S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors

Abstract

S100A6 (Calcyclin) is a calcium-binding protein that has been implicated in a variety of biological functions as well as tumorigenesis. The aim of our study was to investigate the involvement of S100A6 during prostate cancer development and progression. Using immunohistochemistry, the expression of S100A6 was examined in benign (n=66), premalignant (n=10), malignant (n=66) and metastatic prostate (n=5) tissues arranged in a tissue-microarray or whole sections as well as in prostate cancer cell lines. The S100A6 immunostaining pattern in tissues was compared with that of cytokeratin 5 (a basal cell marker) and 18 (a benign luminal cell marker). In all cases of benign epithelium, intense S100A6 expression was seen in the basal cell layer with absent staining in luminal cells. In all cases of prostatic adenocarcinoma (matched), metastatic lesions and 3/10 high-grade prostatic intraepithelial neoplasia lesions, an absence of S100A6 was seen. Western blotting and RT-PCR analysis of cell lines showed S100A6 expression to be absent in LNCaP, LNCaP-LN3 and LNCaP-Pro5 but present in Du145, PC3, PC-3M and PC-3M-LN4. LNCaP cells treated with 5-Azacytidine, caused re-expression of S100A6 mRNA. Sequencing of bisulphite modified DNA showed CpG methylation within the S100A6 promoter region and exon 1 of LNCaP, LNCaP-LN3 and LNCaP-Pro5 cell lines but not in Du145 cells. Our data suggest that loss of S100A6 protein expression is common in prostate cancer development and may occur at an early stage. The mechanism of loss of expression may involve hypermethylation of CpG sites. The finding of intense S100A6 expression in the basal cells of benign glands but loss of expression in cancer could be useful as a novel diagnostic marker for prostate cancer.

Figure 1

S100A6 immunostaining in benign, malignant, metastatic prostate and high-grade PIN samples. (A) Note the intense staining of the basal cells within the epithelium, seen as a single continuously stained cell layer (× 200 magnification). (B) Higher magnification shows intense staining in the cytoplasm and nuclei of basal cells but an absence of staining in luminal cells (× 400). (C) Staining within an area of basal cell proliferation (× 400). Note the intense staining in the hyperproliferative basal cells, with the luminal cells showing absent staining. (D) Adenocarcinoma showing absent S100A6 expression within the malignant cells, but intense expression in basal cells of benign glands (× 200). Absent staining of metastatic cells was seen in (E) bone and (F) lymph node metastasis (× 400). (G) A case of high-grade PIN with a disrupted basal cell layer showing absent staining (× 400). (H) A case of high-grade PIN with an intact basal cell layer, showing intense S100A6 staining within the basal cells (× 400).

Figure 2

Comparison of S100A6 immunostaining with cytokeratin 5 and cytokeratin 18 immunostaining (× 100 magnification). (A) and (B) are serial sections of benign tissue stained with (A) cytokeratin 5 or (B) S100A6. Note that the staining patterns are similar with intense staining of the basal cell layer. In (B), note the weak-moderate staining of the muscle cells within the stroma. (C) and (D) are serial sections of adenocarcinoma stained with (C) cytokeratin 18 or (D) S100A6. Note that the cytokeratin 18 positive cells are not stained for S100A6. Basal cells of benign glands adjacent to carcinoma are stained for S100A6 (D).

Figure 3

Western blot of S100A6, using the polyclonal anti-S100A6 antibody (Dako) on lysates from prostate cancer cell lines. Lane 1. Du145; lane 2, LNCaP; lane 3, PC3. Note the protein expression in Du145 and PC3 cells, seen as an ∼10 kDa band, but its absence in the LNCaP cell line.

Figure 4

RT–PCR of S100A6 using the S100A6 F2/R2 primers ran on a 2.5% agarose/ethidium bromide gel. S100A6 mRNA expression can be seen in lane 1 (Du145), lane 2 (PC3), but not in lane 3 (LNCaP) or lane 4 (using a two-fold excess of LNCaP cDNA). Treatment of LNCaP cells with 5-Azacytidine caused re-expression of S100A6 mRNA using two different doses, lane 5 (1 μM) and lane 6 (5 μM). Nontranscribed total RNA or water used as negative controls showed an absence of PCR product (not shown). GAPDH was used as a loading control in each case and to check RNA integrity.

Figure 5

S100A6 DNA sequence of prostate cancer cell lines. (A) Sequence of the 5′ upstream region of exon 1, which was investigated (Gene bank Accession number J02763). The two CpG sites are shown in bold (B) Chromatographs showing S100A6 exon 1 sequence following bisulphite modification of DNA from prostate cancer cell lines. Du145 showed an absence of methylation as seen by the conversion of all the G residues to A residues. Methylation at the two CpG sites (arrows) can be seen in the LNCaP, LNCaP-LN3 and LNCaP-Pro5 cell lines, since the G residues at these sites were not converted, but remained as G residues.