To understand the molecular mechanisms of metastasis and prognosis of colorectal cancer (CRC), we isolated single cell-derived progenies (SCPs) from SW480 cells in vitro and compared their metastatic potential in an orthotopic CRC tumour model in vivo. Two groups of SCPs with the capability of high and low metastasis, respectively, were obtained. By analysing the gene expression profiles of high (SCP51), low (SCP58) metastatic SCPs, and their parental cell line (SW480/EGFP), we demonstrated that 143 genes were differentially expressed either between SCP51 and SCP58 or between SCP58 and SW480/EGFP. Gene-annotation enrichment analysis of DAVID revealed 80 genes in the top ten clusters of the analysis (gene enrichment score > 1). Of the 80-gene set, 32 genes are potentially involved in metastasis, as revealed by Geneclip. Five putative metastatic genes (LYN, SDCBP, MAP4K4, DKK1, and MID1) were selected for further validations. Immunohistochemical analysis in a cohort of 181 CRC clinical samples showed that the individual expression of LYN, MAP4K4, and MID1, as well as the five-gene signature, was closely correlated with lymph node metastasis in CRC patients. More importantly, the individual expression of LYN, MAP4K4, SDCBP, and MID1, as well as the five-gene signature, was significantly correlated with overall survival in CRC patients. Thus, our five-gene signature may be able to predict metastasis and survival of CRC in the clinic, and opens new perspectives on the biology of CRC.

An accumulated evidence supports that MicroRNAs (miRNAs) have shown a prominent role in pathological processes and different tumor onset. However, to date, the potential functional roles and molecular mechanisms by how microRNA-424-5p(miR-424-5p) affects cancer cell proliferation are greatly unclear, especially in epithelial ovarian cancer(EOC).In this study, we demonstrated that miR-424-5p was significantly down-regulated in EOC tissues and cell lines. The level of miR-424-5p was negatively correlated with tumor size, TNM stage, pathological grade, lymphatic metastasis of EOC. Restoring miR-424-5p expression in EOC cells dramatically suppressed cell proliferation and caused an accumulation of cells in G1 phase, and thus contributed to better prognosis of EOC patients. Mechanistically, miR-424-5p inhibits CCNE1 expression through targeting CCNE1 3'UTR, and subsequent arrest cell cycle in G1/G0 phase by inhibiting E2F1-pRb pathway. This study revealed functional and mechanistic links between miR-424-5p and CCNE1 in the progression of EOC and provide an important insight into that miR-424-5p may serve as a therapeutic target in EOC.

Single cell progenies (SCPs) inherit biological properties from their isogenetic mother cells. If a single cancer cell can give rise to progenies, which can be passaged sustainably in vitro and produce tumor in xenotransplantation, the cell should be cancer initiating cell. CD133 (Prominin-1, Prom1) is the marker of human colorectal cancer (CRC) stem cells and probably a marker of metastatic cancer stem cells (CSCs). Thirty-three SCPs of CRC cell line SW480 were isolated by limited dilution methods, thirty of which are CD133 positive and three negative. All of the CD133(+) SCPs are tumorigenic, and the subcutaneous tumors expanded rapidly, while only 1 of 3 CD133(-) SCPs developed a minimal tumor in nude mice. Orthotopic transplantation experiments showed that CD133(+) SCPs possessed heterogeneity in intestinal wall invasion, lymph node and liver metastases. CD133(+) SCPs varied in cell growth, invasive ability, epithelial-mesenchymal-transition and expression of CSCs markers (CD133, CD44, and CXCR4) associated with metastatic potential. CD133(-) SCPs did not produce secondary transplanted tumor, intestinal invasion and metastasis. The results indicated CD133(+) subpopulation of SW480 SCPs bear heterogeneous invasive and metastatic ability, and CRC-CSCs might be a heterogeous subpopulation.

Purpose: The separation of Luminal A-like from Luminal B-like breast cancer subtypes in surrogate assay relies on Ki67 scores assessed by immunohistochemistry (IHC), a method known to be associated with subjectivity and inconsistency. We attempted to measure Ki67 levels absolutely, quantitatively and objectively in Formalin Fixed Paraffin Embedded (FFPE) specimens, and evaluate its influence on the performance of surrogate assay for breast cancer patients. Methods: The Ki67 protein levels were assessed using both IHC and Quantitative Dot Blot (QDB) methods respectively in 253 specimens. These patients were assigned into Luminal A-like and Luminal B-like subtypes using either Ki67 score of 14% as cutoff in surrogate assay, or 2.31 nmole/g from QDB method as cutoff in adjusted surrogate assay. These two subtyping methods were compared with the Kaplan-Meier, univariate and multivariate survival analyses of the overall survival (OS) of Luminal-like patients. Results: Ki67 levels measured using QDB method was highly correlated with those by IHC analysis (r=0.7, p<0.0001). The survival prediction for Luminal A-like patients was improved significantly in adjusted surrogate assay than surrogate assay (p=0.03 vs p<0.00052). The prediction of Hazard Ratio (HR) was also improve from 2.14 (95%CI: 0.89-5.11, p=0.087) to 6.89 (95%CI: 2.66-17.84, p<0.00001) in multivariate survival analysis. Conclusion: Our study demonstrated that the inherent subjectivity and inconsistency associated with IHC analysis has adverse effect on the performance of surrogate assay. This issue can be improved by objective and quantitative measurement of Ki67 levels with QDB method in daily clinical practice.

BackgroundImmunohistochemistry (IHC)-based surrogate assay is the prevailing method in daily clinical practice to determine the necessity of chemotherapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separate Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC-based Ki67 assessment is known to be plagued with subjectivity and inconsistency to undermine the performance of the surrogate assay. A novel method needs to be explored to improve the clinical utility of Ki67 in daily clinical practice.Materials and MethodsThe Ki67 protein levels in a cohort of 253 specimens were assessed with IHC and quantitative dot blot (QDB) methods, respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan–Meier, univariate, and multivariate survival analyses of the overall survival (OS) of Luminal-like patients.ResultsThe surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmol/g) subtyped the Luminal-like patients more effectively than that based on Ki67 scores (cutoff at 14%) (Log rank test, p = 0.00052 vs. p = 0.031). It is also correlated better with OS in multivariate survival analysis [hazard ratio (HR) at 6.89 (95% CI: 2.66–17.84, p = 0.0001) vs. 2.14 (95% CI: 0.89–5.11, p = 0.087)].ConclusionsOur study showed that the performance of the surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively, and objectively using the QDB method.

BackgroundKi67 is a biomarker of proliferation to be used in immunohistochemistry (IHC)-based surrogate assay to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients. cyclinD1 is another frequently used biomarker of proliferation. A retrospective study was performed here to investigate if these two biomarkers may be combined to improve the prognosis of Luminal-like patients.MethodsBoth Ki67 and cyclinD1 protein levels were measured absolutely and quantitatively using Quantitative Dot Blot method in 143 Luminal-like specimens. Optimized cutoffs for these two biomarkers were developed to evaluate their prognostic roles using Kaplan–Meier overall survival (OS) analysis.ResultscyclinD1 was found as an independent prognostic factor from Ki67 in univariate and multivariate OS analyses. At optimized cutoffs (cyclinD1 at 0.44 μmol/g and Ki67 at 2.31 nmol/g), the subgroup with both biomarkers below the cutoffs (n = 65) had 10-year survival probability at 90% in comparison to those with both biomarkers above the cutoffs (n = 18) with 8-year survival probability at 26% (log-rank test, p &lt;0.0001). This finding was used to modify the surrogate assay using IHC-based cyclinD1 scores, with p-value decreased from 0.031 to 0.00061 or from 0.1 to 0.02, when the Ki67 score of 14 or 20% was used as cutoff, respectively, in the surrogate assay.ConclusionThe current study supports the prospective investigation of cyclinD1 relevance in the clinic.

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