MicroRNA-200b is downregulated in colon cancer budding cells

<div><p>Background</p><p>The microRNA-200 (miR-200) family acts as a major suppressor of epithelial-mesenchymal transition (EMT). Impaired miR-200 expression may lead to EMT initiation and eventually cancer dissemination. The presence of tumor budding cells (TBC) is associated with metastasis and poor prognosis, and molecular similarities to EMT indicate that these cells may reflect ongoing EMT. The aim of this study was to investigate the expression of miR-200b in budding cells of colon cancer and the relationship with the EMT-markers E-cadherin, β-catenin and laminin-5γ2.</p><p>Material & methods</p><p>MiR-200b was investigated by <i>in situ</i> hybridization in 58 cases of stage II (n = 36) and III colon (n = 22) cancers with tumor budding. Expression of E-cadherin, β-catenin and laminin-5γ2 was examined by immunohistochemistry. A multiplex fluorescence assay combining miR-200b with cytokeratin and laminin-5γ2 was employed on a subset of 16 samples.</p><p>Results</p><p>MiR-200b was downregulated in the TBC at the invasive front of 41 out of 58 (71%) cases. The decline was present in both mismatch satellite stable and instable adenocarcinomas. The majority of cases also showed loss of membranous E-cadherin and increased nuclear β-catenin in the TBC, while laminin-5γ2 expression was upregulated at the invasive front and in the tumor buds of approximately half the adenocarcinomas. However, the miR-200b decline was not statistically associated with the expression of any of the EMT-markers. The miR-200b decline was also documented by multiplex fluorescence. Fourteen out of fifteen cases showed a decrease in miR-200b expression in the majority of the TBC, but no obvious relationship between miR-200b and laminin-5γ2 expression was observed. Conclusion: The findings support the assumption of a miR-200b related downregulation in colon cancer budding cells. Whether miR-200b expression may be of clinical significance awaits further studies.</p></div

EMT-protein distribution in tumor center and at the invasive front.

<p>Venn diagram of colon cancers with tumor budding showing differentially expressed E-cadherin, nuclear β-catenin, and laminin-5γ2 in the invasive front compared to the tumor center. Out of 58 cases, a total of 51 cancers showed changes in at least one marker.</p

Clinico-pathological data for the study group.

<p>Clinico-pathological data for the study group.</p

MiR-200b expression in the tumor center and invasive front of colon cancer.

<p><b>(A)</b> High miR-200b expression is present in the central part of the tumor (CT), while <b>(B)</b> expression is decreased in several tumor budding cells (arrows) at the invasive front. High expression is present in only few buds (arrowheads). The image also illustrates that the identification of tumor budding cells in a CISH stain may be challenging. The cells are easily identified, when miR-200b expression is high, but much more difficult, when miR-200b is decreased. Single cells or small groups of cells with large nuclei and distinct nucleoli may suggest tumor buds, but unambiguous identification and quantification on a CISH stain is impossible.</p

Additional file 1: of Does heterogeneity matter in the estimation of tumour budding and tumour stroma ratio in colon cancer?

Table S1. Tumour stroma ratio estimated semi-quantitatively by conventional microscopy and stereology. Table S2. Correlation coefficient for correlations between tumour stroma ratio in the deepest invasive tumour section and random sections A and B. (DOCX 40 kb

Expression of miR-17, miR-21, miR-145, miR-125, miR-200b, and miR-126 as obtained in the study of the test cases.

<p><b>(A & B)</b> miR-17 in normal (N) and adenomatous (A) tissue and in adenocarcinoma (AC); <b>(C & D)</b> miR-21 expression in stroma of the adenoma compared to normal tissue and in the cancer-associated stroma cells(*). A small group of positive tumour epithelial cells is also present (white arrow); <b>(E)</b> miR-145 is seen in smooth muscle cells (arrow) and vascular smooth muscle cells (arrowhead); in the magnification (<b>F</b>) a reduced number of miR-145 positive fibroblast-like cells are found in the adenoma (arrow) compared to normal tissue (arrowhead); <b>(G)</b> Faint miR-125b signal was seen in the muscularis mucosa (mm); <b>(H)</b> miR-200b was seen in the epithelial cells at base of the crypts, but in this case also in the epithelial cancer cells; <b>(I)</b> miR-126 is exclusively seen in the endothelial cells; <b>(J)</b> The scramble probe showed only discrete background staining.</p

Expression of miR-17 in colon cancer development.

<p>Increased expression of miR-17 in low grade adenoma (LGA), high grade adenoma (HGA) and adenocarcinoma (AC) of the colon compared to normal tissue</p

Clinico-pathological data for the two study sets.

<p>^aThis patient had a synchronic T3 colon cancer</p><p>Clinico-pathological data for the two study sets.</p

Results from the validation study of miR-17, miR-21, and miR-145-expression in the normal-adenoma-adenocarcinoma sequence.

<p>^aData missing due to specimens failing to include all three compartments.</p><p>Results from the validation study of miR-17, miR-21, and miR-145-expression in the normal-adenoma-adenocarcinoma sequence.</p

Multivariate mixed effects linear regression of log-miR-17, as obtained by image analysis, in normal, adenomatous and invasive tissue of the colon.

<p>Multivariate mixed effects linear regression of log-miR-17, as obtained by image analysis, in normal, adenomatous and invasive tissue of the colon.</p