT or the 2-ΔCT relative quantification method with DataAssist v.3.01 software. 2.8. Conditioned media preparation, filtration and RNA isolation The cell lines were seeded onto T25 cm2 cell culture flasks and grown to 70% confluency. The medium was changed to medium with low serum supplement 2% FBS (3 ml / flask). Aliquots (1 ml) of conditioned medium (CM) were removed 48 hours post-seeding. CM was prepared according to a previously described method with small modifications [27]. Briefly, 1 ml medium was passed through a 0.22 μm filter (Millex/Sigma-Aldrich, St. Louis, MO, USA), 200 μl aliquots of filtered conditioned medium were added to 500 μl of TriReagent (Sigma; Poole, England) and incubated for 5-10 min on ice prior to snap-freezing/freezing at -80°C, to ensure the complete dissociation of nucleoprotein complexes. A total of 120 ng of total RNA from CM was used for first-strand cDNA synthesis with Maxima First

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ACCEPTED MANUSCRIPT Strand cDNA Synthesis Kit (LifeTechnologies, Waltham, MA, USA), and 3 μl of cDNA from CM was used for amplification. 2.9. Human NMU Elisa Cell lysates and conditioned media were collected as described previously. An ELISA kits for NMU (MyBioSource, San Diego, CA, USA and FineTest, Wuhan City, China) were used according to the

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manufacturer’s instructions.

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2.10. Statistical analysis

The Shapiro-Wilk test was used to confirm the Gaussian distributions of raw data. Data non-departing

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from normal distribution are presented as mean and SD or SEM; otherwise, medians and interquartile ranges are used. For the unpaired comparisons, the appropriate Student’s t test (or the Welch's test for unequal SDs) was performed to test the differences between groups for normally distributed data.

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Respectively the Mann-Whitney’s U test was performed to test the differences between groups of data

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with non-normal distributions.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Characterization and functional effects of Snail overexpression in HT29 cells. To identify early changes in CRC cells induced through the Snail transcription factor, we utilized the

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HT29, the cell line with low endogenous Snail expression. This line has been previously used in our

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studies as a colon cancer model along with human tumor samples [46,47]. Cells overexpressing Snail were generated through the transfection of HT29 cells with pcDNA-Snail. Four positive clones with

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various levels of Snail expression were generated through G418 selection. Two stable clones, reported earlier [47] were selected for subsequent analysis: HT29-Snail clone 3, with moderate Snail overexpression, and HT29-Snail clone 8, with demonstrated higher levels of Snail expression as shown

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by Western blot analysis (Fig. 1A and [47]) and real-time PCR (Fig.S1-A ) and comparable to clones 17 and 40. In both clones E-cadherin and claudin-1 expression was decreased, while mesenchymal vimentin expression was elevated (Fig. 1B and Fig S1-B) confirming the EMT characteristics of these cells.

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Consistent with our previous studies, cells overexpressing Snail grew more scattered, acquired a spindleshaped morphology and lost cell-to-cell contact (Fig. 1B) [47]. This result is consistent with observed in transcriptomic analysis down-regulation of tight-junction, adherence-junction and basement membrane

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adhesion components (Tables S.1-S.3).

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We have also measured the random motion of cells seeded onto various ECM proteins. Clones with Snail overexpression moved faster and covered a longer distance as compared to control cells expressing HT29-pcDNA (Fig. 1C). Interestingly, the extracellular matrix proteins, fibronectin, vitronectin and type I

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collagen, enhanced HT29-Snail cells motility, as compared to poly-L-lysine, to much higher extent than control cells where the increase did not reach statistical significance. The migration rate in both HT29Snail clones also increased over time as compared with control HT29-pcDNA in the wound healing-like

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(scratch) assay (Fig. S.1-C).

Additionally, Snail-expressing clones showed no increased formation of new colonies out of single-cell when examined under adhesive conditions. In opposite, fewer dispersed colonies were formed (Fig. S.2A). Moreover, Snail overexpression did not alter the ability of HT29 cells to survive in suspension (Fig. S.2-B). However, HT29 cells overexpressing Snail proliferated with a slightly higher rate than control cells (Fig. S.2-C). 3.2 Global transcriptional changes in Snail overexpressing HT29 cells. Gene expression microarray analysis of the generated stable cell lines were performed. According to the established criteria (as described in Methods), among the 47 000 transcripts represented in the microarray, 968 and 1685 genes were differentially expressed in HT29-Snail clones 3 and 8, respectively (Fig. 2A, Tables S.1, S.2). A total of 541 genes were commonly altered in both clones. Among those, 340 genes were down-regulated, while 201 genes were upregulated. In a majority of differentially expressed genes (DEGs) the rate of change was higher in HT29-Snail clone 8 than in HT29-Snail clone 3 compared with control cells transfected with the empty vector. Transcriptomic profiles were subjected to hierarchical clustering to order the genes in the structure of a hierarchical tree (dendrogram), where similar 8

ACCEPTED MANUSCRIPT transcriptomes are located in close vicinity to each other, while divergent genes were located far apart in the tree (Fig. 2B). To associate biological functions and diseases with the experimental results and identify the biological processes that might be changed in response to elevated Snail expression, we performed functional enrichment analysis on all differentially expressed genes from HT29-Snail compared with HT29-pcDNA

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microarray data sets using IPA software. After leveraging the HT29-Snail 8 data and complex biological

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interactions stored in the Ingenuity Knowledge Base, we identified significantly (Fisher’s exact test p value < 0.05) overrepresented molecular and cellular functions altered upon Snail overexpression

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associated with cellular movement, cell death, cellular development, cellular growth and proliferation and lipid metabolism (Fig. 2C). Further enrichment analysis showed that the detected DEGs primarily corresponded to transcriptome alterations during cancer and gastrointestinal disease development (Fig.

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2C). Additionally, the IPA Upstream Regulator Analysis predicted the upstream regulators responsible for the changes in gene expression observed in the data sets (Table 1). Table 1. Predicted upstream regulators of the changes resulting from Snail overexpression.

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The p-value overlap, calculated using Fisher’s Exact Test, indicates upstream regulators that explain the changes observed in gene expression. These results represent the significance of the overlap between the data set genes identified here and known targets of transcriptional regulators. The activation z-score was

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additionally used to infer the activation states of predicted upstream regulators (predicted through IPA

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Name

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activation when the z-score is ≥ 2, inhibition when the z-score ≤ ‑2).

IFNL1

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TGFB1

p-value overlap HT29-Snail clone 3 2.95 x 10-54

Activation z-score -7.078

HT29-Snail clone 8 1.62 x 10-14

1.389

For HT29-Snail clone 8 (high expression clone), TGFβ was indicated through IPA as the most significant upstream regulator for the regulation of the transcriptomic changes in response to Snail. Changes in the expression of TGFβ signaling pathway components suggest that this pathway is modestly activated. However, type III interferon was predicted as the primary regulator of the changes in the moderately expressing cell line HT29-Snail clone 3. Thus, HT29-Snail clone 8 was a more interesting model for early EMT studies, as the detected transcriptomic changes resembled those in response to TGFβ, as an early inducer of EMT.

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ACCEPTED MANUSCRIPT 3.3 Snail dependent expression of genes associated with cellular motility in HT29-Snail clones – elevated expression of NMU IPA software analysis of the obtained microarray data set identified 324 (HT29-Snail 8 vs HT29-pcDNA) DEGs associated with changes in cellular motility (Tables 2 and S.4). Fibronectin was the most upregulated gene (fold-change = 87.7) on the list. The second highly overexpressed gene was neuromedin

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U (NMU) (FC=58.9), a neuropeptide that was not previously associated with colon cancer or its

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metastasis.

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Table 2. Selected genes, which are differentially expressed in HT29-Snail 8 vs. HT29-pcDNA (fold change > 20 and/or p < 2 x 10-8) and respective changes in HT29-Snail 3 vs. HT29-pcDNA associated by IPA with cellular movement.

FAS FYN LYN SERPINE1 MAP3K5

Foldchange

p-value

HT29-Snail 3 vs. control

87.776 58.966 55.722 45.666 38.822 34.644

5.06 x 10-11 8.22 x 10-7 1.61 x 10-5 2.85 x 10-7 1.28 x 10-8 1.32 x 10-7

1.015 16.149 7.069 2.625 2.846 1.491

0.74 7.82 x 10-6 9.02 x 10-4 7.73 x 10-4 2.12 x 10-5 0.02

34.266 33.099

3.53 x 10-7 4.63 x 10-6

-1.030 9.635

0.85 5.76 x 10-5

27.103 22.927 17.917

4.38 x 10-7 1.06 x 10-5 1.62 x 10-9

1.501 2.092 -1.023

0.03 0.02 0.65

14.682 11.661 4.24

2.91 x 10-9 1.49 x 10-8 8.00 x 10-9

1.128 1.003 -1.030

0.05 0.98 0.39

2.315

8.87 x 10-9

1.043

0.42

-2.750

1.85 x 10-8

-1.760

5.93 x 10-7

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CXCR4 LGALS1 TGFB1I1

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MAGI2 WNT11

p-value

HT29-Snail 8 vs. control

fibronectin 1 neuromedin U tenascin C matrix Gla protein tubulin, alpha 1a tumor-associated calcium signal transducer 2 membrane associated guanylate kinase wingless-type MMTV integration site family, member 11 chemokine (C-X-C motif) receptor 4 lectin, galactoside-binding, soluble, 1 transforming growth factor beta 1 induced transcript 1 Fas (TNF receptor superfamily) FYN oncogene v-yes-1 Yamaguchi sarcoma viral related oncogene homolog serpin peptidase inhibitor, clade E (plasminogen activator inhibitor type 1) mitogen-activated protein kinase kinase kinase 5

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FN1 NMU TNC MGP TUBA1A TACSTD2

Gene Title

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Foldchange

Gene symbol

The microarray data were validated through real-time PCR, confirming the significant upregulation of NMU transcription in all generated stable clones with Snail-elevated expression (Fig. 3A) however NMU expression in HT29-Snail 17 and 40 was less stable as well as Snail transcription level (Fig. S1A). Observed NMU transcription upregulation was significantly abrogated after silencing of Snail expression through Snail siRNA delivery to HT29-Snail clones (Fig. 3B and S3A and B). Simultaneously in clones with Snail overexpression, the only NMU receptor expressed by HT29 cells, NMUR2 was downregulated (Fig. 3C). The NMUR1 expression was not detected neither in HT29 cells nor in HT29 generated clones. A similar effect of Snail overexpression on NMU increase was detected in LS180, another colon cancer cell line with diverse genetic and epigenetic characterization and Snail overexpression (Fig. S3-C).

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ACCEPTED MANUSCRIPT 3.4 NMU overexpression correlates with NMU release to conditioned media of HT29-Snail cells. The elevated NMU transcription level correlated with protein concentration, analysed by ELISA, in the conditioned medium from HT29-Snail clone 8, but not from HT29-pcDNA (Fig. 4). The rising trend seen in conditioned medium from HT29-Snail clone 3 didn’t reach statistical significance.

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3.5 NMU mRNA detection in conditioned media from HT29 cells overexpressing Snail.

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In conditioned medium (CM) form HT29-Snail clones NMU transcription upregulation correlated also with NMU transcript abundance (Fig. 5 ). In the conditioned media from control cells, NMU transcript

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have not been detected up to 50 cycle by real-time PCR. β-actin mRNA transcript was used as a control gene and detected in all samples. Discussion

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The most successful therapeutic approach in CRC is the elimination of the tumor during the early stages of cancer. However, even when diagnosed early, it remains difficult to predict clinically latent versus

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invasive tumors and qualify patients for more aggressive adjuvant therapy. Advanced systemic post resection therapies for CRC improves the overall survival but once metastasis becomes clinically apparent the prognosis is poor and survival is dramatically shortened.

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The EMT is considered to be an initial and critical process for metastasis, dynamically regulated during

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malignant transformation and its biomarkers are considered to be a predictive factors for cancer development [22,48–51]. Core regulators of early stages of epithelial phenotype conversion, such as

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Snail, remain technically challenging as therapeutic target. Hence, we focused on the transcriptomic changes affected by the Snail, in order to search for still awaited new potential biomarkers of metastasis risk. In our studies we have used the HT29 cell line with epithelial characteristics and well-defined genetic and epigenetic features [52,53], representing 85% of sporadic CRCs with chromosomal instability

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(CIN) and 20-30% CRCs with CpG island methylation phenotypes (CIMP). Consistent with previously reported studies [24,54], we observed that Snail-induced morphology, function and transcriptional changes are characteristics of conversion towards a mesenchymal phenotype. Our data (Fig.1 and S1-S2, tables S.1, S.2) indicate that Snail upregulation results in incomplete phenotype conversion, up to the intermediate epithelial state; cells express mesenchymal markers and become more motile but still well proliferate and have weak capability to form new colonies. These observations imply that EMT process in CRC should be perceived as a spectrum of intermediate states between epithelial and mesenchymal. The existence of these states was recently shown in ovarian and lung cancer cells [55,56]. Transcriptomic analysis of differentially expressed genes in Snail-HT29 clones provided additional information about Snail-regulated genes. In the high expressing clone (HT29-Snail clone 8), we observed almost doubled number of transcripts with altered expression compared with the low-expressing clone (HT29-Snail clone 3). The differences in the transcriptomic results reflect variations in the Snail levels between clones (Fig. 1). These, and the findings shown below are consistent with the previously reported results in epithelial kidney and squamous cell carcinoma, where even small divergences in the Snail level had consequences on the cell phenotype [57]. Unexpectedly, despite the fact that in both established cell 11

ACCEPTED MANUSCRIPT lines Snail expression induced EMT signals, most significant upstream regulators of the two selected clones predicted through IPA analysis were different (Table 1). The HT29-Snail clone 8 data suggest the activation of the TGFβ pathway, while DEGs from HT29-Snail clone 3 indicated the inhibition of type III interferon (IFN)-induced pathways. This observation is interesting in the context of the well-documented antiproliferative and antitumor activity of interferons [58] and adds new information associating IFN-

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signaling proteins with Snail-dependent EMT, as previously observed [59].

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Examination of the results of the functional enrichment analysis showed, as expected, that gene expression altered through Snail in CRC cells contributed with the highest degree to changes in the

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cellular movement characteristics for cancer and gastrointestinal diseases. A total of 324 identified transcripts with altered expression were previously associated with changes in cellular motility but significant number of those genes were not reported as Snail activity-associated. Such as pseudopod-

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specific proteins critical for tumor cell migration and invasion: AHNAK, septins, eIF4E and S100A11 [60] and others (Table S.4).

We report here the new observation that neuromedin U (NMU) is upregulated in cells with Snail

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dependent EMT induction (HT29-Snail clones) and silencing of Snail abrogates this effect. Regulation of NMU expression is potentially associated with the early stages of colon cancer metastasis. NMU mRNA and protein abundance have been detected in conditioned medium of HT29-Snail clones - but not HT29-

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pcDNA (Fig. 4-5 and S4A) thus NMU was not only overexpressed but also released from HT29-Snail

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cells. Our attempt to show Western immunoblotting or immunostaining of NMU failed, most probably because of low levels of protein. Using commercially available ELISA kit we detected NMU protein in whole cell lysate, but there was no changes in its level in Snail-overexpressing clones (Fig.S4B). It is

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possible that NMU expression is combined with NMU release from the cells. Explanation of this observation remains unknown but Mitchell et al. [61] proposed that NMU is a locally acting peptide rather than a circulating hormone. Its concentration detected in human plasma is very low, at least at

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physiological conditions [61]. HT29-Snail cells also express lower level of NMU receptor (NMUR2) (Fig. 3C) which indicates other than autocrine function of released protein. It points to cancer microenvironment cells with NMU receptors expression, like macrophages, vascular smooth muscle or endothelial cells, as potential targets of NMU. By binding to NMUR1 receptor, NMU promotes production of pro-inflammatory IL6 by macrophages in endotoxemia [62] and IL6 and IL8 derived from tumor associated macrophages (TAMs) enhanced invasive activity of colon cancer LOVO cells [63]. Function of NMU receptors on endothelial cells remains unknown but receptors on vascular smooth muscle cells enable NMU to act as a potent endothelium-independent vasoconstrictor of the human artery and vein [61]. Vascular smooth muscle cells cooperate with endothelial cells in angiogenesis process. Besides other proteins regulated by Snail, reported previously and detected in our analysis that are involved in tumor microenvironment modulation such as fibronectin, tenascin C, thrombospondins and collagens, TGFβ, EGF or interleukins (Tables S.1, S.2), NMU, as a paracrine factor, can be an active player in tumor niche rearrangement induced by Snail in colon cancer cells. Our hypothesis is strengthen by observation that NMU is expressed in human colorectal adenoma [64] described as the first stage of colorectal cancerogenic process where cells are exhibiting Snail expression [65]. 12

ACCEPTED MANUSCRIPT NMU mRNA expression was correlated with poor outcome in breast cancer [38]. Similarly to what we found in colon carcinoma cells, it was shown that NMU mRNA was expelled from breast cancer cells and potentially became new independent prognostic factor for disease progression. The rate and importance of this observation in colon cancer warrants the future study. In our preliminary observations, NMU mRNA is released from HT29-Snail clone 8 cells in microvesicles (MV) fraction as there was no transcript

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detection in RNA isolated from exosomes (Fig. S4A). NMU was also detected in microvesicles fraction

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derived from SW480 colon cancer cells [66]. Detection of NMU mRNA in MV is interesting in the context of MV significance in intercellular communication in physiological and cancer conditions [67–

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70] but also points to this molecule as a candidate marker of colon cancer cells phenotype conversion. NMU has never previously been associated with colon cancer progression, thus further analysis of NMU expression and its extracellular abundance in CRC patients, will shed more light on the potential function

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of these molecules as regulators or independent markers of the early stages of the metastatic process.

5. Conclusions

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Our broad transcriptomic observations serve as a base for subsequent investigations of the mechanisms and molecules involved in Snail-mediated EMT and might contribute to discoveries of innovative antimetastasis therapeutic approaches in colon cancer diagnosis and treatment.

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We provide here evidence that Snail, that regulates EMT transition, causes neuromedin U upregulation

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and expulsion both on the protein and transcript levels. Our results provide rationale for extensive investigation of NMU function in colon cancer progression or its utility as a new biomarker of processes

Acknowledgments

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that facilitate cancer cells to escape from the primary location.

This work is dedicated to our mentor and teacher Prof. Czeslaw Cierniewski (1946-2013). Although

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terminally ill, Prof. Cierniewski put an enormous effort in carrying on this project.

Funding sources

This research was financially supported by the National Science Center, Cracow, Poland [Project no DEC-2011/02/A/NZ3/00068].

Competing interests The authors declare that they have no competing interests.

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ACCEPTED MANUSCRIPT Supplementary results Figure S1 – S4 Table S.1. The list of all differentially expressed genes in the HT29-Snail 3 clone vs. HT29-pcDNA detected through microarray gene expression analysis.

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Table S.2. The list of all differentially expressed genes in the HT29-Snail 8 clone vs. HT29-pcDNA

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detected through microarray gene expression analysis.

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Table S.3. Selected genes associated with cell morphology, motility and tumor microenvironment, which are differentially expressed in HT29-Snail clones vs. HT29-pcDNA classified using the Web-based gene ontology tool Panther Classification System

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Table S.4. The list of differentially expressed genes in the HT29-Snail 3 and HT29-Snail 8 clone vs. HT29-pcDNA, previously associated with cellular movement through IPA analysis.

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Supplementary Methods

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Figure 5

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ACCEPTED MANUSCRIPT Highlights Snail regulates phenotype conversion in HT29 up to an intermediate epithelial state

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Neuromedin U (NMU) is upregulated in colon cancer cells with induced EMT signals

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NMU upregulation is Snail dependent

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NMU protein and mRNA is released from HT29-Snail cells

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