Targeting Transforming Growth Factor-Beta1 (TGF-β1) Inhibits Tumorigenesis of Anaplastic Thyroid Carcinoma Cells Through ERK1/2-NFκkB-PUMA Signaling.

BACKGROUND The transforming growth factor-beta (TGF-β) signaling pathway plays a critical role in promoting tumor growth. TGF-β1was found to be overexpressed in anaplastic thyroid cancer (ATC). We therefore tested our hypothesis that targeting TGF-β1 inhibits tumorigenesis of ATC cells. MATERIAL AND METHODS Effects of TGF-β1 stimulation or TGF-β1 inhibition by small interfering RNA (TGF-β1siRNA) on proliferation, colony formation, and apoptosis in 8505C cells in vitro was detected using siRNAs and inhibitors to examine the TGF-β1 signaling pathway. A subcutaneously implanted tumor model of 8505C cells in nude mice was used to assess the effects of TGF-β1 inhibition on tumorigenesis development. RESULTS TGF-β1siRNAs decreased proliferation and colony formation, and increased apoptosis in 8505C cells in vitro and inhibited tumor growth in vivo. TGF-β1siRNA inhibited phosphorylation ERK1/2 (pERK1/2) and increased p65-dependant PUMA mRNA and protein expression. Knockdown of p65 or PUMA by siRNA reduced TGF-β1siRNA-induced apoptosis, as well as caspase-3 and PARP activation. Upregulation of p65 or PUMA expression by TGF-β1siRNA requires pERK1/2 inhibition. TGF-β1 shRNA inhibited tumor growth in vivo. CONCLUSIONS Therapies targeting the TGF-β1 pathway may be more effective to prevent primary tumor formation. The ability of this therapy to decrease tumorigenesis may be related to ERK1/2/NF-κB/PUMA signaling.

Background

Anaplastic thyroid cancer (ATC) is responsible for the majority of deaths from all thyroid malignancies and has a median survival of 6 months. The resistance of ATC to conventional thyroid cancer therapies, including surgery, radioiodine, and thyroid-stimulating hormone [TSH] suppression, contributes to the very poor prognosis of this malignancy [1,2]. Currently, there is no standard or effective therapy for ATC, and patient survival has not improved in over 6 decades [3]. Therefore, investigation of novel antiproliferative and gene therapies has been an ongoing interest.

TGF-β is part of a large family of structurally related cytokines that include bone morphogenetic proteins, growth and differentiation factors, activins, and inhibins. There are 3 isoforms of TGF-β ligand (TGF-β1–3), and as ubiquitous cytokines, they play an important role in numerous cellular processes, including proliferation, adhesion, motility, apoptosis, differentiation, and immune regulation [4]. Many tumor suppressors functions by TGF-β1 signals [5-7], and TGF-β1 functions by various mechanisms, such as ERK1/2 [8,9], NF-κB [10,11], PUMA [12], and p21WAF1[13].

TGF-β1 is highly expressed in various cancers such as prostate cancer [14], ovarian carcinoma [15], hepatocellular carcinoma [16,17], bladder carcinoma [18], breast cancer [19], and cholangiocellular carcinoma [20], and aberrant TGF-β1 expression is associated with more aggressive tumors and poor prognosis. In thyroid cancer, high expression of TGFβ1 was found to be closely related with the occurrence of thyroid cancers [21].

In this study, we used TGFβ1 gene-silenced ATC cells to determine if TGFβ1 silencing can inhibit the proliferation and induce apoptosis of ATC cells in vitro and in vivo. The results of these studies indicate that small interfering RNA (siRNA) -mediated silencing of TGFβ1 in the ATC cells decreased the proliferation and induces apoptosis in vitro, and decreased the growth in an orthotopic model. TGF-β/ERK1/2/NF-κB/PUMA are involved in the mechanisms by which TGFβ1 regulates the growth and apoptosis of ATC cells, providing a novel therapeutic target for the pathogenesis and gene therapy of ATC.

Material and Methods

Cell line and culture

The human anaplastic thyroid cancer cell line 8505C cells were purchased from DSMZ (Beijing, China) and were cultured according to the supplier’s directions. Briefly, the cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, sodium pyruvate, and non-essential amino acids. Adherent monolayer cultures were maintained on plastic and incubated at 37°C in 5% carbon dioxide and 95% air. The cultures were free of Mycoplasma species. In all of the assays, a monolayer of cells that was 50–70% confluent was used. All the methods used were according to the manufacturer’s instructions.

Agents

The following primary antibodies were used from Santa Cruz Biotechnology: pERK (T202/Y204), ERK1/2, TGF-β1–3, TGF-β1 siRNA, SMAD2 siRNA, siRNA, control siRNA, and β-actin. The following primary antibodies were used from Cell Signaling: NF-κBp65 (p65), PUMA, bcl-2, Mcl-1, Bcl-XL, PARP, Bax, Bak, Bid, Bim, and Caspase-3. NF-κBp65 siRNAs and hrTGF-β1 were from Applied Biosystems. MEK/ERK inhibitor U0126 was purchased from Cell Signaling Technology. Anti-Ki67 Ab was from Thermo Scientific. Inhibitors were tested for monotherapy and combination therapy: U0126:10 mM. Cells incubated with culture medium or culture medium with DMSO served as controls.

Plasmids

Short-chain oligonucleotide was designed according to the TGF-β1 mRNA sequence provided by Genebank. The 2 oligonucleotides were selected as:

forward, 5′-GATCCCCTGCCGCTGCTGCTACCttcaagagaGGTAGCA GCGGCAGCATTTTTGGAAA-3′;

reverse, 3′-AGCTTTTCCAAAAATGCTGCTGCCGCTGCTGCTACCtctctt gaaGGTAGCAGCGGCAGCAGGG-5′.

It was chemosynthesized by Shgong.com. It was ligated and then we inserted the 2 oligonucleotides above into the pcDNA3.1 plasmid (which encodes GFP as a reporter protein). The recombinant TGF-β1 shRNA expression vector was evaluated by using enzyme cutting. The negative control plasmid had a sequence inserted at the same place using the following 2 oligonucleotides:

5′-GCTACGCCTTCATAAGGCACGTGCTTCAAACGGGCATGCGCC ATAT GCAGTCTTTTTTGTCGACA-3′;

reverse, 3′-GGCTAAGATTTCCGCGGACGAAGCCTTG CCGTACCCC GAGCACTTCACGAAAAACAGCTGCGAGA-5′.

The recombinant TGF-β1-shRNA plasmid was confirmed by digestion and gene sequencing. Plasmid pcDNA3.1 was as the control plasmid.

Transient/stable siRNA transfection

Cells were seeded in 6-well plates, grown to 50–80% confluence. The cell density is not too high and that the cells are in optimal physiological condition on the day of transfection. 8505C cells were transfected with TGF-β1siRNA (2 μg) in OptiMEM (Gibco, BRL) for 24–72 h using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. To determine the signaling pathways involved in the production of NF-κBp65 (p65) and PUMA, 8505C cells were transfected with p65 siRNA or PUMA siRNA or their control siRNA for 24 h, then transfected with TGF-β1siRNA or its control siRNA for 48 h using Lipofectamine 2000 according to the manufacturer’s instructions above. For stable TGF-β1siRNA transfection, 24 h after TGF-β1siRNA or control siRNA transfection, the cells were split into 96-well plates and subjected to the G418 (1 mg/ml) selection for 2 weeks. The transcriptional silencing TGF-β1 protein and mRNA were screened using reverse transcription-PCR (RT-PCR) and Western blot, as described below. Different agents used in the transfection assay had no effect on target protein expression. All transfection experiments were done at least 3 times.

Western blot assay

For total protein extraction, cells were washed once with phosphate-buffered saline (PBS) and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, supplemented with complete protease inhibitor tablets (Roche Diagnostics) for 30 min on ice. For in vivo study, the tumor tissues were homogenized for tissue lysate extraction. Both cell lysate and tissue lysate were centrifuged and the supernatants were collected. Nuclear and cytoplasmic extracts were prepared using the Nuclear Extract Kit (Active Motif), according to manufacturer’s instructions. Protein concentration was quantified with Coomassie Plus (Bradford) Protein Assay Reagent according to the manufacturer’s instructions. Extracts (40 μg) were resolved on 10% SDS-PAGE and transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare; Germany). Membranes were probed with primary antibodies against TGF-β1, TGF-β2, TGF-β3, ERK1/2, Perk1/2, p65, PUMA, caspase-3, cleaved caspase-3, PARP, cleaved PARP, Noxa, Bak, Bid, Bim, bcl-2, Mcl-1, and Bcl-XL followed by incubation for 1 h at room temperature with HRP-conjugated anti-rabbit IgG or anti-goat IgG, respectively. Immunoblotting for β-actin served as protein loading control. All experiments were performed at least 3 independent times.

Reverse transcriptase polymerase chain reaction (RT-PCR)

RNA from transfection or inhibitors treated cells was extracted using Purification Kit (Roboklon, Germany). We used 2 μg of total RNA for cDNA synthesis with the Reverse Transcriptase Kit (Promega, Shanghai, China). The resulting cDNA was used for RT-PCR analysis with the G-Taq Kit according to the manufacturer’s instructions. RT-PCR was performed using appropriate gene-specific forward and reverse primers, which were selected using the Blast Primer tool. The results were analyzed using the ImageJ software. The relative change in the ratio of the target protein to the DMSO control was determined.

Cell viability assay

8505C cells transfected with TGFβ1 siRNA or control siRNA were seeded in 96-well plates at a density of 5000 cells/well in 100 mL of medium and incubated for 24 h. To determine the effect of p65 and PUMA, 8505C cells were transfected with p65 siRNA or PUMA siRNA or control siRNA for 24 h before the TGFβ1 siRNA or control siRNA transfection, then the cells were also seeded in 96-well plates at a density of 5000 cells/well in 100 mL of medium and incubated for 24 h. Cells incubated with culture medium with or without DMSO served as controls. Determination of viable cells was performed by adding 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich) to a final concentration of 0.5 mg/ml. After 3-h incubation at 37°C and 5% CO2 in a humidified incubator, the formazan crystals were resolved by 10% SDS in 10 mM HCl and the absorption was measured at 560 nm. MTT measurements were performed daily for 5 consecutive days, sometimes at 72 h, as triplicates in 3 independent experiments. The absorption data were used to calculate the doubling time of each cell line.

Apoptosis assay

8505C cells were transfected with TGFβ1 siRNA or control siRNA for 72 h. 8505C cells were transfected with p65 siRNA or PUMA siRNA or control siRNA for 24 h, then transfected with TGFβ1 siRNA or control siRNA for 72 h. After 72 h, transduced cells were collected and fixed overnight at 4°C with 75% ethanol for propidium iodide staining and flow cytometry analysis on a FACSCalibur (Becton Dickinson Immunocytometry Systems) to evaluate sub-G1 cell populations (apoptotic rate).

Soft-agar colony formation assay

8505C, stable 8505C/control siRNA, and stable 8505C/TGFβ1 siRNA cells were resuspended in RPMI-1640, supplemented with 10% FCS and 0.33% (w/v) agarose (Life Sciences Corporation). One ×103 cells/ml were seeded into soft agar and colony growth was analyzed after 7 days by light microscopy. Only colonies with more than 3 cells were counted. Independent colony formation experiments were repeated twice, each in triplicate.

In vivo studies

Animal studies were approved by Animal Care and Use Committee and conducted in accordance with NIH guidelines. 8505C or TGFβ1 shRNA/8505C or control shRNA/8505C cells suspended in Matrigel (5×106 cells/200 mL) were inoculated subcutaneously into the right flank of 4- to 6-week-old female athymic nude (nu/nu) mice. The mice were sacrificed, and tumors were dissected after 7 weeks. The primary tumors were divided into 3 portions for cell lysate production, and for making paraffin blocks for Ki-67 immunohistochemistry and TUNEL staining.

Proliferative index

Paraffin sections (4-μm thick) were routinely stained with hematoxylin and eosin. To assess tumor cell proliferation, immunohistochemical staining for Ki67 antigen (DakoCytomation, Hangzhou, China) was performed. Briefly, after deparaffinization and rehydration, the sections were immersed in 0.3% hydrogen peroxide to quench intrinsic peroxidase activity. The diluted antibodies were then added to the sections and incubated at 37°C for 1 h. The labeled antigen was visualized with the Histofine kit (Bosde, Wuhan, China), followed by reaction with 3,3′-diaminobenzidine. Finally, the sections were counterstained with hematoxylin. Ki67-positive cells were determined by counting about 500 nuclei in randomly selected microscopic fields, and the Ki67 labeling index was expressed as the ratio of Ki67-positive cells to total cells.

TUNEL assay

The TUNEL staining was performed with the In Situ Cell Death Detection kit according to the manufacturer’s instructions. In brief, DNA strand breaks were labeled with fluorescein dUTP and TdT in the dark at 37°C for 1 h. Subsequently, the sections were counterstained with Hoechst 33258 dye. TUNEL-positive (apoptotic) cells were detected as localized bright green cells by using scanning laser confocal microscopy (Leica, Beijing, China). Data were expressed as the ratio of apoptotic cells to total cells.

Statistical analysis

Data are expressed as the mean ±SD. SPSS 11.0 software was used for analysis. The statistical significance of a difference between 2 groups was assessed using the t test (2-tailed). ANOVA was used when more than 2 groups were involved, and then the t test was further applied to analyze difference between groups. P<0.05 was considered to indicate a statistically significant result. All experiments were repeated at least 3 times.

Results

Effect of siRNA on TGF-β1 expression in 8505C cells

8505C cells were transfected with TGF-β1 siRNA and control siRNA for 24–72 h. Western blot and RT-PCR analysis was used to detect the TGF-β1 protein and mRNA level after transfection. Cells transfected with TGF-β1 siRNA displayed a time-dependent reduction in the expression levels of TGF-β1 protein (Figure 1A) and mRNA (Figure 1B). Control siRNA did not exhibit any effect on protein levels of TGF-β1 (Figure 1A) and mRNA levels of TGF-β1 (Figure 1B). These data confirmed the suppression effect of siRNA and established the efficiency of siRNA transfection. We also detected TGF-β2 and TGF-β3 protein and mRNA expression before and after siRNA transfection. Results from our in vitro experiments showed no change in expression for the 2 isoforms, TGF-β2 and TGF-β3, illustrating the specificity of the siRNA sequence designed for this study (Figure 1C, 1D).

Figure 1

TGF-β1 gene knockdown by siRNA transfection in 8505C cells. 8505C cells were transfected with TGF-β1 siRNA and control siRNA for 24–72 h. (A) Representative images showing expression of TGF-β1 protein in control siRNA and TGF-β1siRNA transfected cells as analyzed by Western blot. (B) Representative images showing expression of TGF-β1 mRNA in control siRNA and TGF-β1siRNA-transfected cells as analyzed by RT-PCR. (C) Representative images showing expression of TGF-β2, TGF-β3 protein in control siRNA, and TGF-β1siRNA-transfected cells (72-h transfection) as analyzed by Western blot. (D) Representative images showing expression of TGF-β2 and TGF-β3 mRNA in control siRNA and TGF-β1siRNA-transfected cells (72-h transfection) as analyzed by RT-PCR. β-actin and GAPDH were controls.

Effect of TGF-β1 gene silencing on colony formation, cell survival, and apoptosis

8505C cells were transfected with the siRNA plasmid and selected for stable clones demonstrating inhibition of TGF-β1 expression. Silencing of TGF-β1 transcripts in positive clones was confirmed by RT-PCR, and TGF-β1 protein expression in positive clones was confirmed by Western blot assay. As shown in Figure 2A–2B, TGF-β1 mRNA and protein levels were decreased in both the 8505C/TGF-β1siRNA1 clone and the 8505C/TGF-β1siRNA2 clone when compared with control siRNA transfected cells. To investigate the effect of TGF-β1 gene silencing on the growth of 8505C cells, we performed a soft-agar colony-formation assay. As shown in Figure 2C, TGF-β1siRNA1/2-transfected cells formed a significantly reduced (P<0.01) number of colonies compared with untreated or control siRNA transfected cells.

Figure 2

Effect of TGF-β1 gene targeting on colony formation, cell survival, and apoptosis. (A) Silencing of TGF-β1 transcripts in positive clones was confirmed by RT-PCR. (B) Silencing of TGF-β1 protein in positive clones was confirmed by Western blot assay. (C) Photomicrographs showing soft-agar colony formation and histogram showing number of colonies formed by 8505C cells transfected with siRNA. (D) 8505C cells were transfected with the siRNA plasmid for 120 h, cell survival rate was detected by MTT assay, 8505C cells were transfected with the siRNA plasmid for 120 h, and cell apoptotic rate was detected by flow cytometry assay. (E) 8505C cells were transfected with the siRNA plasmid for 120 h, and cell apoptotic rate was detected by flow cytometry assay. Vs. control, * p<0.05; ** p<0.01.

Next, the effects of TGF-β1 silencing on both cell survival and apoptosis were determined in vitro. Analysis of cell survival using the MTT assay showed a significant decrease in cell survival for TGF-β1siRNA1 and TGF-β1siRNA2 transfected cells when compared with the control siRNA transfected cells, and at 72 h the survival rate reached the lowest value (Figure 2D).

Cell apoptosis was determined using propidium iodine staining followed by flow cytometry analysis. Cells transfected with TGF-β1/siRNA1 and TGF-β1/siRNA2 for 72 h showed significant apoptosis, and the non-transfected cells showed less apoptosis, suggesting TGF-β1 targeting induced cell apoptosis (Figure 2E).

Upregulation of PUMA by TGF-β1 silencing correlates with apoptosis induction in 8505C cells

To study the mechanism of TGF-β1 silencing to induce apoptosis in ATC cells, protein expression of PUMA, caspase-3, PARP, other proapoptotic Bcl-2 family members, and antiapoptotic proteins were detected by Western blot assay. As shown in Figure 3A, TGF-β1siRNA transfection for 48 h in the 8505C cells markedly induced PUMA protein expression in a time-dependent manner. The peaks of PUMA protein induction were detected at 36 h following TGF-β1 siRNA1 transfection. Cleavage of PARP, a 116-kDa protein involved in DNA repair, is a characteristic marker in the detection of apoptotic cells. As shown in Figure 3B, in TGF-β1/siRNA1-transfected cells, full-length PARP was efficiently cleaved, and the characteristic apoptotic 89-kDa cleavage product was detectable 36 h after TGF-β1/siRNA transfection. In addition, proteolytic activation of pro-caspase-3 was detectable by Western blotting with an anti-caspase-3 antibody 36 h after TGF-β1/siRNA1 transfection (Figure 3C). In contrast, TGF-β1/siRNA transfection did not upregulate proapoptotic protein Noxa, Bak, Bid, or Bim, but reduced the expression of the antiapoptotic proteins bcl-2, Mcl-1, and Bcl-XL (Figure 3A).

Figure 3

Upregulation of NF-κB-dependent PUMA by TGF-β1 silencing correlates with apoptosis induction. (A) 8505C cells was transfected with TGF-β1/siRNA or control siRNA for 0–48 h. NF-κBp65, PUMA, or other bcl-2 family member proteins were detected by Western blot assay. (B) Cell lysates (40 ug of protein) were analyzed by Western blotting using a monoclonal anti-PARP antibody at 36 h. Concomitant with the induction of apoptosis, PARP was fragmented, resulting in the characteristic 89-kDa cleavage product. (C) Caspase-3 is activated in TGF-β1/siRNA 8505C cells at 36 h, as shown by the conversion of pro-caspase-3 to activated cleaved caspase-3. (D) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with PUMA siRNA1 and PUMA siRNA2 for 48 h. PUMA mRNA induction by TGF-β1/siRNA was analyzed by RT-PCR. vs. TGF-β1/siRNA, * p<0.01. (E) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with PUMA siRNA or P65 siRNA for 48 h. Cell apoptosis was detected flow cytometry assay. vs. TGF-β1/siRNA, * p<0.01. (F) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with P65 siRNA and PUMA siRNA for 48 h. Cell viability was detected by MTT assay. vs. TGF-β1/siRNA, * p<0.01. (G) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with P65 siRNA1 and P65 siRNA2 for 48 h. P65 and PUMA mRNA induction by TGF-β1/siRNA was analyzed by RT-PCR.

To determine if TGF-β silencing induced cell apoptosis and inhibited proliferation by inducing PUMA upregulation, 8505C-TGF-β1/siRNA1 cells were transfected with siRNAs (siRNA 1 and siRNA 2) targeting PUMA using Lipofectamine 2000. Figure 3D shows that the silencing efficiency of the PUMA protein induced by PUMA siRNA1 and PUMA siRNA2 reached approximately 90%, (p<0.01). 8505C-TGF-β1/siRNA2 cells has the same results as 8505C-TGF-β1/siRNA1 (data not shown).

By flow cytometry analysis, cells transfected with PUMA/siRNA1 and PUMA/siRNA2 showed 5.2–4.6% apoptosis, which was significantly lower compared with TGF-β1/siRNA transfection groups (P<0.01) (Figure 3E). Analysis of cell survival using the MTT assay showed 80–75% cell survival in the PUMA/siRNA1- and PUMA/siRNA2-transfected cells when compared with the TGF-β1/siRNA-transfected cells (P<0.01) (Figure 3F).

TGF-β1 silencing induced NF-κB-dependent PUMA upregulation

We found that the TGF-β1 silencing inhibited proliferation and induced apoptosis, which is correlated to PUMA upregulation. In a previous study, the p65 subunit of NF-κB was recently identified as a transcriptional activator of PUMA in response to TNF-α [22], sorafenib [23], and regorafenib [24]. In the present study we transfected the p65siRNA into the 8505C-TGF-β1/siRNA cells. The results showed that in the presence of p65siRNA, PUMA protein was obviously inhibited in the 8505C-TGF-β1/siRNA cells (Figure 3G), suggesting that p65 by siRNA abrogated PUMA induction by TGF-β1 silencing. Cell apoptosis was also significantly decreased (Figure 3E) and cell survival rate was significantly increased (Figure 3F), which was similar to the PUMA siRNA transfection (Figure 3E, 3F).

TGF-β1-induced NF-κB-PUMA regulation is MEK/ERK-dependent, but SMAD2-independent

TGF-β1 silencing resulted in NF-κB-dependent PUMA upregulation, but the mechanisms remain largely unknown. Recent studies show that targeting Ras/Raf/MEK/ERK signaling induced PUMA upregulation, which is mediated by subsequent NF-κB p65 activation [23,24]. Therefore, we speculated that TGF-β1 silencing could induce inactivation of MEK/ERK signaling, which could promote the subsequent cell apoptosis of ATC cells. To test this, Western blotting analysis was performed, and the results showed that the phosphorylation of ERK1/2(pERK1/2) was inhibited in the 8505C-TGF-β1/siRNA1 cells compared to the control siRNA or non-transfected cells (Figure 4A). 8505C-TGF-b1/siRNA2 cells had the same results as 8505C-TGF-β1/siRNA1 cells (data not shown). TGF-β1 silencing did not have any effect on total ERK1/2 (data not shown).

Figure 4

Knockdown of TGF-β1 activates p65 to induce PUMA through ERK inhibition. (A) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 48 h. Expression of pERK1/2 and PSMAD2 was analyzed by Western blotting. (B) 8505C cells were treated with rhTGF-β1 (1,5 and 10 ng/ml) for 24 h, pSMAD2, pERK1/2, NF-κBp65, and PUMA protein was detected by Western blot assay. (C) 8505C cells were transfected with either a control siRNA or a SMAD siRNA for 24 h, and then treated with 10 ng/ml rhTGF-β1 for 24 h. pSMAD2, pERK1/2, NF-κBp65, and PUMA proteins were detected by Western blot assay.

TGF-β1 silencing can inhibit pERK1/2 level and induce NF-κB-dependent PUMA upregulation, so we next tested whether TGF-β1 could elevate the pERK1/2 level and inhibit the NF-κB-dependent PUMA upregulation. 8505C cells were treated with human recombinant 1,5 and 10 ng/ml TGF-β1(rhTGF-β1) for 24 h, pERK1/2, p65, and PUMA protein was detected by Western blot assay. The results showed that TGF-β1 strongly promoted phosphorylation of ERK1/2. In addition, p65 and PUMA expression was decreased in the 8505C/TGF-β1 cells compared to the control cells (Figure 4B). However, when 8505C cells were treated with U0126 (10 uM) 6 h before TGF-β1 (rhTGF-β1) treatment, p65 and PUMA expression was restored (data not shown), suggesting p65 and PUMA was ERK1/2-dependent.

TGF-β1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, SMAD molecules. The TGF-β1-SMAD signaling pathway is involved in regulation of cell growth, differentiation, migration, apoptosis, and extracellular matrix remodeling. We next studied whether TGF-β1-SMAD signaling is involved in p65-PUMA regulation. To test this, Western blotting analysis was performed. 8505C cells were transfected with TGF-β1 siRNA and control siRNA for 48 h, the pSMAD2 was significantly decreased in the 8505C-TGF-β1/siRNA cells (Figure 4A). When 8505C cells were treated with TGF-β1 for 24 h, the pSMAD2 and ERK1/2 was increased, and p65 and PUMA expression was significantly decreased (Figure 4B). However, when 8505C cells were transfected with SMAD2 siRNA and control siRNA for 24 h before rhTGF-β1 (1,5 and 10 ng/ml) treatment, the pERK1/2, p65, and PUMA expression was not affected (Figure 4C), suggesting that ERK1/2, p65, and PUMA expression were not regulated by SMAD2.

TGF-β1 silencing inhibits tumor growth

TGF-β1 shRNA/8505C cells, control shRNA/8505C cells, and untreated 8505C cells were injected subcutaneously in nude mice and observed for a period of 7 weeks. Xenograft tumor growth and lung metastasis models were obtained as described in the Methods section. As shown in Figure 5A, TGF-β1 shRNA/8505C clones were much smaller compared with control shRNA/8505C cells and untreated 8505C cells groups (p<0.01). By Western blot assay, p65 and PUMA protein were exclusively detected in the TGF-β1 shRNA/8505C groups, and p-ERK1/2 was decreased to almost undetectable levels, compared to the control shRNA/8505C cells and untreated 8505C groups (Figure 5B).

Figure 5

Effects of TGF-β1 silencing on 8505C cell tumor growth in vivo. (A) 8505C ATC cells (6×106) were inoculated subcutaneously into the right flank of 4-week-old female athymic nude (nu/nu) mice. The tumor growth curves represent 8505C cells, TGF-β1 shRNA-transfected 8505C cells, and shRNA-transfected 8505C cells, as labeled. Point, mean tumor volume (calculated from 6 mice); bars, upper 95% confidence intervals. (B) Protein expression of p-ERK1/2, NF-κBp65, PUMA, (PI3K)/Akt, and TGF-β1-3 were detected in the tissues in vivo. (C) Positive Ki67 staining of the tumor tissues. (D) TUNEL staining of the tumor tissues. Data are expressed as the mean ±SEM (* P<0.05).

To determine the effects of TGF-β1silencing on the other 2 isoforms, Western blot analysis was done. As shown in Figure 5B, there was no significant difference in the expression levels of either TGF-β2 or TGF-β2 in the TGF-β1 shRNA/8505C groups when compared with controls.

Xenotransplants of TGF-β1 shRNA/8505C clones revealed fewer cells positive for the proliferation marker Ki67 in the periphery of the tumors than the control shRNA/8505C cells and untreated 8505C cells, which has high positive Ki67 expression (Figure 5C). TUNEL analysis provided in vivo evidence that TGF-β1silencing significantly increased cell apoptosis compared to the non-treated or shRNA/8505C groups (p<0.05) (Figure 5D).

Discussion

In this study we first investigate the effect of reduction of TGFβ1 induced by TGFβ1 siRNA on the proliferation, colony formation, and apoptosis in vitro. We used 2 independent TGFβ1-knockdown clones (8505C/TGFβ1siRNA1 and 8505C/TGFβ1siRNA2) to perform MTT, colony formation, and apoptotic assay. Our results demonstrate that TGFβ1 silencing inhibits growth and colony formation and induces apoptosis of 8505C cells in vitro. In our study, the stable knockdown of TGFβ1 in the ATC cell lines 8505C, by plasmid based shRNA expression, resulted in reduced tumor growth in vivo.

Although the 3 mammalian isoforms of TGFβ1, TGFβ2, and TGFβ3 share 60–80% identity at the amino acid level, the promoter regions of these isoforms are highly variable, suggesting that their expression is regulated by distinct mechanisms [25]. Results from our in vitro and in vivo experiments showed no change in expression for TGFβ2 and TGFβ3 isoforms by TGF-β1sliencing, illustrating the specificity of the siRNA/shRNA sequence designed for this study.

In this study, knockdown of TGFβ1/TGF-βR signaling affected cellular growth and apoptosis, but the central signaling pathway, acting downstream of TGF-β and leading to cell death, is not clear.

Nuclear factor-κB (NF-κB) is a nuclear transcription factor regulating the expression of various genes involved in cell proliferation, tumorigenesis, and inflammation [26]. PUMA (p53 upregulated modulator of apoptosis), a BH3-only Bcl-2 family member, functions as a critical initiator of apoptosis in cancer cells [27]. Our results demonstrate that TGF-β stimulation inhibited NF-κB p65 and PUMA expression, and knockdown of TGF-β activated NF-κB p65 and PUMA. TGF-β silencing-induced apoptosis and growth inhibition in ATC cells was inhibited by NF-κB p65 siRNA transfection. PUMA is necessary for TGF-β1 silencing and induces apoptosis as shown by PARP cleavage and pro-caspase-3 cleavage resulting in caspase-3 activation. To further characterize TGF-β silencing involvement in apoptosis, the expression of apoptosis-related proteins, such as bcl-2, Mcl-1, Bcl-XL, Noxa, Bak, Bid and Bim, was analyzed. Proapoptotic proteins Noxa, Bak, Bid, and Bim were not upregulated, but reduced the expression of the antiapoptotic proteins bcl-2, Mcl-1, and Bcl-XL.

A recent study has shown that PUMA is a direct target of NF-βB and mediates TNF-a-induced apoptosis in vitro and in vivo [22]. Our study found that knockdown of NF-βBp65 reduced TGF-β1siRNA-induced apoptosis and PUMA upregulation. We therefore suggested that knockdown of TGFβ1/TGF-βR signaling induced apoptosis by NF-κBp65-dependant PUMA upregulation. However, the mechanism underlying the relation between TGFβ1/TGF-βR and NF-κBp65-/PUMA is unclear. The targeting of Ras/Raf/MEK/ERK signaling promoted PUMA-dependent apoptosis of tumor cells [23,24], suggesting PUMA was negatively regulated by ERK signaling. In our study, we found that TGF-β1 stimulating activated ERK1/2 and inhibited NF-κBp65/PUMA signaling, and vice versa. However, treatment with ERK inhibitor U0126 inhibited TGFβ1-induced ERK1/2 activity and NF-κBp65/PUMA downregulation. Although SMAD2 activation was shown after TGF-β1 stimulation, knockdown of SMAD2 did not affect the ERK1/2 activity level. We therefore concluded that growth inhibition of ATC cells by TGFβ1 silencing is dependent on the cell apoptosis induction, progressing from ERK inhibition and p65 nuclear translocation, leading to PUMA induction and onset of mitochondria-mediated apoptosis.

Moore et al. [28] has reported that TGFβ1 silencing resulted in a 50% increase in proliferation of breast cancer MDA-MB-435 cells, and has no effect on cell apoptosis in vitro compared to the controls, which was contrary to our study. In Moore’s results, AKT and ERK signaling pathways were activated, suggesting a possible role of these signaling pathways in promoting growth signaling. In Moore’s study, there was no significant difference in the growth kinetics of the primary tumors between the TGFβ1siRNA and control siRNA groups in vivo. However, in our study, significant growth inhibition was found in TGFβ1siRNA groups compared to the control siRNA groups in vivo. In our study, ERK1/2 signal was decreased after the TGFβ1 silencing. ERK inhibition, leading to PUMA and apoptosis induction, may be the main causes of these contrary results.

Conclusions

Results of the present study suggests that therapies targeting TGF-β1 in tumor cells may be effective in decreasing tumorigenesis, which may be related to ERK1/2/NF-κB/PUMA signaling. Future studies on TGF-β signaling in various stages of tumor progression and metastasis may lead to the further development of more tumor-targeted therapies to decrease the incidence of tumorigenesis.