Radiation engenders converse migration and invasion in colorectal cancer cells through opposite modulation of ANXA2/AKT/GSK3β pathway

Radiation therapy is an effective non-surgical means to achieve local control for various solid tumors including colorectal cancer (CRC), but metastasis and recurrences after conventional radiotherapy remains a major obstacle in clinical practice, and the knowledge concerning the changes of metastatic potential after heavy ion radiation is still limited. This study investigated how radiation, including γ- and carbon ion radiation, would change the metastatic capacity of two CRC cell lines, HCT116 and DLD-1, and examined the underlying molecular mechanisms. We found that the migration and invasion was enhanced in DLD-1 cells but impaired in HCT116 cells in vitro and in vivo after radiation of γ-rays or carbons, and radiation induced epithelial mesenchymal transition (EMT) in DLD-1 cells but mesenchymal epithelial transition (MET) in HCT116 cells. The expression of snail, a key inducer of EMT, was significantly enhanced by inhibition of glycogen synthase kinase-3β (GSK3β) in both cell lines, suggesting the modulation of snail was alike in the two CRC cell lines. However, radiation inactivated GSK3β through stimulating the phosphorylation of AKT and GSK3β at Ser473 and Ser9 in DLD-1 cells respectively, but activated GSK3β by decreasing the expression of pAKT^Ser473 and pGSK3β^Ser9 or increasing the phosphorylation of GSK3β at Tyr216 in HCT116 cells. Therefore, the above inverted motility changes was due to the opposite modulation of AKT/GSK3β signaling pathway by radiation, which was further verified in other type of cancer cell lines including MCF-7, U251 and A549 cells. Moreover, it was found that annexin A2 (ANAX2) directly bound with GSK3β and acted as a negative regulator of GSK3β upon radiation. Knocking-down ANXA2 gene reversed the enhanced migration of the irradiated DLD-1 cells and strengthened radiation-impaired migration of HCT116 cells. Collectively, this study reveals that the change of cellular motility after radiation is independent of radiation type but is correlated with the inherent of cells.     

E/N-cadherin switch mediates cancer progression via TGF-β-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma

Background:

Epithelial-to-mesenchymal transition (EMT) is a fundamental process governing not only morphogenesis in multicellular organisms, but also cancer progression. During EMT, epithelial cadherin (E-cadherin) is downregulated while neural cadherin (N-cadherin) is upregulated, referred to as ‘cadherin switch''. This study aimed to investigate whether cadherin switch promotes cancer progression in cholangiocarcinoma (CC).

Methods:

CC cell lines were examined for migration, invasion, and morphological changes with typical EMT-induced model using recombinant TGF-β1. The changes in E-cadherin and N-cadherin expression were investigated during EMT. We also examined E-cadherin and N-cadherin expression in resected specimens from extrahepatic CC patients (n=38), and the associations with clinicopathological factors and survival rates.

Results:

TGF-β1 treatment activated cell migration, invasion, and fibroblastic morphological changes, especially in extrahepatic CC HuCCT-1 cells. These changes occurred with E-cadherin downregulation and N-cadherin upregulation, that is, cadherin switch. Patients with low E-cadherin expression had a significantly lower survival rate than patients with high E-cadherin expression (P=0.0059). Patients with decreasing E-cadherin and increasing N-cadherin expression had a significantly lower survival rate than patients with increasing E-cadherin and decreasing N-cadherin expression (P=0.017).

Conclusion:

Cadherin switch promotes cancer progression via TGF-β-induced EMT in extrahepatic CC, suggesting a target for elucidating the mechanisms of invasion and metastasis in extrahepatic CC.     

Wnt/β-catenin,an oncogenic pathway targeted by H. pylori in gastric carcinogenesis

A section of gastric cancers presents nuclear β-catenin accumulation correlated with H. pylori infection. H. pylori stimulate Wnt/β-catenin pathway by activating oncogenic c-Met and epidermal growth factor receptor (EGFR), or by inhibiting tumor suppressor Runx3 and Trefoil factor 1 (TFF1). H. pylori also trigger Wnt/β-catenin pathway by recruiting macrophages. Moreover, Wnt/β-catenin pathway is found involved in H. pylori-induced gastric cancer stem cell generation. Recently, by using gastroids, researchers have further revealed that H. pylori induce gastric epithelial cell proliferation through β-catenin. These findings indicate that Wnt/β-catenin is an oncogenic pathway activated by H. pylori. Therefore, this pathway is a potential therapy target for H. pylori-related gastric cancer.     

Zipper-interacting protein kinase promotes epithelial-mesenchymal transition,invasion and metastasis through AKT and NF-κB signaling and is associated with metastasis and poor prognosis in gastric cancer patients

Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis.     

Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.     

Thioredoxin 1 mediates TGF-β-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.     

Dihydroartemisinin suppresses proliferation,migration, the Wnt/β-catenin pathway and EMT via TNKS in gastric cancer

Gastric cancer is a common malignancy worldwide. However, the molecular mechanisms underlying this malignancy remain unclear and there are a lack of effective drugs. The present study aimed to investigate the antitumor effect of Dihydroartemisinin (DHA) or inhibition of Tankyrases (TNKS), and determine the underlying molecular mechanisms of gastric cancer. Immunohistochemistry and immunofluorescence analyses were performed to detect the expression levels of TNKS, epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathway-related proteins in gastric cancer tissues and adjacent normal tissues. The Cell Counting Kit-8 assay was performed to assess the viability of HGC-27 and AGS cells following treatment with different concentrations of HLY78 (a Wnt activator) or DHA. Following treatment with HLY78, DHA or small interfering (si)-TNKS1/si-TNKS2, colony formation and migratory abilities were assessed via the colony formation, wound healing and Transwell assays. Furthermore, western blot and immunofluorescence analyses were performed to detect the expression levels of TNKS, EMT- and Wnt/β-catenin-related proteins. The results demonstrated that the expression levels of TNKS, AXI2, β-catenin, N-cadherin and Vimentin were upregulated, whereas E-cadherin expression was downregulated in gastric cancer tissues compared with normal tissues. Furthermore, HLY78 and DHA suppressed the viability of HGC-27 and AGS cells, in a concentration-independent manner. Notably, TNKS knockdown or treatment with DHA suppressed colony formation, migration, TNKS expression, EMT and the Wnt/β-catenin pathway. Opposing effects were observed following treatment with HLY78, which were ameliorated following co-treatment with DHA. Taken together, these results suggest that DHA or inhibition of TNKS can suppress the proliferation and migration of gastric cancer cells, which is partly associated with inactivation of the Wnt/β-catenin pathway and EMT process.     

Inhibitory effect of a TGF�� receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells

Background:

Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.

Methods:

Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.

Results:

TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.

Conclusion:

A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.     

Chemopreventive effect of resveratrol and apocynin on pancreatic carcinogenesis via modulation of nuclear phosphorylated GSK3β and ERK1/2

Despite progress in clinical cancer medicine in multiple fields, the prognosis of pancreatic cancer has remained dismal. Recently, chemopreventive strategies using phytochemicals have gained considerable attention as an alternative in the management of cancer. The present study aimed to evaluate the chemopreventive effects of resveratrol (RV) and apocynin (AC) in N-Nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamster. RV- and AC-treated hamsters showed significant reduction in the incidence of pancreatic cancer with a decrease in Ki-67 labeling index in dysplastic lesions. RV and AC suppressed cell proliferation of human and hamster pancreatic cancer cells by inhibiting the G1 phase of the cell cycle with cyclin D1 downregulation and inactivation of AKT-GSK3β and ERK1/2 signaling. Further, decreased levels of GSK3β^Ser9 and ERK1/2 phosphorylation and cyclin D1 expression in the nuclear fraction were observed in cells treated with RV or AC. Nuclear expression of phosphorylated GSK3β^Ser9 was also decreased in dysplastic lesions and adenocarcinomas of hamsters treated with RV or AC in vivo. These results suggest that RV and AC reduce phosphorylated GSK3β^Ser9 and ERK1/2 in the nucleus, resulting in inhibition of the AKT-GSK3β and ERK1/2 signaling pathways and cell cycle arrest in vitro and in vivo. Taken together, the present study indicates that RV and AC have potential as chemopreventive agents for pancreatic cancer.     

CCR7 enhances TGF-β1-induced epithelial-mesenchymal transition and is associated with lymph node metastasis and poor overall survival in gastric cancer

CCR7 is a G protein-coupled chemokine receptor. In this study, we used immunohistochemistry with tissue microarrays to measure CCR7 expression in tumor specimens from 122 patients with gastric cancer. We show that CCR7 expression is associated with lymph node metastasis (P = 0.022) and overall survival (OS; P = 0.025), and is an independent factor associated with poorer overall survival (P = 0.032). The CCR7 mechanism was predicted based on bioinformatic analysis and verified in gastric cancer cell lines and primary tumor samples. The data show that CCR7 contributes to TGF-β1-induced epithelial-mesenchymal transition (EMT) and that the effects of TGF-β1 are inhibited by a CCR7 neutralizing antibody or a NF-κB inhibitor. Increased TGF-β1 expression was accompanied by nuclear localization of NF-κB-p65 and higher levels of the mesenchymal marker vimentin in human gastric cancer samples. We conclude that the CCR7 axis mediates TGF-β1-induced EMT via crosstalk with NF-κB signaling, facilitating lymph node metastasis and poorer overall survival in patients with gastric cancer. These findings suggest CCR7 is a novel prognostic indicator and a potential target for gastric cancer therapy.     

Histone demethylase RBP2 promotes malignant progression of gastric cancer through TGF-β1-(p-Smad3)-RBP2-E-cadherin-Smad3 feedback circuit

Some feedback pathways are critical in the process of tumor development or malignant progression. However the mechanisms through which these pathways are epigenetically regulated have not been fully elucidated. Here, we demonstrated that the histone demethylase RBP2 was crucial for TGF-β1-(p-Smad3)-RBP2-E-cadherin-Smad3 feedback circuit that was implicated in malignant progression of tumors and its knockdown significantly inhibited gastric cancer (GC) metastasis both in vitro and in vivo. Mechanistically, RBP2 can directly bind to E-cadherin promoter and suppress its expression, facilitating EMT and distant metastasis of GC. RBP2 can also be induced by TGF-β1, a key inducer of EMT, through phosphorylated Smad3 (p-Smad3) pathway in GC. The upregulated RBP2 can be recruited by p-smad3 to E-cadherin promoter and enhance its suppression, contributing to the promotion of metastasis of GC. In addition, the suppression of E-cadherin by RBP2 attenuated inhibition of Smad3 phosphorylation (exerted by E-cadherin), resulting further induction of RBP2 expression, and thus constituting positive feedback regulation during GC malignant progression. This TGF-β1-(p-Smad3)-RBP2- E-cadherin-Smad3 feedback circuit may be a novel mechanism for GC malignant progression and suppression of RBP2 expression may serve as a new strategy for the prevention of tumor distant metastasis.     

Expression of E-cadherin repressors SNAIL,ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer

Background:

There is evidence that tumour–stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial–mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT.

Methods:

mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding.

Results:

Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152).

Conclusions:

In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.     

Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth,invasion, and epithelial-to-mesenchymal transition

Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas.