Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer.


INTRODUCTION
Gastric cancer (GC) is one of the most frequent tumors worldwide and the third leading cause of tumor death according to GLOBOCAN 2012 [1]. Although appropriate surgical resection and adjuvant treatments such as radiotherapy and chemotherapy are used to treat GC, 5-year survival is less than 20% [2] due to local tumor invasion, systemic dissemination, metastases, and recurrence. Therefore, a better understanding of molecular mechanisms accounting for GC development and progression could contribute to prevention, diagnosis, and treatment of GC.
Recently, accumulated evidence indicates that TG2 is involved in tumor formation and progression by organizing the ECM, regulating cancer cell adhesion to the endothelium, as well as controlling migration and invasion of cancer cells and angiogenesis of tumor tissue [16]. Elevated TG2 expression has been observed in breast [17], pancreatic [18], colon [19], lung [20] and ovarian cancers [21], and it has been correlated with cell survival and high tumor invasiveness. For example, in non-small cell lung cancer, progression-free survival (PFS) of high-expressing TG2 patients was shorter than that of low-expressing TG2 patients [20]. In addition, TG2 promoted ovarian tumor metastasis by inducing a cancer stem cell phenotype and epithelial-to-mesenchymal transition (EMT) [22]. Even so, a precise role for TG2 in the development and progression of GC has not been well defined.
Here we measured TG2 expression in GC tissues and corresponding non-tumor mucosal tissues and explored the role and underlying mechanism of TG2 with respect to GC progression using in vitro and in vivo models. TG2 expression was frequently elevated in GC and associated with tumor depth of invasion and late TNM stage. In addition, TG2 promoted GC cell proliferation, migration, and invasion in vitro, as well as tumorigenesis and peritoneal metastasis in vivo through activation of the ERK1/2 pathway in GC cells. Thus, TG2 is a potential therapeutic target for treatment of metastatic GC.

TG2 expression is upregulated in GC cells and associated with clinicopathology
To elucidate the role of TG2 in human GC, we measured TG2 expression in human GC cell lines and GC tissues. TG2 mRNA in six GC cell lines, one normal gastric epithelial cell line (GES-1), and 50 pairs of human GC and matched adjacent non-tumor tissues was measured with qRT-PCR. As shown in Figure 1A, TG2 mRNA expression was significantly up-regulated in GC cell lines compared with normal GES-1 cells. Moreover, TG2 mRNA in GC tissues was higher than in non-tumor tissues ( Figure 1C and 1D) and this finding was confirmed with Western blot ( Figure 1B).
Upregulated TG2 expression was assayed in 127 GC and matched non-tumor tissue pairs using immunohistochemistry (IHC) as depicted in Methods. Of all GC tissues, 82.7% were positive for TG2 protein  Figure 1E; P < 0.05). Typical immunostaining of TG2 in normal and GC tissues was shown in Figure  1F and positive TG2 protein staining occurred in the cytoplasm of GC cells. Elevated TG2 protein expression in tumor tissues was significantly associated with depth of tumor invasion (P = 0.026) and late TNM stage (P = 0.011), but not with other parameters (see Table 1). Thus, TG2 expression is up-regulated in GC cells and is associated with tumor severity.

TG2 promotes GC cell proliferation
Given that TG2 is frequently overexpressed in GC, it may act as an oncogene. To study this, we measured cell growth in cells that variously expressed TG2. TG2 was highly expressed in GC cell lines compared with a normal gastric epithelial cell line GES-1. We also silenced TG2 in MKN45 and NCI-N87 cells using small interfering RNA. TG2-shRNA was transfected into MKN45 and NCI-N87 cells to knock down TG2 expression and Figure 2A showed that TG2 decreased in TG2-shRNA-transfected cells. With TG2 expression modification confirmed, we measured cell proliferation and noted that proliferation in both MKN45/TG2-shRNA and NCI-N87/TG2-shRNA cells was slower than in negative controls and mock groups ( Figure 2C and 2D). Additionally, a TG2expressing plasmid vector was transfected into SGC7901 and AGS cells, which expressed less TG2 ( Figure 2B). We noted that SGC7901/TG2 and AGS/TG2 cells grew faster than vector and mock groups ( Figure 2E and 2F). Thus, TG2 promotes GC cell proliferation.

TG2 enhances migration and invasion of GC cells
We assessed TG2 on GC cell migration and invasion, which are key determinants of malignant C. and D. The effect of TG2 knockdown on MKN45 and NCI-N87 cell proliferation was measured by CCK8 assay. E. and F. The effect of TG2 overexpression on SGC7901 and AGS cell proliferation was measured by CCK8 assay. Data are means ± SD of three independent experiments (*P < 0.05). progression and metastasis, using Transwell assays. As shown in Figure 3A

TG2 regulates cell proliferation, migration, and invasion via activation of the ERK1/2 pathway
The ERK1/2 signaling pathway has been reported to be involved in cell growth [16] and migration [23]. Thus, we measured TG2 and assessed whether it could modulate activation of the ERK1/2 signaling pathway in GC cells. Figure 4A and 4B showed that ERK1/2 phosphorylation in MKN45 and NCI-N87 cells, which overexpressed TG2, was greater than in SGC7901 and AGS cells, which expressed less TG2. In contrast, TG2 knockdown significantly inhibited phosphorylation of ERK1/2 in MKN45/TG2-shRNA and NCI-N87/TG2-shRNA cells ( Figure 4C and 4D) and TG2 overexpression promoted phosphorylation of ERK1/2 in SGC7901/TG2 and AGS/TG2 cells ( Figure 4E and 4F). Therefore, TG2 may be involved in the ERK1/2 pathway regulation as an upstream molecule. We next investigated whether TG2 could regulate cell proliferation, migration, and invasion via the ERK1/2 pathway by treating SGC7901/TG2 and AGS/TG2 cells with an ERK1/2 inhibitor (U0126) (20 μM). Western blot and cell proliferation assays indicated that ( Figure 4G) ERK1/2 was inhibited, TG2 expression did not change, and SGC7901/TG2 and AGS/TG2 cells growth decreased after treatment with U0126 compared with control or mock cells ( Figure 4H and 4I (Figure 4L and 4M). Therefore, TG2 regulates cell proliferation, migration, and invasion via activation of the ERK1/2 pathway, which contributes to the development and progression of human GC.

DISCUSSION
Recently, the significance of TG2 non-enzymatic regulation of its own activity has been established [14,15,24,25], and this regulation affects many physiological processes of cell behavior such as cell growth, adhesion, migration, programmed cell death, differentiation, and ECM remodeling [14]. These cellular processes are vital to wound healing, tissue repair, and tumor growth and metastasis [26]. However, aberrant expression of TG2 and its influence on gastric cancer are unclear. Thus, we confirmed that TG2 was a critical regulator of GC progression and that expression of TG2 was frequently upregulated in GC. In addition, TG2 expression was significantly associated with the depth of tumor invasion and late TNM stages.
Multiple studies reveal that increased TG2 expression in diverse cancers and aberrant expression of TG2 are correlated with poor survival, increased drug resistance, and high tumor invasiveness. Specifically, TG2 can enhance pancreatic cancer cell motility and metastatic potential by regulating the TG2/β1 integrin/ Src/uPAR pathway and EMT [27]. In breast cancer, abnormal TG2 expression can lead to glucose metabolism reprogramming, facilitating metabolic alterations of breast cancer cells, promoting drug resistance, and metastatic competence [28]. Down-regulated TG2 expression has been documented in primary tumors and upregulated expression has been confirmed in secondary metastatic tumors [29,30]. Here, we report that TG2 promoted GC cell proliferation, migration, and invasion in vitro and in vivo with gain-and loss-of-function approaches. Furthermore, TG2 may function as an oncogene in GC and may be associated with GC progression.
TG2 can activate several pathways that contribute to its tumor-promoting properties. Increased TG2 expression is often associated with constitutive activation of NF-kB, which protects against ROS-induced cell damage, inflammatory cytokines, and chemotherapeutic drugs [7]. TG2 can also promote degradation of the phosphatase PTEN, constitutively activating the FAK/AKT pathway for regulation of cell survival and chemotherapeutic drug resistance [31]. In addition, cell surface TG2 could interact with integrins, such as β1, β3 and β5, to promote activation of the FAK/c-Raf/MEK1/2-ERK1/2 pathway [11]. Previous studies indicate that activation of the ERK1/2 pathway is needed for proliferation, migration, and invasion of tumor and non-tumor cells [32][33][34][35]. When ERK1/2 pathway activation is inhibited, the effects of umbilical cord-derived mesenchymal stem cells on breast cancer cell proliferation and migration can be reversed [36].
Here, we report that TG2 also regulated cell proliferation, migration, and invasion in GC cells. Specifically, TG2 knockdown suppressed activation of the ERK1/2 pathway, tumor growth, and peritoneal and other metastases in vivo, whereas TG2 overexpression reversed these changes. In addition, inhibition of the ERK1/2 pathway by U0126, a specific ERK1/2 inhibitor, could partially reverse tumor-promoting effects on proliferation, migration, and invasion caused by high TG2 expression, suggesting an involvement of the ERK1/2 pathway in regulating TG2 function in GC cells. The ERK1/2 pathway is documented to mediate tumor metastasis via regulating matrix metalloproteinases (MMPs) expression and activity and this plays a critical role in ECM degradation [37][38][39], a pivotal step during tumor metastasis [40,41]. Thus, TG2, as an upstream regulator of ERK1/2, regulates GC cell proliferation, migration, and invasion by activating the ERK1/2 pathway. Our data provide a foundation for understanding the mechanism of TG2 overexpression in GC progression and suggest that TG2 may be a promising therapeutic target for treating GC.

Ethical statement
Written consent was acquired from all participants who were fully informed of the experimental procedures during the period of research. The study was approved by the Human Research Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University, School of Medicine. All experiments on animal were given permission by the Experimental Animal Ethics Committee of Ruijin Hospital and carried out according to the Guide for the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University.

Tissue specimens
GC tissues were obtained from 127 patients, none of who received radiotherapy or chemotherapy before surgery, between 2010 and 2013 at the Ruijin Hospital, Shanghai, China. All the patients included 90 men and 37 women, and the average age was 61.1 years old. The TNM-stage of patients was determined by the UICC TNM classification. All the tissue samples were identified by clinical pathologist and then were fixed by formaldehyde and embedded by paraffin to produce tissue chips.

Cell lines
Human GC cell lines, MKN45 and NCI-N87, were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in the RPMI-1640 medium containing 10% calf serum with 100 U/ml penicillin and 100 U/ml streptomycin and were placed in a humidified cell incubator with 5% CO2 at 37°C.

Western blot analysis
Cells were lysed with RIPA cell lysis buffer in the presence of protease inhibitor cocktail (Sigma, USA). The protein concentration of the cell lysates was quantified by a BCA Protein Assay Kit (Pierce, Rockford, USA). The same amount of protein samples were loaded onto 10% SDS-PAGE and then transferred onto PVDF membranes. After blocked by skim milk, the membranes were incubated in the primary antibodies diluted by TBST buffer for overnight at 4°C and then in the HRP-conjugated secondary antibody for 2-3h at room temperature. Finally the protein bands images were captured by a Tanon detection system with ECL reagent (Thermo). The primary antibodies used in the experiments were anti-TG2 (1:1000; Abcam, USA), anti-ERK1/2 (1:1000; Cell Signaling Technology, USA), anti-pERK1/2 (Thr202/Tyr204) (1:1000; Cell Signaling Technology, USA) and anti-GAPDH (1:10000; Abcam, USA).

Cell proliferation assay
Cells (2x10 3 /well) were seeded into 96-well plates for 4 days, and cell proliferation was measured by spectrophotometer at 0h, 24h, 48h, 72h and 96h using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions.

Cell migration and invasion assay
Cell migration and invasion assays were assessed using transwell chambers (Corning Costar, NY, USA). Cells were cultured in serum-free RPMI-1640 for 12-16h, and then 1x10 5 cells in 200ul serum-free medium were seeded into the upper chamber, and 800ul medium with 10% fetal calf serum was added to the lower chamber. For the invasion assay, the insert membranes were coated with diluted matrigel (BD Bioscience, CA, USA). After cultured for 24h, cells that did not move through the membranes were removed and then the membranes were stained with 0.1% crystal violet for 30 min. The stained cells were counted and photographed using an inverted microscope.

In vivo tumorigenesis and metastasis
Four-week-old male BALB/C nude mice, which were purchased from the Institute of Zoology, Chinese Academy of Sciences and housed at a specific pathogenfree environment, were injected subcutaneously with 6x10 5 gastric cancer cells in 100μl PBS or inoculated peritoneally with 2x10 6 cells in 250μl PBS. Tumor length (L) and width (W) were measured every week. Tumor volume was evaluated using the following formula: tumor volume =LW 2 π/6. All mice were sacrificed after 30 days. Subcutaneous tumor grafts were excised and weighed, and peritoneal metastasis nodules were counted and analyzed.

Statistical analysis
The experimental results were analyzed by SPSS 18.0 software and shown as mean ± standard deviation (SD). The relationship between the expression level of TG2 and clinicopathologic parameters was examined by the Pearson χ2 test or Fisher's exact tests. The differences between the two groups were calculated by the Student's t test. The significance level was set at P < 0.05.