Over-expression of AURKA, SKA3 and DSN1 contributes to colorectal adenoma to carcinoma progression

Abstract

Development of colorectal cancer (CRC) involves sequential transformation of normal mucosal tissues into benign adenomas and then adenomas into malignant tumors. The identification of genes crucial for malignant transformation in colorectal adenomas (CRAs) has been based primarily on cross-sectional observations. In this study, we identified relevant genes using autologous samples. By performing genome-wide SNP genotyping and RNA sequencing analysis of adenocarcinomas, adenomatous polyps, and non-neoplastic colon tissues (referred as tri-part samples) from individual patients, we identified 68 genes with differential copy number alterations and progressively dysregulated expression. Aurora A, SKA3, and DSN1 protein levels were sequentially up-regulated in the samples, and this overexpression was associated with chromosome instability (CIN). Knockdown of SKA3 in CRC cells dramatically reduced cell growth rates and increased apoptosis. Depletion of SKA3 or DSN1 induced G2/M arrest and decreased migration, invasion, and anchorage-independent growth. AURKA and DSN1 are thus critical for chromosome 20q amplification-associated malignant transformation in CRA. Moreover, SKA3 at chromosome 13q was identified as a novel gene involved in promoting malignant transformation. Evaluating the expression of these genes may help identify patients with progressive adenomas, helping to improve treatment.

Keywords: AURKA; DSN1; SKA3; colon cancer progression; malignant transformation.

Figure 1. Identification of malignant transformation-related genes

A. Genomic regions with differential copy number alterations in carcinoma samples compared to polyp samples. Purple bars indicate CNAs detected in polyp samples and blue bars indicate CNAs detected in carcinoma samples. Bars on the right side represent amplification-type CNAs while bars on the left side represent deletion-type CNAs. The heights of the bars indicate the numbers of samples with CNAs in that region. Chromosome numbers are indicated at the bottom of each karyogram. B. Venn diagram of genes with differential expression and genes with differential CNAs in carcinoma samples. The number of genes in each list is shown in parentheses. C. Hierarchical clustering of the tissue samples and the 68 candidate genes. The tissue samples include nine carcinomas, nine paired polyps, and nine paired non-neoplastic colon tissues. mRNA expression levels are shifted to a mean of zero and scaled to a deviation of one, with green representing the lowest level and red representing the highest level. The tissue group, MSI status, and CIN level of each sample is indicated under the sample column. Blue dendrogram columns belong to cluster Ia, orange to cluster Ib, and red to cluster II.

Figure 2. Protein expression of candidate genes in tissues

A. Representative immunoblotting analysis of Aurora A, SKA3, UBE2C, and DSN1 in tri-part samples from four patients (CRC01, CRC06, CRC08, and CRC09). Ratios normalized to GAPDH are shown under each lane. (N, non-neoplastic tissue; A, polyp; C, carcinoma) B. Aurora A, SKA3, and DSN1 protein levels in paired adenoma and carcinoma samples. Fold change in protein levels was normalized to paired non-neoplastic samples. (*p<0.01; **p<0.001; ***p<0.0001) C. Representative images of IHC staining of Aurora A, SKA3, and DSN1 in tri-part samples are shown. Each row of images shows the tri-part samples from a single patient. Signal intensity scores are indicated on the bottom left of each image. Magnification: 200X.

Figure 3. ROC analysis of Aurora A, SKA3, and DSN1 for discriminating carcinoma from adenoma

ROC curves show the specificity and sensitivity of each protein by comparing polyps to non-neoplastic tissues (left panel), carcinomas to non-neoplastic tissues (middle panel), and carcinomas to polyps (right panel). The AUC values are indicated in each graph.

Figure 4. Correlations between protein levels and genomic alteration

Each circle represents the proportion of tumor samples in each category. Fold change of protein levels in tumor samples were normalized to paired non-neoplastic tissues, and overexpression is defined as fold change ≥ 1.5. A. Overexpression of Aurora A, SKA3, and DSN1 protein is positively correlated with higher CIN (0 corresponds to CIN-stable, 1 to low degree of CIN, 2 to medium degree of CIN, 3 to high degree of CIN, and 4 to ultra-high degree of CIN). B. Overexpression of SKA3 and DSN1 proteins are correlated with the presence of LOH.

Figure 5. Knockdown of SKA3 reduces cell growth

HT29 and HCT116 cells were transfected with control siRNA or target siRNA. A typical result is shown; each data point represents mean ± SD of cell indexes conducted in triplicate. A-F. Knockdown of SKA3, SKA1, or SKA2 reduced growth rates in HT29 and HCT116 cells. G-H. Knockdown of DSN1 did not affect cell growth rate in either HT29 or HCT116 cells. (**p<0.01; ***p<0.001; N.S., not significant).

Figure 6. Knockdown of SKA3 or DSN1 inhibits cell cycle progression

HT29 and HCT116 cells were transfected with control siRNA or target siRNA. A typical result of cell cycle distribution analysis is presented, with control siRNA in the left column and target RNA in the middle column. Quantification of the results of three independent experiments is presented in the right column. The percentages of cells at each cell cycle phase are shown as mean ± SD. A. SKA3 knockdown decreased the G1 phase population, increased the G2/M population, and increased the sub-G1 population in HT29 cells. B. SKA3 knockdown decreased the S phase population, increased the G2/M population, and increased the sub-G1 population in HCT116 cells. Knockdown of DSN1 increased G2/M arrest in C. HT29 cells and D. HCT116 cells. (*p<0.05; **p<0.01; N.S., not significant).

Figure 7. Knockdown of SKA3 or DSN1 inhibits migration, invasion, and anchorage-independent growth

Transwell migration, invasion, and soft agar colony assays were conducted with HT29 and HCT116 cells transfected with control siRNA or target siRNA. Knockdown of SKA3 or DSN1 inhibited cell migration and invasion in both cell lines A-D. Relative percentages of migrated and invaded cells are presented as mean ± SD. Depletion of SKA3 or DSN1 decreased colony formation in E. HT29 cells and F. HCT116 cells. Representative images of colony formation following each treatment are shown on the left and quantification results are shown on the right. Numbers of colonies are presented as mean ± SD. (*p<0.05; **p<0.01).