S100P, a calcium-binding protein, is preferentially associated with the growth of polypoid tumors in colorectal cancer

Abstract

Colorectal cancer (CRC) is a genetically heterogeneous disease with distinct morphological patterns. It has been shown that polypoid and ulcerative CRC displays different genetic alterations. In the present study, we aimed to investigate genes with differential expression patterns between ulcerative and polypoid CRC. cDNA microarray analysis was performed to compare the gene expression profiles in samples of ulcerative and polypoid CRC with paired normal mucosa samples. Potential candidate genes were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunohistochemistry. The epigenetic regulation of gene expression was investigated using methylation-specific PCR (MSP). cDNA microarray analysis identified 11 upregulated and 14 downregulated genes which were differentially expressed in samples from both tumor types compared to the matched normal mucosa samples. Among these, S100P was the only upregulated gene preferentially associated with polypoid CRC (P=0.032). The samples of polypoid CRC displayed significantly higher S100P protein and mRNA expression levels than the samples of ulcerative CRC (P<0.05, respectively). Using semi-quantitative immunohistochemical analyses, S100P overexpression was found to be preferentially associated with polypoid CRC (24/30 vs. 14/40, P<0.001). The relative methylation level determined by MSP did not differ significantly between the samples of polypoid and ulcerative CRC (43.36 vs. 49.10%, P=0.168), indicating that promoter hypomethylation was not directly related to the upregulation of S100P mRNA. Our results demonstrate that the upregulation of S100P mRNA and protein expression is a predominant characteristic in polypoid CRC, whereas ulcerative CRC presents with a wide range of expression levels, indicating that S100P overexpression is not a key determinant in conferring invasion properties. The clinicopathological significance of S100P in CRC requires further investigation in well-controlled studies.

Figure 1

S100P mRNA and protein expression in polypoid and ulcerative colorectal cancer and matched normal mucosa samples. (A) The S100P mRNA relative expression level in polypoid tumors was significantly higher (−2.5-fold) than that in ulcerative tumors (P<0.05; Student’s t-test). (B) Representative western blot analysis showing S100P protein expression in polypoid and ulcerative tumor and paired normal colon mucosa samples. Pan-cytokeratin (AE1/AE3) was used as an internal control. (C) S100P protein relative expression level in polypoid (n=10) and ulcerative (n=10) tumor and paired normal colon mucosa samples. Data shown are the means ± SE from 3 independent experiments (P<0.05; Student’s t-test).

Figure 2

Representative methylation-specific PCR (MSP) of S100P promoter status. (A) MSP analysis of methylated control DNA (mDNA), unmethylated control DNA (uDNA) and untreated DNA (wDNA) using designed primers sets showing the specificity of primers. (B) MSP analysis of S100P in pancreatic cancer and paired normal tissue. (C) Representative results of MSP analysis of S100P in polypoid and ulcerative colon cancer and paired normal mucosa samples. The PCR products in lanes M and U indicate the presence of methylated and unmethylated templates, respectively. Ma, 100-bp ladder marker.

Figure 3

Immunohistochemical detection of S100P protein expression in placenta and colon tissue. (A) Trophoblasts lining the placental chorionic villi demonstrated uniform and strong staining (magnification, ×200). (B) Normal colonic mucosa showed negative staining (magnification, ×200). (C) Glands with low-grade dysplasia showed heterogeneous nuclear staining and weak cytoplasmic staining (magnification, ×200). (D) Strong nuclear and moderate cytoplasmic staining in glands with high-grade dysplasia (magnification, ×400). (E) Strong staining in the apical region of cytoplasm without nuclear expression (magnification, ×100). (F) Neutrophils forming abscess around invasive cancer displayed very strong immunostaining (magnification, ×200).

Figure 4

Representative S100P protein expression in polypoid tumor samples. (A) Diffuse, strong S100P immunostaining in a polypoid tumor with invasion to the submucosa (panoramic view). (B) Heterogeneous expression of S100P in a polypoid tumor with invasion to the adipose tissue (panoramic view). (C) Magnification of polypoid portion (circle) in (B) showing strong S100P immunoreactivity (magnification, ×100). (D) Magnification of invasive front (square) in (B) displaying diminished S100P expression (original magnification, ×400).

Figure 5

Representative S100P immunostaining in ulcerative tumor samples. (A) An ulcerative tumor showing diffuse, strong positivity (panoramic view). (B) Marked and (C) mild intratumoral heterogeneous immunostaining (panoramic view). (D) A tumor ruptured with abscess formation showing negative staining. Neutrophils scattering within the tumor and forming abscess displaying strong reactivity (magnification, ×200).