Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics

Epithelial and mesenchymal murine liver cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Sigma) supplemented with 10% fetal bovine serum as described [37]. The human HCC cell line Huh7 was provided by Jianming Huh, Penn State College of Medicine and cultured as described [36, 38]. The human HCC The human HCC cell lines MHCC97-L were provided by Xinwei Wang, National Cancer Institute, under agreement with the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China and cultured as described [39].

For Snail1 transient knockdown, cells were transfected with 100 pM of Snail1 Stealth siRNA (Invitrogen) using Lipofectamine 2000 (Invitrogen). For Smad signaling inhibition, cells were transfected with 2 ug of DNA using Fugene 6 (Roche). To generate Snail1 knockdown stable transfectants, mesenchymal cells were transfected with Snail1 Mission shRNA lentivirus (Sigma) and selected with 2 ug/ml of puromycin.

pCMV5-Smad7-HA (Plasmid 11733), pRK-Smad3ΔC (Plasmid 12626), and Nanog-Luc (Plasmid 16337) were provided by Addgene. Cells were plated in 12 well plates, incubated overnight, and transfected with the Nanog-Luc plasmid and Renilla for 24 hours (4:1 Nanog-Luc:Renilla ratio). Cells were washed with 1 × PBS, serum free starved for 2 hours, and treated with 5 ng/ml of TGFβ for 24 hours. Following cell lysis, luciferase activity was measured using the Dual Luciferase Assay Kit (Promega) and a Sirius Luminometer V3.1 (Zylux). Luciferase reading light units (RLU) were normalized to Renilla RLU and a fold change was calculated.

Trizol (Invitrogen) was used to isolate total RNA from cells according to manufacturer's protocol. Isolated RNA was quantified using the ND-1000 spectrophotometer (NanoDrop) and complementary single strand DNA was synthesized using the Omniscript RT Kit according to the manufacturers protocol (Qiagen). qPCR was performed using Taqman Gene Expression Assays and ABI-Prism 7700 Thermal Cycler (Applied Biosystems). Normalization was performed using β-actin or Gapdh as an endogenous control and relative gene expression was calculated using the comparative 2^(-ΔΔCt) method with SDS 2.2.2 software [36].

Cell viability was performed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) kit (Trevigen) according to the manufacturer's protocol. 5 × 10³ cells were plated in 96-well plates, incubated for 24 hours at 37°C, and treated with specified agents at defined time points.

Cells were washed twice with ice cold 1XPBS and cell lysates were harvested by the addition of lysis buffer (40 nM Tris [pH 7.4], 150 mM NaCl, 10 mM ethylene diamine tetraccetic acid, 10% glycerol, 1% Triton X-100, 10 mM glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail tablets (Roche). BCA protein assay (Thermo Fisher Scientific) was used to determine protein concentration as described [40]. 30 ug of protein lysates were separated on a NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Invitrogen). After blocking for 60 min with 5% non-fat dry milk, membranes were incubated with the primary antibody overnight at 4°C followed by incubation with corresponding secondary antibody for 60 min at room temperature. The membranes were developed using enhance chemiluminescence solutions (Thermo Fisher Scientific) [41].

The capability of tumor cell migration was assessed using a wound-healing assay. Confluent cell monolayers were manually wounded by scraping the cells with a 1,000 μL pipette tip down the center of the well. The cell culture medium was replaced and migration was assessed at 24 hours [37].

Cell invasion was assessed using 6-well Transwell permeable inserts with 8-μm pores (Corning) [37]. In brief, 1 × 10⁵ cells were cultured in a serum-free DMEM/F12 medium in an insert coated with Matrigel (BD). Below the insert, the chamber of 6-well plates contained DMEM/F12 supplemented with 10% FBS. Cells were incubated in a 37°C incubator for 48 hours and the number of cells that invaded across the membranes and fallen onto the bottom of the plate was counted.

Using the cell lines from the liver specific Pten ^-/- model described [37] P2E (epithelial) and P2M (mesenchymal) messenger RNA were analyzed using an Illumina mouse gene chip according to the manufacturer's protocol and as described [37]. Housekeeping genes were used as standards to generate expression levels, and data analysis was conducted using 1.4-fold or greater change in expression with p < 0.05 as significant. The full complement of the expression data is available at http://​www.​ncbi.​nlm.​nih.​gov/​geo (Accession number GSE18255).

The capability of self-renewal was assessed using Corning Ultra-Low Attachment Surface (Corning). 5 × 10³ cells were seeded and incubated in a cell culture incubator for 1 week in DMEM/F12 supplemented with 10% FBS or serum free medium and phase-contrast images were obtained.

Cells were counted with trypan blue exclusion and suspended in a 1:3 dilution of Matrigel (Matrigel:DMEM/F12 supplemented with 10% FBS) [36]. 1 × 10⁴ and 1 × 10⁵ cells/50 μL were injected subcutaneously into 10-week-old nude mice. Caliper measurements of tumor volume (length × width × height) were conducted every 2 days. After 3 weeks, mice were sacrificed for tumor analysis. All procedures were in compliance with our institution's guidelines for the use of laboratory animals and approved by the Penn State College of Medicine Institutional Animal Care and Use Committee.

Microarray statistical analysis was performed as describe [37]. Student t test was used comparing two groups. One-way ANOVA was used comparing multiple groups followed by Tukeys post-hoc test. All analysis with a p < 0.05 was considered significant.

In a previous report, we established a model of EMT using liver cancer cell lines derived from Pten ^-/- mice [37]. In this model, we transplanted epithelial liver cancer cells, and from the resulting tumors, harvested epithelial and mesenchymal cells. The epithelial tumor cells were identical to parent cells, labeled P2-Epithelial (P2E), and the mesenchymal, fibroblastoid cells, were labeled P2-Mesenchymal (P2M) (Figure 1A). Both epithelial and mesenchymal cells demonstrated Pten ^-/- genotype [37]. In support of the EMT-metastasis paradigm, mesenchymal cells demonstrated significant metastatic potential [37]. To confirm the persistence of epithelial and mesenchymal phenotypes, we analyzed the expression of key EMT genes and migratory/invasion in vitro. The mesenchymal cells demonstrate loss of E-cadherin, gain of E-box transcription repressors Snail1 and Zeb2, significant migration in wound assay, and increased invasion through Matrigel pores compared to epithelial cells (Figure 1B-E).

In mesenchymal cells, transcriptome profiling demonstrated increased expression of multiple liver TISC markers (Figure 2A). Real-time PCR validated up-regulated Nanog, Oct-4, CD44, and EpCam (Figure 2B). Although CD133 is a strong TISC marker in previous reports, the mesenchymal cells have no detectable CD133 expression, making comparative analysis impossible. In terms of self-renewal assay, the mesenchymal cells were able to form large tumor-spheres in low adherent plates (Figure 2C). Increased stem cell markers and tumor-sphere formation indicates that the mesenchymal cells have a TISC phenotype.

To test the hypothesis that mesenchymal cells are resistant to chemotherapy, a TISC feature, cells were treated with doxorubicin and 5'Fluorouracil. The mesenchymal cells demonstrate increased sensitivity to genotoxic agents compared to epithelial cells (Figure 3A-B). In terms of cell cycle progression, the mesenchymal cells are highly proliferative compared to the epithelial cells (Figure 3E). Thus, we conclude that resistance to chemotherapy is linked to the level of cell proliferation, not mesenchymal status, consistent with the mechanism of action of cytotoxic agents. In addition to rate of proliferation, Abcg2 expression correlated with chemotherapy resistance (Figure 3A & 3B, 2B), indicating that drug resistance may be dependent on the ATP-binding cassette expression as a mechanism of drug efflux. ATP-binding cassette efflux has been highly correlated to epithelial phenotype liver TISCs [14, 42].

In addition to resistance to genotoxic agents, we assessed whether the mesenchymal cells are resistant to TRAIL-induced and TGFβ-induced apoptosis. Although there was no significant difference in response to TRAIL stimulation (Figure 3C), the mesenchymal cells demonstrate resistance to TGFβ-induced apoptosis (Figure 3D), a characteristic of TISCs [40].

During later stages of disease, TGFβ induces EMT and contributes to disease progression [15, 43]. After TGFβ stimulation, epithelial cells undergo a morphological change from cuboidal to fibroblastic-like cells (Figure 4A). In addition to morphology change, TGFβ treatment resulted in increased cell migration and the formation of larger spheroids in low adherent plates (Figure 4B & 4C. This TGFβ-induced change was associated with typical EMT characteristics, including decreased E-cadherin and increased Snail1 and Nanog (Figure 4D & 4E).

In HCC, a TISC phenotype with Snail1 over-expression is associated with poor prognosis [21]. To test the specific role of Snail1 in up-regulating TISC characteristics, we utilized siRNA to knock down Snail1 in mesenchymal cells. After Snail1 siRNA treatment, TISC markers Nanog and CD44 decreased significantly (Figure 5A), which was associated with decreased spheroid formation (Figure 5B) and decreased migration (Figure 5C).

The primary mechanism of TGFβ-induced EMT is through Smad-dependent signaling. Following activation of TGFβ receptors, Smad2 and Smad3 are phosphorylated and form the Smad2/3/4 heterocomplex, which translocates to the nucleus to regulate Snail1 transcription [19, 27, 44]. After TGFβ stimulation in epithelial cells, Snail1 increased (Figure 4D). In order to confirm that TGFβ induces Snail1 through Smad-dependent pathways in our model, we utilized inhibitory Smads, Smad7 and dominant-negative Smad3 (ΔSmad3), which block heterocomplex formation. Epithelial cells were transfected with Smad7 or ΔSmad3 vectors 24 hours prior to TGFβ stimulation. qPCR and western blot analysis demonstrated that inhibitory Smads significantly attenuated TGFβ-induced Snail1 up-regulation (Figure 6A & 6B).

TGFβ regulates Nanog promoter activity through Smad signaling in human embryonic stem cells [31]. To confirm that TGFβ can induce Nanog promoter activity in our model, epithelial cells were co-transfected with Nanog-Luc and Smad7 or ΔSmad3 vectors. Following TGFβ stimulation, Nanog-Luc activity was significantly attenuated by inhibitory Smads (Figure 6C & 6D), indicating that TGFβ stimulates Nanog promoter activity through Smad-dependent signaling.

After transient knock-down of Snail1, Nanog expression is decreased, indicating that Snail1 directly regulates TISC genes in mesenchymal cells (Figure 3B). To further investigate this Snail1-driven TISC expression profile, we established stable Snail1 knock-down in mesenchymal-Snail1-shRNA cells (Figure 7A). In these mesenchymal-Snail1-shRNA cells, down regulation of Snail1 corresponded to decreased Nanog promoter activity and decreased Nanog and CD44 expression (Figure 7A & 7B).

As demonstrated, Snail1 is a key regulator of TISC characteristics in vitro. To investigate the role of Snail1 in tumor initiation, we inoculated 1 × 10⁴ mesenchymal-Snail1-shRNA cells into nude mice. The mesenchymal-Snail1-shRNA cells demonstrate reduced in tumor growth compared to control mesenchymal cells. Analysis of tumors demonstrates that Snail1 expression was down-regulated in 1 × 10⁴ cell initiated tumors from mesenchymal-Snail1-siR cells (Figure 7C). However, tumor initiation was not affected by Snail1 suppression, as evidence by all inoculations forming tumors, even in Snail1 inhibited cells.

In order to investigate SNAIL1 and NANOG expression in human HCC cells, we utilized Huh7 and MHCC97-L cells. Huh7 cells have been described to be epithelial whereas MHCC97-L cells are mesenchymal with metastatic potential [38, 39]. Accordingly, MHCC97-L cells demonstrate significant migration and invasion, increased expression of SNAIL1, NANOG and decreased expression of E-Cadherin (Figure 8B-D). Mesenchymal MHCC97-L cells also demonstrate TISC characteristics including increased NANOG, BMI-1, CD44 and OCT4 mRNA expression as well as increased tumorsphere formation (Figure 8E & 8F).