Thymidylate synthase predicts for clinical outcome in invasive breast cancer

Thymidylate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Whether TS or DPD could be used as valuable parameters for 5-FU sensitivity in clinical patients are largely unknown. We analyzed TS and DPD expression in breast carcinomas to evaluate the clinicopathological significance of these enzymes in patients with invasive breast cancer receiving 5-FU-based chemotherapy. A total of 197 patients with invasive ductal carcinoma were included in our study. Both the TS and DPD expression were analyzed using immunohistochemical method for all the surgical samples. Sixty-three out of 197 (31.97%) patients are positive for TS expression, and 77 out of 197 (39.09%) patients are positive for DPD expression. TS expression was not correlated with DPD expression. Patients with TS-positivity had aggressive phenotype including large tumor size, low differentiation and nodal metastasis. DPD expression is not related with phenotype or prognosis. Multivariate analysis demonstrated that TS expression was an independent prognostic factor for both disease-free and overall survival. The current study demonstrated that TS but not DPD expression was associated with both progression and prognosis in breast cancer receiving 5-FU-based chemotherapy. TS expression in the primary tumor might be useful as a predictive parameter for the efficacy of 5-FU-based chemotherapy for breast cancer.     

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.     

The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas

PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.     

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.     

Orotate phosphoribosyltransferase expression level in tumors is a potential determinant of the efficacy of 5-fluorouracil

Although the intratumoral expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are known to affect the antitumor activity of 5-fluorouracil (5-FU), the importance of orotate phosphoribosyltransferase (OPRT) has remained unclear. This study investigated the relationship between intratumoral OPRT expression and the antitumor activity of 5-FU using human NCI60 cell lines with similar levels of TS and DPD messenger RNAs, as well as 31 tumor xenografts. The OPRT mRNA level was positively correlated with the 5-FU efficacy in these cell lines. In vitro, the 50% growth-inhibitory concentrations of 5-FU were closely correlated with the OPRT mRNA levels in cancer cell lines with similar levels of TS mRNAs when combined with a DPD inhibitor. Moreover, downregulation of OPRT with small-interfering RNA decreased the sensitivities of the cultured tumor cells to 5-FU. These results suggest that the OPRT expression level in tumors is an additional determinant of the efficacy of 5-FU.     

Claudin expression in breast cancer: High or low, what to expect?

The evaluation of claudins (CLDNs) expression pattern in tumours can be important to understand breast carcinogenesis. The study of CLDNs became more appealing since it was found that CLDN3 and CLDN4 are putative therapeutic targets for Clostridium perfrigens enterotoxin (CPE), as well as for monoclonal antibody-based therapy. Moreover, the recently characterized CLDN-low molecular subgroup of breast tumours increased the interest in these molecules. Based on these facts, our aim was to explore the pattern of expression of CLDNs among a large series of invasive breast carcinomas. We also analysed the correlation between the combinatorial expression of CLDN3/CLDN4 and classical prognostic factors and biological markers. In addition, we also compared the characteristics of tumours with low expression of CLDN3, CLDN4 and CLDN7, assessed by immunohistochemistry (IHC), and the ones from CLDN-low subgroup of tumours previously defined by genomic assays. The combinatorial analysis of the expression of CLDN3/CLDN4 showed a significant association between high CLDN3/CLDN4 levels and triple-negative tumours, as well as with worse patient outcome. This combined analysis may provide useful information for breast carcinomas, since these two CLDN members are putative therapeutic targets. Comparing tumours with low expression of CLDN3, CLDN4 and CLDN7 with tumours previously referred to as CLDN-low by genomic assays, we demonstrated that the single IHC evaluation of these three specific CLDNs is insufficient to identify the CLDN-low molecular subtype of breast tumours. The analysis of several other molecular markers, such as EMT (epithelial-to-mesenchymal transition) and CSC (cancer stem cell) markers should probably be added to improve the identification of this subgroup of tumours by IHC, which probably are enriched in carcinomas with metaplastic differentiation.     

Identification of BCOX1, a novel gene overexpressed in breast cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.     

SYK expression in human breast cancer

BACKGROUND: Syk (Splenic Tyrosine Kinase) is an intracellular receptor protein kinase involved in cell proliferation, differentiation and phagocytosis. It has been studied in T and B lymphocytes, NK cells and platelets. The strong expression of Syk in mammary gland prompted research into its potential role in mammary carcinogenesis. There have been very few studies about its role in breast cancer with conflicting results. This study aims to investigate the hypothesis that Syk expression is down-regulated in breast cancer compared with ANCT and the association between its expression and clinicopathological parameters. MATERIALS AND METHODS: mRNA was extracted from 48 breast cancer specimens. Relative Syk to ribosomal RNA expression was determined by RT-PCR and Taqman methodology. Mann-Whitney U test was used to examine the association between Syk expression in cancer and ANCT. Spearman's rank correlation test was used to examine the association between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion and clinical outcome. RESULTS: The median for the relative value of Syk expression was 0.17 and 0.18 (range: 0.12 - 0.56 and 0.0 - 1.77) for tumours and ANCT respectively. There was no significant association between Syk expression in cancers and ANCT (p= 0.598) nor between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion or prognosis. CONCLUSION: This study shows that Syk mRNA expression does not seem to vary between breast tumours and ANCT. Furthermore, we observed no significant association between Syk expression and clinicopathological parameters.     

An alpha2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas

The MUC1 mucin is expressed on the luminal surface of most simple epithelial cells but in carcinomas, especially those of the breast and ovary, MUC1 is upregulated and aberrantly glycosylated. MUC1 contains a large amount of O-linked glycans which, in the mucin expressed by normal mammary epithelial cells, consist mainly of core 2 based structures carrying polylactosamine chains. However, the mucin expressed by breast carcinomas has shorter side-chains, often consisting of sialylated core 1 (Galbeta1-3GalNAc). in situ hybridization of primary breast tissue showed that a sialyltransferase (ST3Gal I), responsible for adding sialic acid to core 1 thereby terminating chain extension, is elevated in primary breast carcinomas when compared to normal or benign tissue. Furthermore, the level of mRNA expression encoding ST3Gal I is correlated to the intensity of staining seen with the antibody SM3, which specifically recognises underglycosylated, tumour associated MUC1. Thus, the aberrant glycosylation of MUC1 seen in breast carcinomas appears to be due, at least in part, to the elevation of ST3Gal I.     

Absence of differentiation-related expression of keratin 10 in earlystages of vulvar squamous carcinoma

Using specific monoclonal antibodies (DE-K10 and DE-SCK respectively), the expression of some differentiation-related epidermal keratins was studied in 38 human vulvar squamous carcinomas. In the epidermis, expression of keratin 10 (K10) strictly paralleled the extent of differentiation; it was absent in the basal layer, appeared in the first suprabasal layers and increased in concentration towards the granular layer. However, K10 was rarely detected (1 case out of 12) in early stages of vulvar squamous carcinomas (tumours less than 2 cm, clinical stage I) regardless of the tumour grade. In larger and more advanced tumours (greater than 2 cm, clinical stages II and III), K10 was detected in 21 out of 26 cases. Its expression appeared to be related to maturation of malignant keratinocytes, being preferentially detected in more-differentiated parts. Occasionally however, cells that did not show histological signs of keratinisation were also K10-positive. Modified stratum corneum keratins (recognized specifically by monoclonal antibody DE-SCK) were detected in the most keratinized areas (horn pearls and their close vicinity) of some K10-positive tumours, i.e., in a pattern close to their normal expression in terminally differentiated epidermal cells. These data suggest differences in the regulation of K10 expression during the differentiation processes in the normal keratinising squamous epithelium and in squamous carcinomas. While the normal pattern of vulvar epithelial differentiation is accompanied by an increasing expression of K10, malignant keratinocytes, also when these are histologically moderately or well differentiated, cease expressing this keratin in the early stages of tumour development.     

乳腺癌中丝／苏氨酸蛋白激酶Plk1mRNA的表达及其预后价值

目的：检测乳腺癌细胞和组织中丝／苏氨酸蛋白激酶Plk1基因mRNA的表达情况并分析其预后价值。方法：应用半定量RT-PCR方法分析3株人乳腺癌细胞和1株正常乳腺上皮细胞中Plk1基因mRNA的表达水平。同时分析84例乳腺癌及对应的癌旁正常乳腺上皮组织中Plk1mRNA的表达水平。统计学分析Plk1mRNA表达水平与乳腺癌患者年龄、肿瘤大小、组织分化程度、淋巴结转移状况、TNM分期和雌激素受体（ER）等临床病理参数之间的关系,以及与预后之间的关系。结果：Plk1基因mRNA在乳腺癌细胞中的相对表达水平显著高于其在正常乳腺上皮细胞中的相对表达水平（P值均小于〈0．05）。另外,Plk1mRNA在乳腺癌组织中平均表达水平（0．88±0．18）显著高于其在癌旁正常乳腺上皮组织中平均表达水平（0．22±0．10;P〈0．01）。统计学分析结果袁明：Plk1mRNA表达水平和乳腺癌患者的淋巴结转移状况及TNM分期密切相关（P=0．009或0．007）。Kaplan—Meier生存曲线分析结果表明：高Plk1mRNA表达水平的乳腺癌患者的5年无疾病进展率及总体生存率均显著低于低Plk1mRNA表达水平的乳腺癌患者（P=0．0026及0．0136）。COX模型的多因素预后分析结果表明：Plk1基因mRNA表达水平是乳腺癌患者的一个独立的预后因素（HR=4．764,95％CI：1．341-6．123,P=0．0025）。结论：Plk1在乳腺癌组织呈现高表达水平,其mRNA表达水平有望成为临床乳腺癌患者一个重要的预后判断分子指标。     

Distribution of Microsomal Epoxide Hydrolase in Humans: An Immunohistochemical Study in Normal Tissues, and Benign and Malignant Tumours

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.     

Changes in BRCA1 and BRCA2 expression produced by chemotherapeutic agents in human breast cancer cells

We have investigated the effects of chemotherapeutic agents such as adriamycin (ADR), camptothecin (CPT), mitomycin-C (MYC-C) and methotrexate (MTX) on the regulation of expression of the tumor susceptibility genes (BRCA1 and BRCA2), and the association of cell cycle progression in human breast cancer and normal breast epithelial cells. Results revealed that the mRNA and protein expression levels of BRCA1/2 were reduced by the treatment of chemotherapeutic agents used in the breast cancer cell lines tested, with ADR being the most effective. The regulation of the cell cycle was dose-dependent and low doses of ADR (1.5 microM) induced G2/M phase arrest whereas a late S phase arrest was observed with a higher dose of ADR (15 microM) in both breast cancer cells (MCF-7 and MDA-MB-231) tested. In addition, a negative correlation was observed between BRCA1/2 mRNA and expressions of the proteins with the cell cycle alterations being regulated by chemotherapeutic agents.     

Levels of expression of lipoxygenases and cyclooxygenase-2 in human breast cancer

Lipoxygenases and cyclooxygenase are key mediators of arachidonic acid metabolism. The eicosanoids metabolites from these oxygynases have been shown to regulate the growth and death of cancer cells. This study determined the level of expression of 5-, 12-, 15-lipoxygenase and cyclooxygenase-2 expression in a cohort of breast cancer patients and their correlation with clinical outcomes. Compared with normal breast tissues, tumour tissues exhibited a significantly higher levels of 12-lipoxygenase and cyclooxygenase-2 (P<0.05), and significantly lower level of 15-lipoxygenase (P=0.05). Lobular carcinomas had a higher level of cyclooxygenase-2 and lower level of 15-lipoxygenase than ductal carcinomas. The lowest level of 15-lipoxygenase was seen in TNM3 and TNM4 tumours and from patients who died of breast cancer. Levels of 12- and 5-lipoxygenases were also particularly high in tumours from patients who died of breast cancer. This study shows that human breast tumours aberrantly express lipoxygenases and cyclooxygenase-2 and that decreased level of 15-lipoxygenase and raised level of cyclooxygenase-2 and 12-lipoxygenase has prognostic value in patients with breast cancer.     

Thymidine phoshorylase expression in breast cancer: the prognostic significance and its association with other angiogenesis related proteins and extracellular matrix components

Thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor, stimulates chemataxis of endothelial cells and is involved in the angiogenesis of human solid tumours. In this study we investigated tissue sections from 93 breast carcinomas for the immunohistochemical expression of thymidine phosphorylase protein and in relationship to several clinicopathological parameters. The possible relationship to tumour neovascularization, VEGF expression, extracellular matrix components (tenascin, fibronectin, collagen type IV and laminin) and cathepsin D was also estimated. Nuclear and/or cytoplasmic TP expression was observed in tumour cells. Immunoreactivity was also often present in the stroma, endothelium and tumour-associated macrophages. High cytoplasmic TP expression, was observed in 35.5%, moderate in 30.1%, mild in 18.3%, while 16.1% of the cases were negative for TP expression. Moderate and high nuclear TP expression was observed in 30.1% of the tumours, low in 43%, while 26.9% did not show nuclear TP expression. High tumour stroma TP expression was expressed in 23.7% of the cases, moderate in 21.5%, mild in 45.2%, while 9.7% did not show stromal TP expression. TP expression did not correlate with the conventional clinicopathological features as well as with the microvessel density and the VEGF expression. Patients with high levels of tumour cell TP expression were significantly associated with a favorable outcome in univariate method of analysis. A positive correlation of TP expression with Cathepsin D expression was noticed. In addition, tumour cell TP expression was correlated with the extracellular matrix component tenascin, while stromal cell TP expression was correlated with the growth fraction of the tumour. Our data suggests that TP expression does not seem to affect directly the neovasculatur of breast carcinoma, although it seems to be implicated in the remodeling of breast cancer tissue, through the interaction with other extracellular matrix components or proteolytic enzymes. In addition, tumour cell TP expression could be considered as a prognostic indicator of breast cancer patients.