Lumican and versican protein expression are associated with colorectal adenoma-to-carcinoma progression

<div><p>Background</p><p>One prominent event associated with colorectal adenoma-to-carcinoma progression is genomic instability. Approximately 85% of colorectal cancer cases exhibit chromosomal instability characterized by accumulation of chromosome copy number aberrations (CNAs). Adenomas with gain of chromosome 8q, 13q, and/or 20q are at high risk of progression to cancer. Tumor progression is also associated with expansion of the extracellular matrix (ECM) and the activation of non-malignant cells within the tumor stroma. The glycoproteins versican and lumican are overexpressed at the mRNA level in colon carcinomas compared to adenomas, and are associated with the formation of tumor stroma.</p><p>Purpose</p><p>The aim of this study was to characterize versican and lumican protein expression in tumor progression and investigate their association with CNAs commonly associated with adenoma-to-carcinoma progression.</p><p>Methods</p><p>Tissue microarrays were constructed with colon adenomas and carcinomas that were characterized for MSI-status and DNA copy number gains of chromosomes 8q, 13q and 20q. Sections were immunohistochemically stained for lumican and versican. Protein expression levels were evaluated using digitized slides, and scores were finally dichotomized into a positive or negative score per sample.</p><p>Results</p><p>Lumican and versican expression were both observed in neoplastic cells and in the tumor stroma of colon adenomas and carcinomas. Lumican expression was more frequently present in epithelial cells of carcinomas than adenomas (49% versus 18%; <i>P</i> = 0.0001) and in high-risk adenomas and carcinomas combined compared to low-risk adenomas (43% versus 16%; <i>P</i> = 0.005). Versican staining in the tumor stroma was more often present in high-risk adenomas combined with carcinomas compared to low-risk adenomas (57% versus 36%; <i>P</i> = 0.03) and was associated with the presence of gain of 13q (71% versus 44%; <i>P</i> = 0.04).</p><p>Conclusion</p><p>Epithelial lumican and stromal versican protein expression are increased during colorectal adenoma-to-carcinoma progression.</p></div

Lumican and versican expression in colon adenomas and carcinomas analyzed by immunohistochemistry.

<p>Representative examples show lumican staining (A-C) and versican staining (D-F) in the stroma and epithelium of the same tissue cores of one adenoma (A,D) and two carcinomas (B,C,E,F). These examples were classified as negative (0), weak (1), moderate (2), and strong (3), with expression for epithelium (E) and stroma (S) indicated between brackets [E,S] as follows; A [0.2], B [3.2], C [1.1], D [2.2], E [0.2], F [3.1]. Scale bars indicate 50 μm.</p

Lumican and versican expression in MSS low-risk adenomas, high-risk adenomas and carcinomas.

<p>Epithelial lumican staining was more frequently detected in high-risk adenomas combined with carcinomas compared to low-risk adenomas (A). The frequency of stromal lumican staining was similar for each type of lesion (B). The frequency of epithelial versican staining did also not differ between lesion types (C) while stromal versican staining was more frequently observed in high-risk adenomas combined with carcinomas compared to low-risk adenomas (D).</p

Distribution of lumican staining according to histological and molecular characteristics of the colorectal adenoma and carcinoma samples.

<p>MSS: microsatellite stable, MSI: microsatellite instable, CS: chromosomal stable, CIN: chromosomal instable, CNA: copy number aberration.</p

Schematic overview of in silico analysis pipeline used in this study.

<p>First level (association 1) shows a graphical representation of integration analysis of miRNA expression with copy number (CN) data. CN values for miRNAs on 13q were determined by combining values within a 2 Mb window surrounding the start of the miRNA. These values defined the covariate set used to determine association with miRNA expression. Second level (association 2) gives a graphical representation of the association of miRNA expression with predicted target mRNA expression. For each miRNA on 13q target mRNAs were determined by combining three or more target prediction tools. Expression levels of these target mRNAs defined the covariate set used to determine association with miRNA expression.</p

Functional analysis of the effect of <i>miR-15a-5p</i>, <i>-3p</i> and <i>miR-17</i> silencing in CRC cell line SW480.

<p>A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).</p

Analysis of miRNA expression in colorectal adenomas and carcinomas.

<p>A) Box plots comparing relative expression determined by qRT-PCR in adenomas (n = 48) versus carcinomas (n = 52) of four top candidate miRNAs, <i>miR-15a-3p</i>, <i>miR-15a-5p</i>, <i>miR-17</i> and <i>miR-20a</i>. B) Box plots comparing miRNA expression between tumours (adenomas and carcinomas) with (n = 24) or without (n = 73) 13q gain. P-values, determined by Mann-Whitney U-test, ≤ 0.05 are considered significant (* p ≤ 0.05; ** p < 0.01).</p

Analysis of target gene expression in colorectal adenomas and carcinomas.

<p>A) Box plots comparing relative mRNA expression (determined by expression microarray) in adenomas (n = 37) versus carcinomas (n = 31) of 5 target genes, <i>TYRO3</i>, <i>UCP2</i>, <i>RNF125</i>, <i>RASGEF1B</i> and <i>COPS2</i>. B) Box plots comparing mRNA expression between tumours (adenomas and carcinomas) with (n = 18) or without (n = 44) 13q gain. C) Box plots comparing mRNA expression in adenomas without 13q gain (n = 30) to carcinomas with 13q gain (n = 14). P-values, determined by Mann-Whitney U-test, ≤ 0.05 are considered significant (* p ≤ 0.05; ** p < 0.01; ns, not significant).</p

Association of miRNA expression with 13q copy number status and target mRNA expression.

<p>MiRNAs are ordered by significance of FDR of miRNA expression versus copy number. Chr, Chromosome; bp, base pair; exp, expression; n.d., no data; Significance considered at FDR<0.05.</p><p>Association of miRNA expression with 13q copy number status and target mRNA expression.</p

Analysis of mRNA expression levels of target genes in colorectal cancer cell line SW480.

<p>Relative expression of <i>UCP2</i> and <i>COPS2</i> in SW480 cells silenced for <i>miR-15a-5p</i>, <i>-3p</i> and <i>miR-17</i> compared to a non-targeting control (* p<0.05).</p