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.     

Tumour-suppressive function of SIRT4 in human colorectal cancer

Background:

SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers.

Methods:

We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry.

Results:

SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis.

Conclusions:

SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.     

Reply: Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

Background:

Emerging evidence has demonstrated that lysine-specific demethylase 1 (LSD1) has an important role in many pathological processes of cancer cells, such as carcinogenesis, proliferation and metastasis. In this study, we characterised the role and molecular mechanisms of LSD1 in proliferation and metastasis of colon cancer.

Methods:

We evaluated the correlation of LSD1, CDH-1 and CDH-2 with invasiveness of colon cancer cells, and investigated the roles of LSD1 in proliferation, invasion and apoptosis of colon cancer cells. We further investigated the mechanisms of LSD1-mediated metastasis of colon cancer.

Results:

Lysine-specific demethylase 1 was upregulated in colon cancer tissues, and the high LSD1 expression was significantly associated with tumour-node-metastasis (TNM) stages and distant metastasis. Functionally, inhibition of LSD1 impaired proliferation and invasiveness, and induced apoptosis of colon cancer cells in vitro. The LSD1 physically interacted with the promoter of CDH-1 and decreased dimethyl histone H3 lysine 4 (H3K4) at this region, downregulated CDH-1 expression, and consequently contributed to colon cancer metastasis.

Conclusion:

Lysine-specific demethylase 1 downregulates the expression of CDH-1 by epigenetic modification, and consequently promotes metastasis of colon cancer cells. The LSD1 antagonists might be a useful strategy to suppress metastasis of colon cancer.     

Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion

Background:

Focal adhesion kinase (FAK) is overexpressed in a variety of cancers, such as breast, colon, prostate, ovary, and lung cancers. However, the mechanism by which extracellular matrix fibronectin stimulates lung cancer cell migration and invasion through FAK remains to be investigated.

Methods:

The signalling pathways in fibronectin-mediated lung cancer cell migration and invasion were examined using western blotting. The metastasis function was detected by wound healing, migration and invasion assays. Further, RNA interference and kinase inhibitors were also used to study the downstream signals.

Results:

In this study, we examined the FAK signalling pathways in relation to calpain-2 and RhoA in fibronectin-mediated lung cancer cell migration and invasion. We found that A549 lung epithelial cells stimulated by fibronectin showed increased phosphorylation of FAK and its downstream targets, Src, ERK1/2, phosphatidylinositol 3′-kinase (PI3K), and Akt. Consistent with this observation, depletion of FAK by siRNA resulted in the inhibition of Src, ERK1/2, PI3K, and Akt activity. In addition, the Src inhibitor, PP2, blocked the phosphorylation of FAK, ERK1/2, PI3K, and Akt. Conversely, inhibition of MEK1/2 using PD98059 reduced the expression of matrix metalloproteinase-9 (MMP9) and calpain-2. The PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, further blocked the expression of MMP9 and RhoA. Inhibition of both MEK1/2 and PI3K caused reduced cell migration and invasion.

Conclusion:

Our data suggest that fibronectin-mediated activation of FAK that leads to lung cancer metastasis could occur through ERK or PI3K/Akt regulation of MMP9/calpain-2 or MMP9/RhoA activity, respectively.     

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.     

Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells

Background:

Breast cancer stem cells (BCSCs) are characterized by high aldehyde dehydrogenase (ALDH) enzyme activity and are refractory to current treatment modalities, show a higher risk for metastasis, and influence the epithelial to mesenchymal transition (EMT), leading to a shorter time to recurrence and death. In this study, we focused on examination of the mechanism of action of a small herbal molecule, psoralidin (Pso) that has been shown to effectively suppress the growth of BSCSs and breast cancer cells (BCCs), in breast cancer (BC) models.

Methods:

ALDH⁻ and ALDH⁺ BCCs were isolated from MDA-MB-231 cells, and the anticancer effects of Pso were measured using cell viability, apoptosis, colony formation, invasion, migration, mammosphere formation, immunofluorescence, and western blot analysis.

Results:

Psoralidin significantly downregulated NOTCH1 signaling, and this downregulation resulted in growth inhibition and induction of apoptosis in both ALDH⁻ and ALDH⁺ cells. Molecularly, Pso inhibited NOTCH1 signaling, which facilitated inhibition of EMT markers (β-catenin and vimentin) and upregulated E-cadherin expression, resulting in reduced migration and invasion of both ALDH⁻ and ALDH⁺ cells.

Conclusion:

Together, our results suggest that inhibition of NOTCH1 by Pso resulted in growth arrest and inhibition of EMT in BCSCs and BCCs. Psoralidin appears to be a novel agent that targets both BCSCs and BCCs.     

Ubiquitin D is correlated with colon cancer progression and predicts recurrence for stage II-III disease after curative surgery

Background:

Our recent study observed that the expression of ubiquitin D (UBD), a member of ubiquitin-like modifier family, was upregulated in colon cancer parenchymal cells. The present study further investigated the clinical signicance of UBD in colon cancer.

Methods:

Using quantitative PCR, tissue microarray (TMA), western blot analysis and immunohistochemical stain, we evaluated UBD mRNA and protein levels in tumour tissues from patients with colon cancer at different stages and in paired adjacent normal epithelium.

Results:

Immunohistochemical detection of UBD on a TMA containing 203 paired specimens showed that increased cytoplasmic UBD was signicantly associated with depth of cancer invasion, lymph node metastasis, distant metastasis, tumour histologic grade, advanced clinical stage and Ki-67 proliferative index. Patients with UBD-positive tumours had a significantly higher disease recurrence rate and poorer survival than patients with UBD-negative tumours after the radical surgery. Stratification analysis according to tumour stage revealed UBD as an independent predictor for tumour recurrence in patients with stage II and III tumours.

Conclusion:

UBD may contribute to the progression of colon carcinogenesis and function as a novel prognostic indicator of forecasting recurrence of stage II and III patients after curative operations.     

Wnt3a expression is associated with epithelial-mesenchymal transition and promotes colon cancer progression

Introduction

Epithelial–mesenchymal transition (EMT) contributes to the progression and metastasis of cancer cells and is associated with a more invasive phenotype of cancer. The Wnt/β-catenin signaling pathway is one of the major pathways involved in EMT regulation. Many studies provide evidence that β-catenin, the key regulator of the canonical Wnt signaling pathway, is important in regulating EMT in cancer. However, the roles of Wnt3a, the representative canonical Wnt ligand, in EMT and colon cancer progression have not yet been fully explored.

Methods

The expression levels of Wnt3a and EMT-associated proteins (E-cadherin, vimentin, and β-catenin) were assessed by immunohistochemistry in human colon cancer tissues to evaluate the clinicopathological significance of Wnt3a, as well as the correlation between Wnt3a and EMT. We then upregulated Wnt3a expression in HCT116 colon cancer cells, established a nude mouse xenograft model, detected the expression of EMT and Wnt/β-catenin signaling-associated proteins, and observed invasion and clone-initiating abilities.

Results

In 203 human colon cancer tissue samples, Wnt3a protein overexpression was related to colon cancer histological differentiation (P = 0.004), clinical stage (P = 0.008), presence of metastasis and recurrence (P = 0.036), and survival time (P = 0.007) of colon cancer patients. Wnt3a expression was notably concomitant with EMT immunohistochemical features, such as reduced expression of the epithelial marker E-cadherin (P = 0.012), increased expression of the mesenchymal marker vimentin (P = 0.002), and cytoplasmic distribution of β-catenin (P = 0.021). Results of in vitro and in vivo experiments showed that Wnt3a overexpression could alter cell morphology, regulate EMT-associated protein expression, and enhance clone-initiation and invasion. Dkk1 (antagonist of Wnt/β-catenin signaling) could also partially reverse the expression of EMT-associated proteins in Wnt3a-overexpressing cells.

Conclusions

Wnt3a expression was associated with EMT and promoted colon cancer progression. The EMT-inducing effect was partially due to the stimulative effect of Wnt3a on the Wnt/β-catenin pathway.     

Down-regulation of frizzled-7 expression decreases survival, invasion and metastatic capabilities of colon cancer cells

Background:

The canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.

Methods and results:

In this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40–50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).

Conclusion:

These data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway.     

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis

Background:

TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown.

Methods:

miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC.

Results:

miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G₀/G₁ cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels.

Conclusion:

We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.     

Akt/Ezrin Tyr353/NF-κB pathway regulates EGF-induced EMT and metastasis in tongue squamous cell carcinoma

Background:

Epithelial–mesenchymal transition (EMT) is a crucial programme in cancer metastasis. Epidermal growth factor (EGF) is a key inducer of EMT, and Ezrin has an important role in this process. However, how Ezrin is activated and whether it mediates EGF-induced EMT in tongue squamous cell carcinomas (TSCCs) through activating NF-κB remains obscure.

Methods:

We used two TSCC cell lines as a cell model to study invasion and EMT in vitro, and used nude mice xenografts model to evaluate metastasis of TSCC cells. Finally, we evaluated the level of pEzrin Tyr353, nuclear p65 and EMT markers in TSCC clinical samples.

Results:

Ezrin Tyr353 was phosphorylated through Akt (but not ERK1/2, ROCK1) pathway, and lead to the activation of NF-κB in EGF-treated TSCC cells. Akt and NF-κB inhibitors blocked EGF-induced EMT, and suppressed invasion and migration of TSCC cells. In vivo, silencing Ezrin significantly suppressed EGF-enhanced metastasis of TSCC xenografts. Finally, high levels of expression of pEzrin Tyr353, nuclear p65, vimentin and low level of expression of E-cadherin were correlated with cancer metastasis and poor patient prognosis.

Conclusion:

Our data suggest that Akt/Ezrin Tyr353/NF-κB pathway regulates EGF-induced EMT and metastasis inTSCC, and Ezrin may serve as a therapeutic target to reverse EMT in tongue cancers and prevent TSCC progression.     

P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells

Background:

Our previous study demonstrated that extracellular adenosine 5′-triphosphate (ATP) stimulated prostate cancer cell invasion via P2Y receptors. However, the purinergic receptor subtype(s) involved in this process remains unclear. Here we aimed to determine whether P2Y2, one subtype of P2Y receptors, was involved in the invasion and metastasis of prostate cancer cells, and elucidated the underlying mechanism.

Methods:

RNAi was introduced to silence the expression of P2Y2. In vitro invasion and migration assays and in vivo experiments were carried out to examine the role of P2Y2 receptor in cell invasion and metastasis. cDNA microarray was performed to identify the differentially expressed genes downstream of ATP treatment.

Results:

P2Y2 was significantly expressed in the prostate cancer cells. Knockdown of P2Y2 receptor suppressed cell invasion and metastasis in vitro and in vivo. Further experiments identified that ATP could promote IL-8 and Snail expression and inhibit E-cadherin and Claudin-1 expression. Knockdown of P2Y2 receptor affected the expression of these EMT/invasion-related genes in vitro and in vivo.

Conclusion:

P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes. Thereby, P2Y2 receptor could be a potential therapeutic target for the treatment of prostate cancer.     

Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells

Background:

Human colorectal cancer is caused by mutations and is thought to be maintained by a population of cancer stem cells. Further phenotypic changes occurring at the invasive edge suggest that colon cancer cells are also regulated by their microenvironment. Type I collagen, a promoter of the malignant phenotype in pancreatic carcinoma cells, is highly expressed at the invasive front of human colorectal cancer.

Methods:

This study investigates the role of type I collagen in specifying the colorectal cancer cell phenotype. The effect of type I collagen on morphology, localisation of cell–cell adhesion proteins, differentiation and stem cell-like characteristics was examined in a panel of human colorectal carcinoma cell lines.

Results:

Human colorectal carcinoma cells grown on type I collagen in serum-free medium show an epithelial–mesenchymal-like transition (EMT-like), assuming a more flattened less cohesive morphology. Type I collagen downregulates E-cadherin and β-catenin at cell–cell junctions. Furthermore, type I collagen inhibits differentiation, increases clonogenicity and promotes expression of stem cell markers CD133 and Bmi1. Type I collagen effects were partially abrogated by a function-blocking antibody to α2 integrin.

Conclusion:

Together, these results indicate that type I collagen promotes expression of a stem cell-like phenotype in human colorectal cancer cells likely through α2β1 integrin.     

TBLR1 is a novel prognostic marker and promotes epithelial–mesenchymal transition in cervical cancer

Background:

Invasion and metastasis remain a critical issue in cervical cancer. However, the underlying mechanism of it in cervical cancer remains unclear. The newly discovered protein, TBLR1, plays a crucial role in regulating various key cellular functions.

Methods:

In this study, western blot, real-time RT–PCR, immunohistochemical staining, 3D morphogenesis Matrigel culture, wound healing and Boyden chamber invasion assays, xenografted tumour model, luciferase assays, and chromatin immunoprecipitation assays were used.

Results:

The expression of TBLR1 in cervical cancer cell lines and tissues was significantly upregulated at both the RNA and protein levels compared with that in normal cervical cells. Statistical analysis suggested that TBLR1 as an independent prognostic factor was significantly correlated with the clinical stage, survival time and recurrence. Moreover, overexpression of TBLR1 in Hela and Siha cell lines promoted invasion in vitro and in vivo with the increases of the mesenchymal factors vimentin and fibronectin and decreases of the epithelial marker α-catenin. In contrast, RNAi-mediated knockdown of TBLR1 inhibited epithelial–mesenchymal transition in vitro and in vivo. Further study indicated that this might be mediated via the NF-κB and Wnt/β-Catenin signalling pathway, and involve regulation of Snail and Twist.

Conclusions:

The TBLR1 protein may be a prognostic marker in cervical cancer and play an important role in the invasion and metastasis of human cervical cancer.     

Identification of tumour suppressive microRNA-451a in hypopharyngeal squamous cell carcinoma based on microRNA expression signature

Background:

Hypopharyngeal squamous cell carcinoma (HSCC) has a very poor prognosis because of its high rates of regional and distant metastasis. Identification of differentially expressed miRNAs and their regulated molecular targets in tumour cells might enhance our understanding of the molecular mechanisms of metastasis in human cancers.

Methods:

A HSCC miRNA signature was constructed by array-based methods. Functional studies of microRNA-451a (miR-451a) and target genes were performed to investigate cell proliferation, migration and invasion by cancer cell lines. To identify miR-451a-regulated molecular targets, we adopted gene expression analysis and in silico database analysis.

Results:

Our miRNA signature revealed that miR-451a was significantly downregulated in HSCC. Restoration of miR-451a in cancer cell lines revealed that this miRNA significantly inhibited cancer cell migration and invasion. Our data demonstrated that the gene coding for endothelial and smooth muscle cell-derived neuropilin-like molecule (ESDN/DCBLD2) was a direct target of miR-451a regulation. Silencing of ESDN inhibited cell migration and invasion by cancer cells.

Conclusions:

Loss of tumour suppressive miR-451a enhanced cancer cell migration and invasion in HSCC through direct regulation of ESDN. Our miRNA signature and functional analysis of targets regulated by tumour suppressive miR-451a provide new insights into the potential mechanisms of HSCC oncogenesis and metastasis.