Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers

Background:

Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC.

Methods:

Van-Gogh-like 2 expression and promoter methylation after 5-aza-2′-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression.

Results:

Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels.

Conclusion:

Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling.     

The influence of survivin shRNA on the cell cycle and the invasion of SW480 cells of colorectal carcinoma

Background

The objective was to understand the influence of Survivin plasmid with short hairpin RNA (shRNA) on the cell cycle, invasion, and the silencing effect of Survivin gene in the SW480 cell of colorectal carcinoma.

Methods

A eukaryotic expression vector, PGCH1/Survivin shRNA, a segment sequence of Survivin as target, was created and transfected into colorectal carcinoma cell line SW480 by the non-lipid method. The influence on the Survivin protein was analyzed by Western blotting, while the cell cycle, cell apoptosis were analyzed by flow cytometry, and invasion of the cell was analyzed by Transwell''s chamber method.

Results

After the transfection of PGCH1/Survivin shRNA, the expression of Survivin protein in SW480 cells was dramatically decreased by 60.68%, in which the cells were stopped at G2/M phase, even though no apoptosis was detected. The number of transmembranous cells of the experimental group, negative control group, and blank control group were 14.46 ± 2.11, 25.12 ± 8.37, and 25.86 ± 7.45, respectively (P <0.05).

Conclusion

Survivin shRNA could significantly reduce the expression of Survivin protein and invasion of SW480 cells. Changes in cell cycle were observed, but no apoptosis was induced.     

Paired related homoeobox 1, a new EMT inducer,is involved in metastasis and poor prognosis in colorectal cancer

Background:

Paired related homoeobox 1 (PRRX1) has been identified as a new epithelial-mesenchymal transition (EMT) inducer in breast cancer. However, the function of PRRX1 in colorectal cancer (CRC) has not been elucidated.

Methods:

We utilised ectopic PRRX1-expressing cell lines to analyse the function of PRRX1 in CRC. The clinical significance of PRRX1 was also examined on three independent CRC case sets.

Results:

PRRX1 induced EMT and the stem-like phenotype in CRC cells. In contrast to studies of breast cancer, abundant expression of PRRX1 was significantly associated with metastasis and poor prognosis in CRC.

Conclusion:

PRRX1 is an indicator of metastasis and poor prognosis in CRC cases. Further investigation is required to uncover the signalling network regulating PRRX1.     

Colorectal cancer,comorbidity, and risk of venous thromboembolism: assessment of biological interactions in a Danish nationwide cohort

Background:

Venous thromboembolism (VTE) is a major source of morbidity and mortality in cancer patients. Incident colorectal cancer (CRC) and comorbidity both predict VTE, but potential synergy between these factors has not been explored.

Methods:

Danish nationwide cohort study of CRC cases diagnosed in 1995–2010 and a matched general population reference cohort of subjects without CRC who matched cases on age, sex, and comorbidities. We calculated the Charlson Comorbidity Index using diagnoses recorded in the Danish National Patient Registry. We calculated standardised incidence rates (SIRs) and interaction contrasts (IC) to measure additive interaction between comorbidity and CRC status with respect to 5-year VTE incidence.

Results:

Among 56 189 CRC patients, 1372 VTE cases were diagnosed over 145 211 person-years (SIR=9.5 cases per 1000 person-years). Among 271 670 reference subjects, 2867 VTE cases were diagnosed over 1 068  860 person-years (SIR=2.8 cases per 1000 person-years). CRC and comorbidity were positively and independently associated with VTE, but there was no evidence for biological interaction between these factors (e.g., comparing the ‘severe comorbidity'' stratum with the ‘no comorbidity'' stratum, IC=0.8, 95% CI: −3.3, 4.8).

Conclusions:

There is neither a deficit nor a surplus of VTE cases among patients with both comorbidity and CRC, compared with rates expected from these risk factors in isolation.     

Inactivation of miR-34a by aberrant CpG methylation in Kazakh patients with esophageal carcinoma

Background

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported.

Methods

To determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation.

Results

We found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis.

Conclusions

Our findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.     

Prognostic impact of histological categorisation of epithelial–mesenchymal transition in colorectal cancer

Background:

The crosstalk between cancer cells and stroma is involved in the acquired capability for metastasis through the induction of epithelial–mesenchymal transition (EMT). We aimed to clarify the prognostic value of the histological category of EMT in colorectal cancer (CRC).

Methods:

Tumour EMT was graded into one of three histological categories on the basis of integrated assessment of poorly differentiated clusters and pro-EMT desmoplasia at the leading edge of the primary tumour (Histology^EMT). Stage II and III CRC patients (cohort 1, N=500) and stage IV patients (cohort 2, N=196) were retrospectively analysed.

Results:

In cohort 1, patients were stratified into three groups with widely different disease-free survival rates (95%, 83% and 39%) on the basis of Histology^EMT (P<0.0001). In cohort 2, Histology^EMT significantly stratified overall survival of patients irrespective of metasectomy. Multivariate analyses indicated that Histology^EMT had a strong prognostic impact independent of staging factors. Statistically, Histology^EMT had a better prognostic stratification power than T and N stages; however, in cohort 2, the power of M substage was superior.

Conclusions:

A histological model to categorise EMT by integrated assessment of dedifferentiation and desmoplastic environment is a potent prognostic index independent of staging factors.     

The mutation rates of EGFR in non-small cell lung cancer and KRAS in colorectal cancer of Chinese patients as detected by pyrosequencing using a novel dispensation order

Background

The purpose of this study was to develop a cost-effective approach for the determination of EGFR and KRAS mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) samples from Chinese patients based on a sensitive pyrosequencing (PS) technique.

Methods

The NSCLC and CRC cell lines were tested to determine the limitation of detection and reproducibility of the PS method. In addition, 494 NSCLC and 1099 CRC patient samples were assayed by PS to evaluate the EGFR or KRAS mutation patterns according to the clinicopathological features.

Results

The PS assay was able to reproducibly detect as few as 2 % mutant alleles with excellent linearity. EGFR mutations were detected in 35.63 % of the NSCLC samples, and KRAS mutations were detected in 39.76 % of the CRC samples. EGFR mutations were more frequently observed to be significant by multivariate analysis in NSCLC patients who were 65 years old or younger (OR = 2.51), had a nonsmoking history (OR = 3.63), and adenocarcinoma (OR = 3.57), but not in females (OR = 0.64). KRAS mutations were more frequently detected in CRC patients who were female (OR = 1.64) and 50 years old or older (OR = 4.17), and had adenocarcinoma (OR = 2.41).

Conclusions

This is the first extensive validation of PS on FFPE samples using the detection of EGFR exons 18–21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of PS analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and provide important clinical and molecular characteristics of NSCLC and CRC from Chinese patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0179-9) contains supplementary material, which is available to authorized users.     

Elevated kinesin family member 26B is a prognostic biomarker and a potential therapeutic target for colorectal cancer

Background

Kinesins play a key role in the development and progression of many human cancers. The present study investigated the expression and clinical significance of kinesin family member 26B (KIF26B) in colorectal cancer (CRC).

Methods

Using quantitative real-time PCR and Western blot analyses as well as immunohistochemical staining of a tissue microarray we examined KIF26B mRNA and protein levels in CRC tumor tissues and paired adjacent normal mucosa. Moreover, the effect of KIF26B knockdown on CRC cell proliferation was investigated using Cell Counting Kit-8 assays.

Results

Expression of KIF26B was found to be elevated in CRC. Suppression of KIF26B inhibited CRC cell proliferation. Furthermore, upregulated expression of KIF26B was significantly correlated with tumor size (P = 0.020), American Joint Committee on Cancer (AJCC) stage (P = 0.018), T stage (P = 0.026), N stage (P = 0.013), and differentiation histology (P = 0.047). KIF26B was also shown to be an independent prognostic indicator of overall survival for CRC patients (HR 5.621; 95% CI 2.302–13.730; P < 0.001).

Conclusion

Our data indicate that KIF26B plays an important role in colorectal carcinogenesis and functions as a novel prognostic indicator and a potential therapeutic target for CRC.     

Nomogram for predicting lymph node metastasis rate of submucosal gastric cancer by analyzing clinicopathological characteristics associated with lymph node metastasis

Background

To combine clinicopathological characteristics associated with lymph node metastasis for submucosal gastric cancer into a nomogram.

Methods

We retrospectively analyzed 262 patients with submucosal gastric cancer who underwent D2 gastrectomy between 1996 and 2012. The relationship between lymph node metastasis and clinicopathological features was statistically analyzed. With multivariate logistic regression analysis, we made a nomogram to predict the possibility of lymph node metastasis. Receiver operating characteristic (ROC) analysis was also performed to assess the predictive value of the model. Discrimination and calibration were performed using internal validation.

Results

A total number of 48 (18.3%) patients with submucosal gastric cancer have pathologically lymph node metastasis. For submucosal gastric carcinoma, lymph node metastasis was associated with age, tumor location, macroscopic type, size, differentiation, histology, the existence of ulcer and lymphovascular invasion in univariate analysis (all P<0.05). The multivariate logistic regression analysis identified that age ≤50 years old, macroscopic type III or mixed, undifferentiated type, and presence of lymphovascular invasion were independent risk factors of lymph node metastasis in submucosal gastric cancer (all P<0.05). We constructed a predicting nomogram with all these factors for lymph node metastasis in submucosal gastric cancer with good discrimination [area under the curve (AUC) =0.844]. Internal validation demonstrated a good discrimination power that the actual probability corresponds closely with the predicted probability.

Conclusions

We developed a nomogram to predict the rate of lymph node metastasis for submucosal gastric cancer. With good discrimination and internal validation, the nomogram improved individualized predictions for assisting clinicians to make appropriated treatment decision for submucosal gastric cancer patients.     

DAL-1 attenuates epithelial-to mesenchymal transition in lung cancer

Background

Epithelial-to mesenchymal transition (EMT) involves in metastasis, causing loss of epithelial polarity. Metastasis is the major cause of carcinoma-induced death, but mechanisms are poorly understood. Here we identify differentially expressed in adenocarcinoma of the lung-1 (DAL-1), a protein belongs to the membrane-associated cytoskeleton protein 4.1 family, as an efficient suppressor of EMT in lung cancer.

Methods

The relationship between DAL-1 and EMT markers were analyzed by using immunohistochemistry in the clinical lung cancer tissues. Quantitative real-time PCR and western blot were used to characterize the expression of the EMT indicator mRNAs and proteins in DAL-1 overexpressed or knockdown cells. DAL-1 combined proteins were assessed by co-immunoprecipitation.

Results

DAL-1 levels were strongly reduced even lost in lymph node metastasis and advanced pathological stage of human lung carcinomas. Overexpression of DAL-1 altered the expression of numerous EMT markers, such as E-cadherin, β-catenin Vimentin and N-cadherin expression, meanwhile changed the morphological shape of lung cancer cells, and whereas silencing DAL-1 had an opposite effect. DAL-1 directly combined with E-cadherin promoter and regulated its expression that could be the reason for impairing EMT and decreasing cell migration and invasion. Strikingly, HSPA5 was found as DAL-1 direct binding protein.

Conclusions

These results suggest that tumor suppressor DAL-1 could also attenuate EMT and be important for tumor metastasis in the early transformation process in lung cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-014-0117-2) contains supplementary material, which is available to authorized users.     

Overexpression of forkhead box protein M1 (FOXM1) plays a critical role in colorectal cancer

Background

The forkhead box M1 (FOXM1), an important regulator of cell differentiation and proliferation, is overexpressed in a number of aggressive human carcinomas. However, the clinical significance of FOXM1 signaling in human colorectal cancer (CRC) pathogenesis remains unknown. The aim of this study was to evaluate the role of FOXM1 in CRC tumorigenesis.

Methods

We investigated FOXM1 expression in 103 cases of primary CRC and matched normal tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on CRC proliferation and metastasis using in vitro models of CRC.

Results

The results showed that high expression of FOXM1 staining was 85.44 % (88/103) in 103 cases of CRC and 20.39 % (21/103) in 103 cases of adjacent non-cancerous tissue samples; the difference of FOXM1 expression between two groups was statistically significant (P < 0.001). Silencing of FOXM1 inhibited the proliferation of CRC cells, and the invasion and migration of CRC cells were distinctly suppressed. Furthermore, FOXM1 knockdown led to substantial reductions in VEGF-A levels in CRC cell lines.

Conclusions

Our data suggest that the pathogenesis of CRC maybe mediated by FOXM1, and FOXM1 could represent selective targets for the molecularly targeted treatments of CRC.

     

Alcohol intake and risk of colorectal cancer: Results from the UK Dietary Cohort Consortium

Background:

Epidemiological studies have suggested that excessive alcohol intake increases colorectal cancer (CRC) risk. However, findings regarding tumour subsites and sex differences have been inconsistent.

Methods:

We investigated the prospective associations between alcohol intake on overall and site- and sex-specific CRC risk. Analyses were conducted on 579 CRC cases and 1996 matched controls nested within the UK Dietary Cohort Consortium using standardised data obtained from food diaries as a main nutritional method and repeated using data from food frequency questionnaire (FFQ).

Results:

Compared with individuals in the lightest category of drinkers (>0–<5 g per day), the multivariable odds ratios of CRC were 1.16 (95% confidence interval (95% CI): 0.88, 1.53) for non-drinkers, 0.91 (95% CI: 0.67, 1.24) for drinkers with 5–<15 g per day, 0.90 (95% CI: 0.65, 1.25) for drinkers with 15–<30 g per day, 1.02 (95% CI: 0.66, 1.58) for drinkers with 30–<45 g per day and 1.19 (95% CI: 0.75, 1.91) for drinkers with ⩾45 g per day. No clear associations were observed between site-specific CRC risk and alcohol intake in either sex. Analyses using FFQ showed similar results.

Conclusion:

We found no significantly increased risk of CRC up to 30 g per day of alcohol intake within the UK Dietary Cohort Consortium.     

Combination of platelet count and neutrophil to lymphocyte ratio is a useful predictor of postoperative survival in patients with colorectal cancer

Background:

This study investigated the usefulness of a novel inflammation-based prognostic system, named the COP-NLR (COmbination of Platelet count and Neutrophil to Lymphocyte Ratio), for predicting the postoperative survival of patients with colorectal cancer (CRC).

Methods:

The COP-NLR was calculated on the basis of data obtained on the day of admission: patients with both an elevated platelet count (>30 × 10⁴ mm⁻³) and an elevated NLR (>3) were allocated a score of 2, and patients showing one or neither were allocated a score of 1 or 0, respectively.

Results:

Four-hundred and eighty patients were enrolled. Multivariate analysis of clinical characteristics selected by univariate analysis showed that the COP-NLR (1, 2/0) (odds ratio, 0.464; 95% confidence interval, 0.267–0.807; P=0.007) had an association with cancer-specific survival, along with pathology, lymph node metastasis, the serum levels of carcinoembryonic antigen, C-reactive protein and albumin, and the Glasgow Prognostic Score. Kaplan–Meier analysis and log-rank test revealed that the COP-NLR was able to divide such patients into three independent groups (P<0.001).

Conclusion:

The COP-NLR is considered to be a useful predictor of postoperative survival in patients with CRC.     

Fucosylated TGF-β receptors transduces a signal for epithelial–mesenchymal transition in colorectal cancer cells

Background:

Transforming growth factor-β (TGF-β) is a major inducer of epithelial–mesenchymal transition (EMT) in different cell types. TGF-β-mediated EMT is thought to contribute to tumour cell spread and metastasis. Sialyl Lewis antigens synthesised by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilised as tumour markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-β is unclear.

Methods:

Colorectal cancer cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion and migration assays. The expression and phosphorylation status of TGF-β downstream molecules were analysed by western blot. Fucosylation of TGF-β receptor (TβR) was examined by lectin blot analysis.

Results:

Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TβR and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT.

Conclusion:

Fucosyltransferase 3/6 has an essential role in cancer cell adhesion to endothelial cells by upregulation of sialyl Lewis antigens and also by enhancement of cancer cell migration through TGF-β-mediated EMT.