Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all the common malignancies and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with cancer cells usually representing a relatively small proportion of the cells in the tumor mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by silver staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with silver stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired non-malignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in tumor cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in tumor cells was identified as the calcium-binding protein, S100A6. To determine the incidence of S100A6 overexpression in pancreatic cancer, we carried out immunohistochemical analysis on sections from a pancreas cancer tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 pancreas cancer patients. Normal pancreatic ductal epithelia were either devoid of detectable S100A6 or showed weak expression only. Moderately or poorly differentiated tumors, by contrast, showed a higher incidence and a higher level of S100A6 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in PDAC.

Background: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. Aims: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. Methods: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays.Results: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p,0.001) and cytoplasmic (p,0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p,0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. Conclusions: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Recent studies have reported elevated levels of S100A6 in pancreatic ductal adenocarcinoma cells. Here, we describe a detailed analysis of S100A6 expression in benign (n = 32), malignant (n = 60), and premalignant pancreatic ductal cells [96 pancreatic intraepithelial neoplasias (PanIN) from 46 patients]. S100A6 staining was more intense in malignant cells than in benign cells (P = 0.0001). In malignant cells, staining was higher in the nucleus than in the cytoplasm (P = 0.003). Univariate analysis revealed a significant decrease in survival time for patients with high levels of nuclear (P = 0.01) but not cytoplasmic (P = 0.20) S100A6. No evidence was found for an association between nuclear S100A6 expression and other variables, including gender, age at surgery, tumor size or grade, nodal metastases, resection margin, vascular invasion, perineural invasion, p53 or Smad4 levels (both linked to survival in previous studies), or the p65 subunit of nuclear factor-KB (a potential regulator of S100A6). Although nodal metastases and resection margin involvement were also associated with poor survival (P = 0.06 in both cases), multivariate analysis suggests that nuclear S100A6 is a significant independent indicator of survival (P = 0.003). Whereas PanIN 1a lesions showed a general absence of S100A6 staining, there was a progressive increase in the proportion of positively stained PanINs with increasing PanIN grade. In particular, we observed an increase in the frequency and intensity of nuclear staining. Our results suggest that upregulation of S100A6 is an early event in pancreatic cancer development and that elevated levels of nuclear S100A6 may affect clinical outcome. (Cancer Res 2005; 65(8): 3218-25)

No abstract

The presence of the EF-hand-calcium-binding protein S100A4 in the carcinoma cells of the primary tumour is associated with a shorter survival time of a group of breast cancer patients. In colon cancer, primary tumours as well as metastases to the liver can be studied. Here we show, using quantitative PCR applied to RNA from 24 normal colon, four liver tissues, 24 colon carcinoma specimens, and 24 livers containing colonic carcinoma metastases, that the level of S100A4 mRNA was significantly higher in the carcinomas compared to normal specimens (Mann-Whitney U-test, P=0.05), and in liver metastases compared to carcinoma specimens (P=0.039). The latter comparison included seven liver metastases and their matched primary carcinomas (P50.001) from the same patient. In situ hybridization and immunocytochemistry techniques have localized S100A4 to both carcinoma cells and lymphocytes in the malignant specimens. The percentage of specimens stained for S100A4 in the epithelial cells is significantly higher for those isolated from carcinomas and metastases than from the corresponding normal tissue, and from metastases than from corresponding carcinoma (Fisher Exact text, P50.0016, P=0.04, respectively). In most specimens, S100A4 is present in clusters of T lymphocytes and this distribution is also found in the lymphoid, uninflamed appendix. Keywords: p9Ka; S100A4; T-lymphocytes; colon carcinoma; metastasisThe EF-hand-containing protein, S100A4 (p9Ka) is closely linked with the ability of mammary tumour cells to metastasize in experimental model systems. Transfection of the gene for rat (Davies et al, 1993) or human (Lloyd et al, 1998) S100A4 into benign rat mammary cells, and the expression of S100A4 protein at an elevated level leads to the expression of a metastatic phenotype. Similar results have been obtained by over-expressing the mouse S100A4 in human MCF-7 cells (Grigorian et al, 1999). Mice, transgenic for the S100A4 gene, which over express S100A4, show no phenotype, but when these mice are mated with mice bearing, as an expressed transgene, an activated oncogene, such as c-erbB-2, which produces benign mammary tumours, offspring which express both the onco-transgene and the S100A4 transgene, exhibit tumours which also metastasize to the lungs (Davies et al, 1996). A similar result was obtained when transgenic mice bearing the mouse S100A4 transgene were mated with a strain of mice which yielded MMTV-induced mammary tumours (Ambartsumian et al, 1996).In a study of 349 patients with breast cancer, the presence of immunocytochemically-detectable S100A4 in the primary tumours is associated with their early demise when followed up for 19 years Rudland et al, 2000). This result suggests a direct link, at least in this group of patients, between the presence of S100A4 in the carcinoma cells of the primary tumour and the process of metastasis, since it is commonly believed that breast cancer patients die of metastatic spread to sites other than that of the lymph nodes. Thus it is important to establish whether the cell...

No abstract

The cross-talk between tumour cells and the surrounding supporting host cells (stroma) is a key regulator of cancer growth and progression. By undertaking 2-DE analysis of laser capture microdissected malignant and stromal components of pancreatic tumours and benign ductal elements, we have identified high levels of S100A8 and S100A9 in tumour-associated stroma but not in benign or malignant epithelia. Immunohistochemical analysis (n = 71 patients) revealed strong expression of both proteins in stromal myeloid cells, subsequently identified as CD14(+)/CD68(- )monocytes/macrophages. Co-immunofluorescence revealed that S100A8 was expressed in a subset of S100A9-positive cells. Correlation of the expression of S100A8 and S100A9 to patient parameters revealed that the microenvironments of tumours which lacked expression of the tumour suppressor protein, Smad4, had significantly reduced numbers of S100A8-immunoreactive (p = 0.023) but not S100A9-immunoreactive (p = 0.21) cells. The ratio of S100A8- to S100A9-positive cells within individual tumours was significantly lower in Smad4-negative tumours than in Smad4-positive tumours (p<0.003). Pancreatitic specimens also contained S100A8- and S100A9-expressing cells, although this was not observed in regions displaying extensive fibrosis. In conclusion, our study provides an extensive analysis of S100A8 and S100A9 in pancreatic disease and highlights a potentially important relationship between pancreatic cancer cells and their surrounding microenvironment.

No abstract