Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer

Abstract

Background: Over the past years, some members of the family of suppressor of cytokine signalling (SOCS) proteins have emerged as potential tumour suppressors. This study aimed at investigating the clinical significance of SOCS proteins in colorectal carcinoma (CRC).

Methods: We integrated publicly available microarray expression data on CRC in humans, analysed the expression pattern of SOCSs and assessed the predictive power of SOCS2 and SOCS6 for diagnostic purposes by generating receiver operating characteristic curves. Using laser microdissected patient material we assessed SOCS expression on RNA and protein levels as well as their methylation status in an independent CRC patient cohort. Finally, we investigated the prognostic value of SOCS2 and SOCS6.

Results: The meta-analysis as well as the independent patient cohort analysis reveal a stage-independent downregulation of SOCS2 and SOCS6 and identify both molecules as diagnostic biomarkers for CRC. We demonstrate a different methylation pattern within the SOCS2 promoter between tumour tissue and normal control tissue in 25% of CRC patients. Furthermore, early CRC stage patients with low expression of SOCS2 display significantly shorter disease-free survival.

Conclusions: Our data offers evidence that SOCS2 and SOCS6 levels are reduced in CRC and may serve as diagnostic biomarkers for CRC patients.

Figure 1

Bioinformatic analysis reveals SOCS2 and SOCS6 downregulation in CRC. (A) Bar plot showing the log2 FC values of the SOCS family genes and marker genes in adenoma and CRC samples compared to normal colorectal mucosa. Error bars correspond to 95% confidence intervals for mean log FC; *FDR<0.05, **FDR<0.01 and ***FDR<0.001. (B) Heatmap representation of SOCS (highlighted in yellow) and STAT (highlighted in grey) family genes as well as marker genes in normal tissues, IBD, adenomas, and CRC stages (A–D).

Figure 2

SOCS2 and SOCS6 are potential biomarkers in human CRC. ROC curves with corresponding AUC values for (A) SOCS2 and (B) SOCS6 when classifying CRC patients and healthy donors. All grades of CRC were pooled. Distributions of gene expression values for healthy and CRC patients are shown in the insets.

Figure 3

Expression of SOCS family members in primary human CRC samples. (A) Marker gene expression levels in CRC vs normal tissue of two patients. (B) SOCS1, SOCS2, SOCS3 and SOCS6 mRNA levels in CRC compared to normal mucosal tissue. Data are presented as mean±s.e.m.; *P<0.05, **P<0.001 and ***P<0.0001.

Figure 4

Protein levels of SOCS2 and SOCS6 in human CRC. (A) Western blot detection of SOCS2 and SOCS6 in CRC and distant normal counterpart tissues. Caco-2 and Hela cells served as positive control for SOCS2 and SOCS6, respectively. The samples were run on the same gel but the membrane was cut for representative purposes (dotted line). (B) Immunohistochemical staining for SOCS2 and SOCS6 in CRC and normal counterpart for two different patients, magnification × 200.

Figure 5

Methylation analysis of the SOCS2 promoter in primary human CRC samples. (A) Treatment with 5-aza-dC/DAC increases basal SOCS2 expression in LS174t and HT-29 colon cancer cell lines whereas SOCS6 levels are unchanged. Data are representative of four independent experiments and presented as mean±s.d. (B) Representative histogram of the methylation pattern of four primary human CRC samples compared to their respective normal counterpart in a sequence −158 to +334 relative to the start codon. Each CpG site analysed is represented by a dot. (C) Representation of the SOCS2 promoter region that shows differential methylation between tumour and normal control samples. The sequence analysed by mass array is underlined and the CpG sites are highlighted in red. Analysis of the methylated sequence shows the presence of 2 STAT GAS motifs (TTCnnnGAA) highlighted in yellow. The start codon is indicated in green. The primers used in other SOCS2 methylation studies are indicated (Liu et al: pink arrows; Fiegl et al: green arrows; Sutherland et al: blue arrows).

Figure 6

Prognostic value of SOCS2 and SOCS6 in CRC. Disease-free survival curves for patients with high and low expression of SOCS2 or SOCS6 in primary CRC including (A) all stages or (B) stages I and II only. The number of patients included in each group is mentioned within brackets. Significant P-values are indicated.