Differential Expression of SPARC in Intestinal-type Gastric Cancer Correlates with Tumor Progression and Nodal Spread.

AIMS: Nodal spread is the single most important prognostic factor of survival in gastric cancer patients. In this study, genes that were upregulated in the lymph node metastases of gastric cancer were identified and may serve as putative novel therapeutic target. METHODS: Complementary DNA (cDNA) microarray analysis and quantitative real-time polymerase chain reaction of primary gastric carcinomas and matched lymph node metastasis were carried out. Immunohistochemistry with anti-SPARC antibodies was performed on large tissue sections of 40 cases with primary gastric carcinoma (20 diffuse, 20 intestinal) and the corresponding lymph node metastases, as well as on tissue microarrays of 152 gastric cancer cases. RESULTS: A cDNA microarray identified SPARC as being upregulated in primary gastric carcinoma tissue and the corresponding lymph node metastasis compared with the nonneoplastic mucosa. SPARC was expressed in fibroblasts and, occasionally, in tumor cells. However, the level of immunoreactivity was particularly strong in stromal cells surrounding the tumor. The level of expression of SPARC, determined by immunohistochemistry, correlated in intestinal-type gastric cancer with the local tumor growth, nodal spread, and tumor stage according to the International Union Against Cancer. CONCLUSIONS: Our study provides transcriptional and translational evidence for the differential expression of SPARC in gastric cancer tissue. On the basis of our observations and those made by others, we hypothesize that SPARC is a promising novel target for the treatment of gastric cancer.

Angiotensin II-generating enzymes, angiotensin-converting enzyme (ACE) and mast cell chymase (CMA1), in gastric inflammation may be regulated by H. pylori and associated cytokines.

BACKGROUND: The local angiotensin II system (LAS) has numerous functions, including the regulation of growth and differentiation in the gastrointestinal tract. Angiotensin II (AngII) may be generated by angiotensin-I-converting enzyme (ACE) or mast cell chymase (CMA1) and plays an important role in inflammatory processes, although opinions differ as to which AngII-generating enzyme is primarily associated with AngII-mediated effects. ACE inhibitors have been shown to have a protective or healing effect on gastric ulcers and colitis in animal models, which could be related to the local expression of ACE. METHODS: The localisation of ACE and CMA1 was examined immunohistochemically in Helicobacter pylori gastritis, non-H. pylori gastritis, gastric ulcers and non-lesional gastric tissues. Using real-time qRT-PCR, ACE- and CMA1-mRNA expression in gastric cell lines were examined and changes in ACE levels after exposure to H. pylori or cytokines (IL-1beta, IL-6, IL-8, TNF, TGFbeta1) were quantified. RESULTS: ACE and CMA1 were not expressed in the non-lesional foveolar epithelium. Cytoplasmic staining for ACE in fundic chief cells, and apical membranous expression of ACE in the mucin-secreting cells of the antral and pyloric region was observed. ACE was found in endothelial cells of the gastric ulcer granulation tissue and CMA1 was strongly expressed in mast cells. ACE but not CMA1 was expressed in the MKN28, N87 and MKN45 gastric cell lines, and ACE mRNA expression was regulated by both H. pylori and the cytokines. CONCLUSIONS: ACE in the gastric mucosa and the microvasculature of granulation tissue may represent a novel therapeutic target for the promotion of healing processes in gastritis and ulceration using ACE inhibitors or AT1R antagonists.

Proteomics of pancreatic cancer.

Pancreatic cancer is a devastating disease, with a mortality rate almost identical with its incidence. Late diagnosis and limited therapeutic options make early detection of pancreatic cancer a pressing clinical problem. In this context, the investigation of the pancreatic cancer proteome has recently gained considerable attention because profiles of proteins may be able to more accurately identify disease states, such as cancer. Recent pancreatic cancer proteome studies may be categorized into basic studies cataloguing the pancreatic proteome, studies investigating differential protein expression patterns, and studies searching for proteome-based biomarkers for early cancer detection and differentiation. Although these studies clearly demonstrate that a range of biological samples are suitable for proteomic analyses, comparison of different studies is problematic due to the diversity of methodologies, sample sources, and characterization of patient populations. Reproducibility between studies has rarely been investigated, and no investigation has compared the different methods of proteomic research. The results of this review have shown that more stringent requirements concerning the design and the analysis of future studies should be implemented. These include an adequate patient number, obligatory histological examination of tissues, appropriate control groups, identification of proteins and peaks, validation of differential expression using independent cohorts and/or a second methodology, and, finally, demonstration of result reproducibility. This will hopefully lead to the discovery of prognostic and predictive biomarkers that help to improve prognosis of pancreatic cancer patients.

Differential expression and localisation of gasdermin-like (GSDML), a novel member of the cancer-associated GSDMDC protein family, in neoplastic and non-neoplastic gastric, hepatic, and colon tissues.

AIM: Gasdermin-like (GSDML) is a novel member of the cancer associated gasdermin-domain containing (GSDMDC) protein family. The GSDMDC family has been linked to cancer development and progression, and this is the first study analysing the expression and intracellular localisation of GSDML. METHODS: GSDML gene transcription was analysed using quantitative real-time RT-PCR. Anti-peptide antibodies against GSDML were developed in rabbits, and an in vitro transcription-translation reaction was used to verify specificity. The Protein-G affinity purified antibodies were used in immunohistochemistry and immunoblotting on hepatocellular, gastric, and colorectal carcinomas and non-lesional tissues. RESULTS: The GSDML gene was transcribed in human gastric, liver and colon cell lines, carcinomas and non-lesional tissues. The GSDML protein was localised to the cytoplasm of cells in both tumour and non-lesional tissues. The GSDML protein splicing variants range in molecular weight from 35 to 50 kDa, and the expression profile varies between tumour and non-tumour. A distinctive vesicular staining pattern was exhibited by GSDML in the apical region of gastric chief cells and colonic surface mucous cells, and the basal region of neuroendocrine cells. CONCLUSION: GSDML may be a secretory or metabolic product involved in a secretory pathway, and changes in the regulation of GSDML splicing variant transcription and translation may be seen in the development and/or progression of gastrointestinal and hepatic cancers.

Expression of the local angiotensin II system in gastric cancer may facilitate lymphatic invasion and nodal spread.

PURPOSE: In this study we investigated the putative pathophysiological mechanism, by which angiotensin converting enzyme (ACE), and angiotensin II receptor (ATR) type 1 and 2 might contribute to cancer progression and lymph node metastasis in gastric cancer. MATERIALS AND METHODS: Local expression of ACE, AT1R and AT2R was investigated immunohistochemically in non-lesional tissue, primary tumors and lymph node metastases from 45 gastric cancer patients. The distribution of the ACE genotypes was studied in gastric cancer cell lines. In vitro cell proliferation, apoptosis and invasion assays were carried out in the presence of ACE, AT1R and AT2R inhibitors. RESULTS: ACE and AT2R were significantly upregulated in tumors and metastases, and expressed in the lymph node metastases of 26 (58%) and 40 (89%) gastric cancer patients, respectively. AT1R expression was higher in all tissues of metastatic cancers than in previous investigations. ACE, but not AT1R or AT2R, occasionally exhibited an increased expression in tumor cells directly surrounding lymph follicles. All three possible combinations of the ACE gene insertion/deletion polymorphism were found in gastric cancer cell lines, i.e., the DD- (AGS, MKN28), the II- (MKN45) and the ID-genotype (N87). ACE, AT1R and AT2R inhibition resulted in a significantly increased proliferation and a significant reduction in invasive ability of the N87 and MKN45 cell lines, with N87 exhibiting reduced apoptosis. CONCLUSIONS: Our study provides evidence of the expression of the local angiotensin II system in lymph node metastases, and that ACE-, AT1R- and AT2R-activity promotes tumor cell invasion.

The gene polymorphism of the angiotensin I-converting enzyme correlates with tumor size and patient survival in colorectal cancer patients.

We studied the putative significance of angiotensin I-converting enzyme (ACE) in colorectal cancer (CRC) biology. Local expression of ACE was investigated by quantitative reverse transcription-polymerase chain reaction and by immunohistochemistry in CRCs and adenomas. ACE insertion (I)/deletion (D) polymorphism was studied in 141 CRC patients and 189 controls. ACE mRNA was upregulated in CRCs compared to corresponding nonlesional tissues (2.5-fold; P = .009). ACE protein was more commonly expressed in adenomas [17 (81%)] and cancer epithelial cells [22 (100%)] than in corresponding non-neoplastic crypt and surface epithelium [2 (10%) and 2 (9%), respectively]. Thirty-seven CRC patients (26%) carried II genotype, 69 (49%) carried ID genotype, and 35 (25%) carried DD genotype. The distribution of the genotypes did not differ from that of controls. Female CRC patients more commonly carried the ID genotype and less frequently the II and DD genotypes compared with male patients (P = .033). Men heterozygous or homozygous for the D-allele had larger tumors compared to carriers of the II genotype (P < .01). Women homozygous for the D-allele lived longer than carriers of the ID and II genotypes. Our study shows that ACE is differentially expressed in CRCs and that gene polymorphism is associated with gender-specific differences in primary tumor size and patient survival.

The angiotensin II/angiotensin II receptor system correlates with nodal spread in intestinal type gastric cancer.

We aimed to substantiate the putative significance of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) for gastric cancer biology by investigating the correlation of their expression with various clinicopathologic variables and patient survival. Local expression of AT1R, AT2R, and angiotensin-converting enzyme (ACE) was investigated by immunohistochemistry in tumor and corresponding nontumor specimens obtained from 100 patients with gastric cancer, and compared with the ACE insertion/deletion gene polymorphism. AT1R and AT2R were found in the tumor epithelial cells of 26 (26%) and 95 (95%) patients, respectively. AT1R was significantly more prevalent (P < 0.001) in intestinal type gastric cancer than in diffuse type gastric cancer. In intestinal type gastric cancer, its expression correlated with the N category (P = 0.009) and the International Union Against Cancer tumor stage (P = 0.024). AT1R+ intestinal type gastric cancers had a larger number of lymph node metastases (P = 0.026), a higher International Union Against Cancer tumor stage (P = 0.032), and a shorter survival time (P = 0.009) than AT1R- tumors. Multivariate analysis with lymph nodes as a dependent variable showed that AT1R status and ACE-I/D gene polymorphism are independent risk factors. Irrespective of the genotype, AT1R+ gastric cancers had a relative risk of lymph node metastases of 4.40 (95% confidence interval, 1.30-14.86). When the ACE genotype was included, the relative risk of having lymph node metastases increased considerably in AT1R+ tumors being heterozygous or homozygous for the ACE D allele (odds ratio, 19.00; 95% confidence interval, 1.45-248.24). Our study shows that AT1R and AT2R are expressed locally in gastric cancer and that the combination of AT1R expression and ACE I/D gene polymorphism correlates with nodal spread in intestinal type gastric cancer.

Protective role of heat shock protein 27 in gastric mucosal injury.

Heat shock protein 27 (HSP27) mediates cytoprotective effects through its function as a molecular chaperone and through the phosphorylation-dependent stabilization of actin filaments. The role of HSP27 in gastric ulcer formation and healing is, however, unknown. The expression of HSP27 was studied in human gastric tissue specimens obtained from patients with gastric ulcers and from healthy Helicobacter pylori-negative individuals, who received low-dose aspirin, rofecoxib, and the combination in a prospective study. The susceptibility of the gastric mucosa to indomethacin-induced lesions was further studied in transgenic mice overexpressing human HSP27 (Tg(huHSP27)) and compared with wild-type mice (Wt). The expression of HSP27, COX-1, and COX-2 was investigated in Tg(huHSP27) mice and Wt mice by immunohistochemistry and western blot analysis. While no specific changes in HSP27 expression were found following exposure of healthy human gastric mucosa to oral administration of aspirin or refocoxib, chronic gastric ulcers showed strong HSP27 expression at the ulcer base and margins. Here it was expressed by granulation tissue and regenerating surface epithelium. In Tg(huHSP27) mice, overexpression of HSP27 led to a significant decrease of indomethacin-induced erosions and ulcers compared with Wt mice. COX-1 and COX-2 levels did not change. HSP27 is involved in chronic gastric ulcer repair mechanisms in humans, while overexpression of human HSP27 in gastric epithelial cells in mice reduces the susceptibility to non-steroidal anti-inflammatory drug (NSAID)-induced gastric ulceration. This indicates that HSP27 expression is critical for mucosal protection in the stomach.

The number of lymph node metastases in gastric cancer correlates with the angiotensin I-converting enzyme gene insertion/deletion polymorphism.

PURPOSE: In the present study, we aimed to substantiate the putative significance of angiotensin I-converting enzyme (ACE) on gastric cancer biology by investigating the influence of its gene polymorphism on gastric cancer progression. EXPERIMENTAL DESIGN: Genomic DNA was purified from peripheral blood mononuclear cells or tissue specimens. Amplified ACE gene fragments were separated on agarose gels. D or I alleles were identified by the presence of 190- or 490-bp fragments, respectively. Local expression of ACE was investigated by immunohistochemistry. RESULTS: Twenty-four of 113 (21%) gastric cancer patients had the II, 57 (51%) the ID, and 32 (28%) the DD genotype. The distribution of the ACE genotypes did not differ significantly from the control group of 189 patients without gastric cancer. However, the ACE genotypes correlated with the number of lymph node metastases and the Unio Internationale Contra Cancrum (UICC) tumor stage. Patients with the II genotype had a highly significantly smaller number of lymph node metastases (P < 0.001) and a significantly lower UICC tumor stage (P = 0.01) than patients with the DD genotype. No correlation was found between tumor type, tumor location, local tumor growth, distant metastases, and the ACE genotype. The expression of ACE in gastric cancer was investigated by immunohistochemistry in 100 of 113 patients. ACE was expressed by endothelial cells in all (100%) specimens and by tumor cells in 56 (56%) specimens. CONCLUSIONS: Our study shows that ACE is expressed locally in gastric cancer and that the gene polymorphism influences metastatic behavior.

Increased expression of ADAM family members in human breast cancer and breast cancer cell lines.

PURPOSE: ADAMs (A Disintegrin and Metalloprotease) are multifunctional, membrane-bound cell surface glycoproteins, which have numerous functions in cell growth, differentiation, and motility. We wished to investigate the expression of ADAM 9, 10, 12, 15, and in human breast cancer. METHODS: Expression of ADAMs was determined in breast cancer specimens and the corresponding non-neoplastic breast tissue from 24 patients, and in the MCF-7 and MDA-MB 453 breast cancer cell lines via quantitative RT-PCR and immunohistochemistry. The effects of anti-ADAM antibodies on cell proliferation were assessed by measuring DNA-synthesis. RESULTS: Breast cancer tissue samples showed increased mRNA expression of ADAM 9, 12, and 17, whereas ADAM 10 and 15 were not differently expressed. Protein expression was studied by immunohistochemistry. All ADAMs were expressed in MCF-7 and MDA-MB453 cell lines, with the highest expression levels being observed for ADAM 9, 12, and 17. Application of anti-ADAM 15 and anti-ADAM 17 antibodies significantly inhibited the proliferation of both MCF-7 and MDA-MB453 breast cancer cell lines. In contrast, the growth of MCF-7 cells appeared to be stimulated by the administration of anti-ADAM 12 antibody. CONCLUSION: The results of this study suggest that ADAMs are differentially expressed in human breast cancer and are capable of modulating tumour cell growth.

The disintegrin-metalloproteinases ADAM9, ADAM12, and ADAM15 are upregulated in gastric cancer.

The ADAMs (A disintegrin and metalloproteinase) are a family of cell-surface membrane glycoproteins, whose multidomain structure enables diverse roles in a wide range of cellular processes. Accumulating evidence associates an increased expression of individual ADAM family members with various types of cancer, and we investigated the possible involvement of ADAM9, 12 and 15 in the pathogenesis of gastric cancer (GC). Using immunohistochemistry and quantitative RT-PCR, we examined the transcription and expression pattern of ADAM9, 12, and 15 in GCs and the corresponding non-tumor tissue, and in GC cell lines (AGS, MKN45, MKN28, NCI-N87, KATOIII). All three ADAMs were found to be significantly upregulated in GC compared to non-neoplastic foveolar epithelium, with ADAM12 expression being higher in intestinal- than in diffuse-type tumors. In vitro proliferation assays were used to evaluate the effects of ADAM-specific antibodies on the growth of GC cell lines. The administration of anti-ADAM9 and anti-ADAM15 antibodies inhibited cell growth, whereas anti-ADAM12 enhanced the proliferation of the GC cell lines. ADAM9, 12 and 15 are implicated in the malignant growth of GC cells, perhaps via the interaction with adhesion molecules, or the proteolytic 'shedding' of signaling molecules and the consequent transactivation of their receptors, such as the epithelial growth factor receptor and its ligands. The resultant modulation of the tumor-host interface may contribute to the pathogenesis, development or progression of gastric cancer.

The ectopeptidases CD10, CD13, CD26, and CD143 are upregulated in gastric cancer.

Due to their extracellular orientation, the ectopeptidases CD10, CD13, CD26, and CD143 have numerous functions, including the post-secretory processing of the neuropeptides and peptide hormones involved in the regulation of growth and differentiation in the gastrointestinal tract. We investigated the transcription and expression pattern of these four ectopeptidases in gastric carcinomas (GC), the corresponding non-neoplastic epithelium, a selection of lymph node metastases (LNM), and the MKN28, AGS, NCI-N87, KATO III gastric cancer cell lines. The gastric foveolar epithelium did not express CD10, CD13, or CD143, but the intestinal metaplasia demonstrated strong immunoreactivity at the brush border for all four ectopeptidases. CD10, CD13, and CD143 were significantly up-regulated in GCs and the lymph node metastases, confirming that they are important for the tumor cell biology. However, there is a lack of correlation between expression in intestinal metaplasia and tumor, as well as in tumor and LNM. Cell proliferation assays were performed with MKN28 and AGS, in which inhibition of CD10 significantly reduced the growth of both cell lines, and inhibition of CD13 significantly increased the proliferation of the AGS cells, indicating that the ability to degrade gastrointestinal peptides may play an important role in the pathobiology of gastric cancer.

Ectopeptidases are differentially expressed in hepatocellular carcinomas.

We investigated the expression pattern of neprilysin (CD10), aminopeptidase N (CD13) and angiotensin-I converting enzyme (CD143) in hepatocellular carcinomas (HCC), and their putative roles in hepatocarcinogenesis. Tissue samples were obtained from 31 patients with HCC. Tissue samples obtained from non-neoplastic liver, fetal livers and focal nodular hyperplasias (FNH) were used by comparison. Transcription and expression of CD10, CD13, and CD143 were studied by quantitative RT-PCR, Western blotting, and immunohistochemistry. Cell proliferation assays were performed with the C3A hepatoma cell line. The mRNA and protein of each of CD10, CD13 and CD143 were differentially expressed in HCCs. CD10 was decreased in HCCs as compared to non-neoplastic liver tissue, while CD13 and CD143 were mildly increased. In fetal liver and FNHs, the expression of CD10 was less intense than in the surrounding non-tumorous liver. The expression patterns of CD13 and CD143 in fetal livers and FNHs were similar to HCCs and were predominantly localized in bile canaliculi (CD13) and endothelial cells (CD143). CD10 and CD13 mRNAs were expressed by C3A cells and blocking either CD10 or CD13 ectopeptidase activity retarded cell growth significantly in vitro. We demonstrate that ectopeptidases are differentially expressed in HCCs and may have influence on tumor biology. Overall, expression of CD10 in non-neoplastic and neoplastic hepatocytes appears to correlate inversely with their state of proliferation or differentiation. CD13 shows a characteristic canalicular distribution pattern and may be important for cell polarization and bile compartmentalization in HCCs, while CD143 may influence angiogenesis.

Angiotensin I-converting enzyme (CD143) is down-regulated in focal nodular hyperplasia of the liver.

In surgical pathology, focal nodular hyperplasia (FNH) is sometimes difficult to diagnose, and liver cirrhosis (LC) and hepatocellular adenoma (HA) are often among the differential diagnoses. Recently, we found a reduced expression of angiotensin I-converting enzyme (CD143) in FNH compared with cirrhotic and noncirrhotic liver. Intrigued by this observation, we investigated the expression pattern of CD143 in FNH, LC, and HA and its possible diagnostic value. The expression of CD143 was studied by immunohistochemistry in 20 FNHs, 13 corresponding extralesional noncirrhotic liver parenchyma, 20 patients with LC, and four HAs. Endothelial cells were identified with antibodies directed against CD31 and CD34. CD31+, CD34+, and CD143+ sinusoidal endothelial cells were found in extralesional liver, LC, HA, and FNH. However, the number of CD143+ sinusoidal endothelial cells and the intensity of immunostaining were significantly reduced in FNH compared with extralesional liver, LC, and HA. The expression of CD143 was further assessed using a numerical scoring system ranging from 0 to 6. The mean immunoreactivity score for CD143 was 2.4 +/- 1.7 for sinusoidal endothelial cells in FNH, 5.7 +/- 0.6 in extralesional liver, 4.8 +/- 1.1 in LC, and 5.8 +/- 0.5 in HA. The differences between the mean immunoreactivity scores for CD143 were highly significant. The difference between FNH and extralesional liver was confirmed on transcriptional level by fluorescence-mediated real-time RT-PCR, which also showed a significantly decreased level of CD143 mRNA in FNH. Our study provides evidence that CD143 is down-regulated in FNHs and that the phenotype of endothelial cells lining the sinusoids in FNH differs from those in non-neoplastic liver, LC, and HA. The observed variations in expression patterns for CD143 might be of diagnostic use in surgical pathology.