miR-142-3p Suppresses Cell Growth by Targeting CDK4 in Colorectal Cancer.

BACKGROUND/AIMS: Deregulation of microRNAs (miRNAs) has been associated with a variety of cancers, including colorectal cancer (CRC). Here, we investigated anomalous miR-142-3p expression and its possible functional consequences in primary CRC samples. METHODS: The expression of miR-142-3p was measured by quantitative RT-PCR in 116 primary CRC tissues and adjacent non-tumor tissues. The effect of miR-142-3p up- or down-regulation in CRC-derived cells was evaluated in vitro by cell viability and colony formation assays and in vivo by growth assays in xenografted nude mice. RESULTS: Using quantitative RT-PCR, we found that miR-142-3p was down-regulated in 78.4 % (91/116) of the primary CRC tissues tested when compared to the adjacent non-tumor tissues. We also found that the miR-142-3p mimic reduced in vitro cell viability and colony formation by inducing cell cycle arrest in CRC-derived cells, and inhibited in vivo tumor cell growth in xenografted nude mice. Inversely, we found that the miR-142-3p inhibitor increased the viability and colony forming capacity of CRC-derived cells and tumor cell growth in xenografted nude mice. In addition, we identified CDK4 as a potential target of miR-142-3p by predictions and dual-luciferase reporter assays. Concordantly, we found that miR-142-3p mimics and inhibitors could decrease and increase CDK4 protein levels in CRC-derived cells, respectively. CONCLUSION: From our results we conclude that miR-142-3p may act as a tumor suppressor in CRC and may serve as a tool for miRNA-based CRC therapy.

Development and validation of a novel classification scheme for combining pathological T stage and log odds of positive lymph nodes for colon cancer.

AIM: Log Odds of Positive Lymph Nodes (LODDS) have a better predictive ability than N stage for colon cancer. However, the prognostic value of developing a novel prognostic classification by combining T stage and LODDS (TLODDS) for colon cancer remains unknown. Therefore, in the present study, we aimed to develop a TLODDS classification for colon cancer, and assess whether or not the novel TLODDS classification could improve survival stratification by comparing its discrimination, model-fitting, and net benefits, with the American Joint Committee on Cancer (AJCC) Tumor/Node/Metastasis (TNM) classification. METHODS: 45,558 Western colon cancers were identified in the Surveillance, Epidemiology, and End Results database as a training set. A novel LODDS stage was established and patients with similar survival rates were grouped by combining T and LODDS stages to develop a novel TLODDS classification. The TLODDS classification was further assessed in a Chinese validation set of 3,515 colon cancers and an application set of 3,053 rectal cancers. RESULTS: We developed a novel TLODDS classification that incorporated 7 stages: stage I (T1LODDS1), IIA (T2LODDS1, T1LODDS2, T1LODDS3), IIB (T2LODDS2-3, T3LODDS1, T1LODDS4), IIC (T3LODDS2, T2LODDS4, T4aLODDS1), IIIA (T3LODDS3, T1-2LODDS5, T4bLODDS1, T4aLODDS2), IIIB (T3LODDS4-5, T4aLODDS3-4, T4bLODDS2) and IIIC (T4bLODDS3-5, T4aLODDS5). In the training set, it showed significantly better discrimination (area under the receiver operating characteristic (ROC) curve, 0.691 vs. 0.664, P < 0.001), better model-fitting (Akaike information criteria, 265,644 vs. 267,410), and superior net benefits, than the latest AJCC TNM classification. The predictive performance of the TLODDS classification was further validated in colon cancers and was successfully applied in rectal cancers with regards to both overall and disease-free survival. CONCLUSIONS: The TLODDS classification has better discriminatory ability, model-fitting, and net benefits than the existing TNM classification, and represents an alternative to the current TNM classifications for colon and rectal cancers.

Screening and validation of a novel T stage-lymph node ratio classification for operable colon cancer.

BACKGROUND: Lymph node ratio (LNR) has advantages in predicting prognosis compared with American Joint Committee on Cancer (AJCC) pathological N stage. However, the prognostic value of a novel T stage-lymph node ratio (TLNR) classification for colon cancer combining LNR and pathological primary tumor stage (T stage) is currently unknown. METHODS: We included 62,294 patients with stage I-III colon cancer from the Surveillance, Epidemiology, and End Results Program as a training cohort. External validation was performed in 3,327 additional patients. A novel LNR stage was established and combined with T stage in a novel TLNR classification. Patients with similar survival were grouped according to T and LNR stages, with T1LNR1 as a reference. RESULTS: We developed a novel TLNR classification as follows: stages I (T1LNR1-2, T1LNR4), IIA (T1LNR3, T2LNR1-2, T3LNR1), IIB (T1LNR5, T2LNR3-4, T3LNR2, T4aLNR1), IIC (T2LNR5, T3LNR3-4, T4aLNR2, T4bLNR1), IIIA (T3LNR5, T4aLNR3-4, T4bLNR2), IIIB (T4aLNR5, T4bLNR3-4), and IIIC (T4bLNR5). In the training cohort, the novel TLNR classification had better prognostic discrimination (area under receiver operating characteristic curve, 0.621 vs. 0.608, two-sided P<0.001), superior model-fitting ability for predicting overall survival (Akaike information criteria, 561,129 vs. 562,052), and better net benefits compared with the AJCC 8th tumor/node/metastasis classification. Similar results were found in the validation cohort for predicting both overall and disease-free survival. CONCLUSIONS: This novel TLNR classification may provide better prognostic discrimination, model-fitting ability, and net benefits than the AJCC 8th TNM classification, for potentially better stratification of patients with operable stage I-III colon cancer; however, further studies are required to validate the novel TLNR classification.

Sp1 contributes to overexpression of stanniocalcin 2 through regulation of promoter activity in colon adenocarcinoma.

BACKGROUND: Aberrant expression of stanniocalcin 2 (STC2) is implicated in colon adenocarcinoma (COAD). A previous study identified that STC2 functions as a tumor promoter to drive development of some cancers, but the role of its overexpression in the development of COAD remains unclear. AIM: To evaluate the regulation mechanism of STC2 overexpression in COAD. METHODS: The expression of STC2 in COAD was assessed by TCGA COAD database and GEO (GSE50760). Methylation level of the STC2 promoter was evaluated with beta value in UALCAN platform, and the correlation between STC2 expression and survival rate was investigated with TCGA COAD. Transcription binding site prediction was conducted by TRANSFAC and LASAGNA, and a luciferase reporter system was used to identify STC2 promoter activity in several cell lines, including HEK293T, NCM460, HT29, SW480, and HCT116. Western blotting was performed to evaluate the role of Sp1 on the expression of STC2. RESULTS: The central finding of this work is that STC2 is overexpressed in COAD tissues and positively correlated with poor prognosis. Importantly, the binding site of the transcription factor Sp1 is widely located in the promoter region of STC2. A luciferase reporter system was successfully constructed to analyze the transcription activity of STC2, and knocking down the expression of Sp1 significantly inhibited the transcription activity of STC2. Furthermore, inhibition of Sp1 remarkably decreased protein levels of STC2. CONCLUSION: Our data provide evidence that the transcription factor Sp1 is essential for the overexpression of STC2 in COAD through activation of promoter activity. Taken together, our finding provides new insights into the mechanism of oncogenic function of COAD by STC2.

Role of serine/threonine kinase 33 methylation in colorectal cancer and its clinical significance.

Serine/threonine kinase 33 (STK33) is a novel protein that has been the focus of an increasing number of studies in recent years; however, the role of STK33 in tumorigenesis remains controversial. Previous studies have demonstrated that STK33 is overexpressed in several human cancers and exerts a pro-tumorigenic effect through the promotion of cell proliferation. However, the role of STK33 in colorectal cancer (CRC), which is one of the most aggressive human malignancies, remains unclear. The aim of the current study was to investigate the methylation status of STK33 in CRC and to determine its clinical significance. The results demonstrated that STK33 was hypermethylated in CRC cell lines and promoted the proliferation of CRC cells. In addition, the methylation status and expression of STK33 in 94 pairs of cancer and noncancerous tissues obtained from patients with CRC was investigated. STK33 methylation was significantly increased in cancer tissues when compared with adjacent noncancerous tissues (P<0.001). STK33 methylation was associated with lymph node metastasis (P<0.05), tumor invasion (P<0.05), distant metastases (P<0.01) and tumor stage (P<0.01). Reduced STK33 mRNA and protein expression in CRC was associated with STK33 hypermethylation (P<0.001). In addition, patients with hypermethylated STK33 exhibited shorter overall survival rates when compared with those with unmethylated STK33 (P<0.01). In conclusion, the results of the current study suggest that STK33 hypermethylation may be a promising novel biomarker for the diagnosis, prognosis and suitable treatment of CRC.

Pleomorphic adenoma gene like-2 induces epithelial-mesenchymal transition via Wnt/ß-catenin signaling pathway in human colorectal adenocarcinoma.

Epithelial-mesenchymal transition (EMT) is a critical step in the acquisition of metastatic and invasive power for tumor cells. Colorectal adenocarcinoma (CRC) is a common cancer where metastasis is directly linked to patient survival. Recent studies show that pleomorphic adenoma gene like-2 (PLAGL2) could induce tumor EMT and is an independent predictive factor associated with poor prognosis in cancer. In the present study, we confirmed the role of PLAGL2 in the prognosis of CRC patients and provide molecular evidence of PLAGL2 promoted EMT in CRC cell line SW480. We found that PLAGL2 expression was upregulated in the paraffin-embedded CRC tissues compared to borderline or benign tissues. Experimental EMT induced by PLAGL2 plasmid transfection proved PLAGL2 protein overexpression could enhance the cell scratch wound-healing and transwell ability and significantly upregulated mesenchymal marker proteins, N-cadherin and vimentin and concurrently downregulated epithelial marker of E-cadherin. Subsequently, through western blot assay, we found that PLAGL2 could activate the wnt-signaling component ß-catenin in the nuclei. More CRC cell metastasis to the lungs was observed when the PLAGL2 overexpressing SW480 cells were injected into the tail vein of rats, compared with the cell control and PLAGL2 silence group. Our findings indicated that PLAGL2 might be a very upstream key molecule regulating EMT involved in Wnt/ß­catenin signaling pathway.

Macroscopic serosal classification of colorectal cancer and its clinical significance.

BACKGROUND: Macroscopic serosal classification of gastric cancer has been reported in previous studies, but rarely reported about it of colorectal cancer. The purpose of this study was to propose a macroscopic serosal classification of colorectal cancer and to investigate clinical significance of this classification. MATERIALS AND METHODS: Morphologic features of colorectal cancer were analyzed according to the macroscopic serosal appearance and clinicopathologic characteristics of these patients were retrospectively reviewed. Microscopic serosal structure was compared between different types under light microscope and transmission electron microscope. RESULTS: Macroscopic serosal classification was divided into normal type, reactive type, nodular type and colloid type according to the macroscopic serosal appearance and microscopic structure. There were significant differences in tumor size, tumor gross type, histological type, histological grade, tumor necrosis, pT stage, number of nodes metastasis, lymph node metastasis ratio, pN stage, M stage and peritoneal metastasis between patients with different serosal types. Univariate analysis of prognosis revealed macroscopic serosal classification as one of factors significantly correlated with patient survival. However, multivariate analysis only revealed TNM stage significantly correlated with patient survival, while macroscopic serosal classification did not, maybe due to insufficient samples. CONCLUSIONS: Macroscopic serosal classification of colorectal cancer is preliminarily defined and divided into four types. Different macroscopic serosal types indicate different clinicopathologic features and correlate with prognosis of patients with colorectal cancer, but still cannot be proven as an independent factor.

Effects of allogeneic blood transfusion in patients with stage II colon cancer.

The aim of the present study was to determine whether allogeneic red blood cell transfusions showed a deleterious effect and what might be preoperative risk factors for blood transfusion in patients with TNM stage II colon cancer. Total 470 patients who fulfilled inclusion criteria were selected for a further 10-year follow-up study. We found that there were statistical significance between non-transfused and transfused group in mortality (P=0.018), local recurrence (P=0.000) and distant metastasis (P=0.040). Local recurrence and distant metastasis between 1 to 3 units and more than 3 units group did not show any significant differences. There was no difference in survival rate between non-transfused and 1 to 3 units group (log rank =0.031, P=0.860). The difference between different blood transfusion volume in transfused patients was found (78.77% vs 63.83%, P=0.006). Meanwhile, the significant difference of survival rate was existed between non-transfused group and more than 3 units group (84.83% vs 63.83%, P=0.002 ). Univariate analysis showed the following 3 variables to be associated with an increased risk of allogeneic blood transfusions: preoperative CEA level (P<0.05), location of tumor (P<0.01) and diameter of tumor (P<0.01). Multivariate analysis revealed that location of tumor and diameter of tumor are two independent factors for requirement of perioperative transfusions. Therefore, allogeneic transfusion increase the postoperative tumor mortality, local recurrence and distant metastasis in patients with stage II colon cancer. The postoperative tumor mortality, local recurrence and distant metastasis were not associated with the blood transfusion volume. The blood transfusion volume was associated with the survival rate. Location of tumor and diameter of tumor were the independent preoperative risk factors for blood transfusion.