Differential expression of the Ca2+ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa

doi:10.1016/S0959-8049(00)00043-5

European Journal of Cancer

Volume 36, Issue 8, May 2000, Pages 1050-1059

https://doi.org/10.1016/S0959-8049(00)00043-5 Get rights and content

Abstract

The expression of calcium-binding protein S100A6 was investigated in normal colon tissue, in colon adenomas and in colorectal carcinomas. Using an immunoblotting approach we detected four S100A6 variants with Mwt of 10 kDa and pI of 5.05 (isoform I), 5.15 (isoform II), 5.23 (isoform III) and 5.32 (isoform IV) that were differentially expressed in the analysed samples. The quantitative examination of S100A6 variant expression in 25 pairs of colorectal carcinoma and matched control mucosa proved a statistically significant increased abundance of S100A6 isoforms I (P=0.004) and III (P=0.025) in malignant tissue, and conversely, an increased level of S100A6 isoform IV in healthy tissue (P=0.022). The expression of isoforms I and III and the loss of isoform IV were also observed in colon cancer cell lines. In addition, the immunohistochemical study of 16 primary colorectal carcinomas revealed both in the non-paired Student t-test and in the Mann–Whitney test the statistically significant accumulation of S100A6 protein (P<0.001) in the invasive margin of the tumour. The immunohistochemical analysis of S100A6 protein in polyps differing in clinical severity gave a strong staining that was maximal in dysplastic lesions. Thus, our results indicate a possible, statistically significant correlation (non-paired Student t-test P=0.036) between S100A6 expression and colon carcinoma progression.

Introduction

Colorectal tumorigenesis is generally assumed to be a multistep process in which the role of calcium ions in the control of the proliferation and differentiation of epithelial cells is increasingly appreciated [1]. The members of the S100 family of small acidic Ca²⁺-binding proteins that are expressed in a cell-type specific fashion seem to be excellent candidates to function as cell-type specific mediators of Ca²⁺ signalling. Furthermore, the participation of S100 proteins in the control of cell growth is also supported by the disregulation of their levels in various neoplastic diseases 2, 3.

The S100A6 protein, formerly designated as calcyclin, was the first S-100 protein specifically identified as being related to the state of cellular proliferation. The deregulation of S100A6 gene expression during malignant transformation has, so far, been described for human malignant melanoma [4], squamous cell carcinoma of the oral mucosa [5] and human breast cancer tissues [6]. Additionally, mRNA encoding the S100A6 protein and mRNAs for S100A10 (calpactin I light chain) and S100A4 (calvasculin) were found to be expressed in most human epithelial tumour cells and their levels increased in parallel with increases in the S phase population of cells [7]. The expression of the S100A6 protein can be, at least in some cases, under the positive control of oncogenes since increased expression of S100A6 protein was observed in metastatic ras-transformed cells [8].

Knowledge of the expression of calcium-binding proteins in the course of colon carcinogenesis is rapidly expanding. Studies carried out using human colon cancer cell lines revealed the overexpression of S100A11 and S100A10 mRNAs, as well as the presence of an alternatively spliced mRNA for calretinin 9, 10, 11. Recently, the expression of calretinin by a large proportion of undifferentiated colorectal carcinomas was described. Additionally, the degree of its expression coincided with an increase in the number of metastases in the regional lymph nodes and in other organs [12]. Similarly, an association between an increased level of S100A4 mRNA in colon adenocarcinomas and the invasion and metastasis of tumour cells has been reported by Japanese authors [13].

We recently reported the upregulation of S100A9 and S100A8 proteins in neoplastic colon mucosa, using two-dimensional gel electrophoresis (2-DE) for the separation of proteins directly extracted from surgically resected colorectal carcinomas [14].

In the present study, we used a new polyclonal antihuman recombinant S100A6 antibody [15] to explore S100A6 protein expression in normal, preneoplastic and neoplastic colon mucosa.

Section snippets

Tissue specimens

Matched sets of 25 colorectal carcinomas and normal colon mucosa used for 2-DE analysis were obtained within 30 min after surgical resection. Non-necrotic tumour tissues and control mucosa samples that were taken from each patient at 5–10 cm from the tumour mass were immediately homogenised in double the volume of 9.0 M urea lysis buffer (9.0 M urea, 2.0% Triton X-100, 3.0% CHAPS, 70 mM DTT and 2% carrier ampholytes, pH 8.5–10). Then cell lysates were centrifuged at 15 000g for 5 min, 4°C and

Results

The aim of our study was to investigate S100A6 protein expression in normal, preneoplastic and neoplastic colon mucosa. We applied two-dimensional gel electrophoresis followed by immunoblotting as a superior technique for the detection of possible post-translational protein processing [21]. Fig. 1 shows the S100A6 immunostaining in normal and malignant colon mucosa collected from patient No. 1 suffering from colon adenocarcinoma, Dukes' B. Three variants of S100A6 protein (I, II and IV), with

Discussion

Ca²⁺ ions are suggested to play an important role in the development of colorectal cancer. In addition to reducing the amount of free secondary bile acids and ionised fatty acids in the lumen of the colon, they directly control proliferation and terminal differentiation in normal colon crypts probably via surface Ca²⁺ receptors [1]. The cellular actions of Ca²⁺ ions are mediated by site-specific binding to specialised proteins. From this point of view, monitoring the expression of selected

Acknowledgements

The authors thank Jana Michaličková, Alena Firychová, Jitka Žáková and Šárka Průchová for their excellent technical assistance. This work was supported by grants from the Ministry of Health, Czech Republic (project no. 4075-3), from the Grant Agency of Czech Republic (project no. 310/98/P238) and by the Swiss National Science Foundation (project no. 31-50510.97).

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It was proposed that high expression of β-catenin correlated with the aggressive biological potential of cancer cells such as invasiveness and metastasis. Interestingly, the expression of S100A6 is also up-regulated in various cancer types [3] and has been frequently associated with increased metastatic properties of cancer cells [5,6,23]. However, neither the cause of such up-regulation nor its significance for the process of tumorigenesis has been, as yet, sufficiently explained.
In this paper we document an increased expression of S100A6, a calcium binding protein of the S100 family, and its co-localization with β-catenin in colorectal cancer tissues and in metastatic, SW620, versus non-metastatic, SW480, human colorectal cancer cell lines. Moreover, we show up-regulation of the S100A6 protein level in non-metastatic SW480 cells due to overexpression of β-catenin as well as the activation of the S100A6 gene promoter upon cell transfection with β-catenin and the TCF-Lef1 transcription factor. Since we found a high level of S100A6 in metastatic SW620 cells we searched for its interacting partners in the protein extract prepared from these cells. Using several methods we found that S100A6 interacts with lamin A/C, a protein known to be implicated in colon carcinogenesis. Our results reveal a novel and important network of relations and interactions between proteins potentially involved in colorectal cancer development and progression.
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Concerning MLL-AF4 leukemogenesis, previous mouse models suggest that the tumorigenesis capacity of MLL-AF4 alone is insufficient for causing leukemia. Based on the finding that an Fms-like tyrosine kinase 3 (Flt3) gene mutation in the tyrosine kinase domain (TKD) was observed in approximately 15% of mixed lineage leukemia (MLL), we investigated synergistic leukemogenesis effects of the two genes in vitro.
In a mouse interleukin-3 (IL-3)−dependent cell line, 32Dc, expression of MLL-AF4 and mutant Flt3 was induced using a lentiviral vector. We analyzed apoptosis induction in the absence of IL-3 and the granulocyte colony-stimulating factor − related induction of differentiation, gene expression profiling, and the mechanism involved in the synergistic effects of MLL-AF4 and Flt3-TKD.
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S100A6 (calcyclin) is a calcium-binding protein implicated in many cellular processes and often up-regulated in cancer. Its various biological effects possibly originate from the fact that it may bind to other proteins and modulate their function by inducing conformational changes or interfering with posttranslational modifications. Thus, to elucidate the biological role of S100A6 it is important to identify its targets. Here, we report, based on affinity chromatography and co-immunoprecipitation results that S100A6 interacts with p53 in the presence of calcium ions. We investigated functional implications of the S100A6–p53 interaction by comparing various aspects of p53 activity in HEp-2 cells with either unaltered or diminished S100A6 content due to stable expression of siRNA. We found that the presence of S100A6 results in higher p53 transcriptional activity which is also reflected by higher cell susceptibility to apoptosis evoked by hydrogen peroxide. As revealed by electrophoretic mobility shift assay (EMSA) S100A6 does not affect p53 binding to DNA. On the other hand, we observed that the presence of S100A6 coincides with more efficient nuclear accumulation of p53 under stress conditions. Collectively, our results indicate that S100A6 interacts with p53 and affects its biological activity.

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Differential expression of the Ca2+ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa

Abstract

Introduction

Section snippets

Tissue specimens

Results

Discussion

Acknowledgements

Trends Biol. Sci.

Cancer Lett.

Cell. Calcium

Analyt. Biochem.

Analyt. Biochem.

Analyt. Biochem.

FEBS Lett.

Curr. Opin. Biotechnol.

Curr. Biol.

Calcium — cell cycle regulator, differentiator, killer, chemopreventor, and maybe, tumor promoter

J. Cell. Biochem.

New perspectives on S100 proteinsa multi-functional Ca2+-, Zn2+- and Cu2+-binding protein family

BioMetals

Differential expression patterns of S100A2, S100A4 and S100A6 during progression of human malignant melanoma

Int. J. Cancer

Enhancement of calcyclin gene RNA expression in squamous cell carcinoma of the oral mucosa, but not in benign lesions

J. Oral. Pathol. Med.

Expression of Ca2+-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples

Int. J. Cancer

Modulation of S-100 genes response to growth conditions in human epithelial tumor cells

Pathol. Int.

Ras-responsive genes and tumor metastasis

Crit. Rev. Oncogenesis

Expression of the gene for the light chain of calpactin 1 (annexin II) in cell lines DiFi, HT-29, and WI-38

Anticancer Res.

Differential expression of the Ca²⁺ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa

New perspectives on S100 proteinsa multi-functional Ca²⁺-, Zn²⁺- and Cu²⁺-binding protein family

Expression of Ca²⁺-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples