Analysis of normal epithelial cell specific-1 (NES1)/kallikrein 10 mRNA expression by in situ hybridization, a novel marker for breast cancer.

PURPOSE: Normal epithelial cell specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary and prostate epithelial cells, but the expression of NES1 mRNA and protein is markedly reduced in established breast and prostate cancer cell lines although the NES1 gene is intact. Here, we wished to assess whether NES1 expression is down-regulated in primary breast cancers. EXPERIMENTAL DESIGN: We developed and used an in situ hybridization technique with an antisense NES1 probe to detect NES1 mRNA in sections of normal breast specimens, typical and atypical ductal hyperplasia, ductal carcinoma in situ, and infiltrating ductal carcinoma. RESULTS: All of the 30 normal breast specimens showed high NES1 expression. Notably, 18 (75%) of 24 typical and atypical breast hyperplasia specimens showed high NES1 expression, with weak-to-moderate expression in 6 (25%). Significantly, 13 (46%) of 28 ductal carcinoma in situ specimens lacked NES1 expression, and the remaining 15 (54%) showed weak-to-moderate expression. Finally, 29 of 30 (97%) infiltrating ductal carcinoma grades I-III samples lacked NES1 mRNA, with weak expression in the remaining one sample. CONCLUSIONS: Our results demonstrate that NES1 mRNA is expressed in normal breast tissue and benign lesions, with loss of NES1 expression during tumor progression. We suggest that NES1 expression may serve as a molecular tool in the study of breast cancer progression. Studies with larger series of specimens should help assess whether NES1 expression can be a diagnostic and/or prognostic marker in breast and other cancers.     

Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin

Curcumin has anti-inflammatory, antiproliferative, and antitumor effects. To understand the chemopreventive mechanism of curcumin against human malignancies, the cellular and molecular changes induced by this agent in human mammary epithelial (MCF-10A) and breast carcinoma (MCF- 7/TH) cell lines were investigated. The human multidrug- resistant breast cancer cell line was 3.5 fold more sensitive to curcumin than the mammary epithelial cell line. Even though both cell lines accumulated a similar amount of curcumin, a significantly higher percentage of apoptotic cells was induced in breast cancer cells compared to a very low percentage of apoptosis in mammary epithelial cells. Incubation of breast cancer cells with 20 and 40 microM curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively. Curcumin treatment caused a reduction in the expression of Ki67, PCNA, and p53 mRNAs in breast cancer cells. The human mammary epithelial cell line showed a down-regulation of p21 mRNA and an up-regulation of Bax mRNA expression with curcumin treatment. The results suggest that apoptosis is involved in the curcumin-induced inhibition of tumor cell growth, and genes associated with cell proliferation and apoptosis may be playing a role in the chemopreventive action of curcumin.     

Loss of immunoreactivity for human calmodulin-like protein is an early event in breast cancer development

Cell proliferation requires calmodulin, a protein that regulates calcium-dependent enzymes involved in signal transduction pathways in eukaryotic cells. Calmodulin-like protein (CLP) is found in certain epithelial cell types, including normal breast epithelium, and, although it closely resembles calmodulin in amino acid sequence, CLP interacts with different proteins than does calmodulin. The observation that CLP mRNA expression is dramatically reduced in transformed breast epithelial cells led to two hypotheses: (1) CLP helps to maintain the differentiated state in epithelial cells; and (2) downregulation of CLP accompanies malignant transformation of breast epithelial cells. The objective of this study was to determine if the expression of CLP in human breast cancer specimens is reduced in comparison to its expression in normal breast tissue. Eighty human breast cancer biopsy specimens were analyzed immunohistochemically for CLP expression by using a polyclonal rabbit antihuman CLP antibody. CLP expression was reduced in 79% to 88% of the invasive ductal carcinoma and lobular carcinoma specimens and in a similar fraction of the ductal carcinoma in-situ specimens, compared with normal breast specimens. None of the breast cancer specimens showed an increase in CLP expression. These findings support the hypotheses that CLP behaves as a functional tumor suppressor protein and is downregulated early in breast cancer progression.     

Two monoclonal antibodies selective for human mammary carcinoma

Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with the MCF-7 human mammary carcinoma cell line. Among hybridomas, two (3B18 and 15A8) were selected and cloned. Hybridoma 3B18 produces kappa-IgG1 antibodies that react with a cytoplasmic component of MCF-7 cells. In immunoperoxidase assays, 3B18 reacts with 27 of 31 specimens of human mammary carcinoma. It reacts most consistently with poorly differentiated and infiltrating ductal breast cancers, but it also reacts with isolated cells in 3 of 5 benign mammary pathological lesions with a variable distribution. The antibody does not react with normal mammary epithelium. It does not react with any normal human tissues, and it reacts with only one of 19 other cancers tested. Hybridoma 15A8 produces kappa-IgG1 antibodies that react with the surface membranes of the cells of two human breast cancer cell lines but not with a human fibroblast cell line. In immunoperoxidase assays, the antibody reacted with 28 out of 31 human mammary carcinomas. The antibody also reacts more weakly with normal human epithelial cells of breast, renal proximal tubule, skin, esophagus, and salivary gland, but no other normal tissue. The antibody was unreactive with 14 of 18 other malignant tissues tested. Since 3B18 and 15A8 detect antigens found predominantly in human mammary carcinomas and, possibly, distinguish overlapping categories of human mammary carcinomas, they may prove useful in determining the cellular lineage from which human mammary carcinomas arise, or they may have other clinical applications in breast cancer.     

乳腺癌还氧合酶-2表达的研究现状

还氧合酶-2(COX-2)过表达与很多肿瘤发生发展有关。最近研究显示COX-2在乳腺癌细胞株和组织标本中表达增加,提示COX-2可能直接参与乳腺癌的形成,并且与预后有关。因此,应用COX-2抑制剂治疗乳腺癌成为目前的研究热点。     

KAI1 protein is down-regulated during the progression of human breast cancer.

The KAI1 gene was identified as a metastasis suppressor gene for human prostate cancer. Recently, we showed that KAI1 mRNA levels were higher in an immortal, normal-like breast epithelial cell line and nonmetastatic breast cancer cell lines but lower substantially in highly metastatic breast cancer cell lines. In this study, we examined KAI1 protein expression in breast cancer cell lines by Western blot and immunohistochemical study. KAI1 protein levels paralleled KAI1 mRNA levels and were inversely correlated with the metastatic potential of breast cancer cells. Furthermore, we examined KAI1 protein expression immunohistochemically in specimens from 81 patients with breast cancer and then correlated the findings with the clinical and histopathological parameters of the patients. High levels of KAI1 protein expression were found in normal breast tissues and noninvasive breast cancer (ductal carcinoma in situ). In contrast, KAI1 expression was reduced in most of the infiltrating breast tumors. We found that, in general, more malignant tumors demonstrated significantly lower KAI1 expression (P = 0.004). Additionally, among 29 specimens demonstrating multiple stages of malignancy within a single specimen, 23 demonstrated significant differences in KAI1 expression between benign breast tissue, ductal carcinoma in situ, and invasive carcinoma. The higher the incidence for malignancy within a given specimen, the lower the KAI1 expression (P < 0.001). These data suggest that in advanced breast cancer, KAI1 expression is down-regulated. Therefore, KAI1 may be a potentially useful indicator of human breast cancer progression.     

Localisation of COX-2 protein is different in breast ductal carcinoma and adjacent non-tumour ductal epithelium

Introduction A number of findings suggest that cyclooxygenase-2 (COX-2) is overexpressed in breast tumours. However, there is a lack of consensus in the literature regarding the pattern of expression of this protein in invasive breast ductal carcinoma and in the adjacent non-tumour ductal epithelium. This study compares the expression of COX-2 mRNA and protein in breast ductal carcinoma relative to non-tumour breast tissue. Material and methods We analysed the expression of COX-2 mRNA by quantitative PCR, and COX-2 protein by immunohistochemistry in invasive ductal carcinoma as well as in non-tumour adjacent ductal epithelium from 54 breast biopsies diagnosed as being invasive ductal carcinoma. As control, we analysed expression of COX-2 protein by immunohistochemistry in surgically-resected benign breast lesions. Results Our results show that COX-2 mRNA and protein are overexpressed in non-tumour ductal epithelium compared with invasive ductal carcinoma. However, the pattern of the protein expression is different in tumour and non-tumour tissue: COX-2 protein is expressed predominantly in the membrane of the non-tumour ductal epithelium (including in benign breast lesions) while, in invasive ductal carcinoma cells, it is localised in the cytoplasm. Conclusions The non-tumour ductal epithelium adjacent to invasive ductal carcinoma shows a higher COX-2 expression than does the invasive ductal carcinoma. However, the different localisation of the immunohistochemically-detected protein suggests a possible post-translational regulation of the protein.     

TRAF4蛋白在乳腺癌组织中的表达及意义

背景与目的:对肿瘤坏死因子受体相关因子4(tumor necrosis factor receptor-associated factor 4,TRAF4)在乳腺癌中表达的研究存在争议.本研究探讨TRAF4在正常乳腺组织、乳腺癌组织及不同侵袭能力乳腺癌细胞系中的表达情况.方法:应用免疫组化方法检测TRAF4在70例乳腺癌组织和14例正常乳腺组织中的表达.应用Western blot方法检测TRAF4在乳腺癌细胞系MDA-MA-231(高侵袭)和MCF-7(低侵袭)中的表达.结果:TRAF4在正常乳腺组织中呈浆、核阳性表达.其浆阳性率在正常乳腺组织(78.57%)、非浸润性导管癌(88.57%)和浸润性导管癌(91.43%)中逐渐增高,但差异无统计学意义(P＞0.05).而核阳性率在正常乳腺组织(64.28%)中显著高于非浸润性导管癌(28.57%,P＜0.01),在非浸润性导管癌中高于浸润性导管癌(5.7%,P＜0.05).TRAF4总蛋白在乳腺癌高侵袭细胞系中的表达量高于低侵袭细胞系,但差异无统计学意义(P＞0.05).结论:TRAF4在乳腺癌细胞核中表达明显降低,与乳腺癌侵袭性相关.     

A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells

Introduction

Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.

Methods

To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.

Results

We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E₂. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.

Conclusion

Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.     

The prostaglandin E2 receptor EP2 is required for cyclooxygenase 2-mediated mammary hyperplasia

Expression of cyclooxygenase 2 (COX-2) in breast cancer correlates with poor prognosis, and COX-2 enzyme inhibitors reduce breast cancer incidence in humans. We recently showed that COX-2 overexpression in the mammary gland of transgenic mice induced mammary cancer. Because prostaglandin E2 (PGE2) is the major eicosanoid and because the EP2 subtype of the PGE2 receptor is highly expressed in the mammary tumors, we tested if this G protein-coupled receptor is required for tumorigenesis. We crossed the MMTV-COX-2 transgenic mice with Ep2-/- mice and studied tumor development in bigenic mice. Lack of EP2 receptor strongly suppressed COX-2-induced effects such as precocious development of the mammary gland in virgins and the development of mammary hyperplasia in multiparous female mice. Interestingly, the expression of amphiregulin, a potent mammary epithelial cell growth factor was down regulated in mammary glands of Ep2-/- mice. Total cyclic AMP (cAMP) levels were reduced in Ep2-/- mammary glands suggesting that PGE2 signaling via the EP2 receptor activates the Gs/cAMP/protein kinase A pathway. In mammary tumor cell lines, expression of the EP2 receptor followed by treatment with CAY10399, an EP2-specific agonist, strongly induced amphiregulin mRNA levels in a protein kinase A-dependent manner. These data suggest that PGE2 signaling via the EP2 receptor in mammary epithelial cells regulate mammary gland hyperplasia by the cAMP-dependent induction of amphiregulin. Inhibition of the EP2 pathway in the mammary gland may be a novel approach in the prevention and/or treatment of mammary cancer.     

Isolation of genes overexpressed in freshly isolated breast cancer specimens

A number of approaches have been used to identify genes important in breast cancer. In one approach the genes already shown to be involved in other tumors, such as p53 and Her2neu, were examined. A second approach examined genes detected through genetic screening of families with a high incidence of breast cancer, for example, BRCA-1 and BRCA-2. We used a third approach, subtractive hybridization, to identify and clone genes that were preferentially expressed in breast cancer cells compared to normal mammary epithelium. Instead of analyzing breast cancer cell lines, we examined fresh human breast cancer specimens. By subtracting normal mammary epithelial cDNA from breast cancer cDNA, we were able to clone several genes overexpressed in breast cancer. Two of these genes, L19 and MLN70, were previously reported to be overexpressed in breast cancer. Three of these genes, L19, L34, and MLN70, were localized to a region on chromosome 17 where Her2/neu and BRCA-1 are found. In addition, we isolated a gene we call breast cancer associated gene-1 that was expressed almost exclusively in fresh breast cancer tissue and not in normal mammary epithelium or breast cancer cell lines. We were unable to detect expression of breast cancer associated gene-1 in cell lines from melanoma, renal cell carcinoma, lymphoma, or leukemia. The full-length sequence from two separate breast cancer specimens revealed one amino acid difference compared to the sequence from normal breast epithelial tissue. Further studies are necessary to determine whether these genes contribute to breast cancer development or can be used as therapeutic targets.     

Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer

Expression of Secreted frizzled related protein 1 (SFRP1), a recently identified tumor suppressor gene encoding a WNT signaling antagonist, has been found to be frequently down-regulated in breast cancer and is associated with disease progression and poor prognosis. Here, we investigated the role of epigenetic silencing of SFRP1 in breast cancer cell lines and primary breast tumors. Through analyses by methylation-specific PCR and bisulfite sequencing, promoter methylation of SFRP1 was detected in 88% (7/8) of breast cancer cell lines, 17% (1/6) of grade 1 of ductal carcinoma in situ (DCIS), 69% (9/13) of grade 2 and 3 of DCIS, 68% (19/28) of invasive ductal carcinomas (IDC) and 33% (6/18) of lobular carcinomas but not in any (0/14) of normal mammoplasty specimens and mammary epithelial organoids examined. Real-time RT-PCR studies indicated that loss or downregulation of SFRP1 expression in tumors is frequently associated with promoter hypermethylation. In addition, breast cancer cell lines with SFRP1 promoter hypermethylation reexpressed SFRP1 mRNA after treatment with 5-azaC, implying that DNA methylation is the predominant epigenetic mechanism for SFRP1 gene silencing. These findings suggest that frequent downregulation of SFRP1 expression in breast cancer can be attributed, in large part, to aberrant promoter hypermethylation in conjunction with or without histone deacetylation. Based on the frequency of tumor-specific hypermethylation in this gene, SFRP1 could provide a valuable marker for breast cancer.     

Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy

It is well known that dysfunction of the apoptotic pathway confers apoptosis resistance and results in a low sensitivity of human cancer cells to therapeutic agents. A novel strategy to overcome the resistance is to target the apoptotic pathway directly. To identify molecular targets in the apoptotic pathway that are differentially regulated in cancer and normal cells, we have examined the levels of apoptotic effectors and inhibitors in human tumor and normal cell lines as well as in cancer and normal tissues. These include three pancreatic cancer lines (BXPC-3, MIA PaCa-2, and Panc-1), four breast cancer cell lines (MDA-MB-231, MDA-MB-435, MDA-MB-361, and MCF-7), and colon carcinoma line (SW620). Additionally, breast carcinoma tissue specimens were examined. Compared with normal human fibroblast and mammary epithelial cell lines, we detected high basal levels of caspase-3 and caspase-8 activities and active caspase-3 fragments in the tumor cell lines and cancer tissues in the absence of apoptotic stimuli. Furthermore, the tumor cells expressed high levels of survivin and XIAP, two members of the inhibitor of apoptosis (IAP) protein family. When the activity of these IAPs was blocked by expression of dominant-negative mutant survivin (survivinT34A) and XIAP-associated factor 1, respectively, apoptosis was induced in tumor but not normal cell lines. Moreover, down-regulation of both survivin and XIAP significantly enhanced tumor-cell apoptosis as compared with inhibition of either survivin or XIAP alone. These results suggest that up-regulated IAP expression counteracts the high basal caspase-3 activity observed in these tumor cells and that apoptosis in tumor cells but not normal cells can be induced by blocking IAP activity. Therefore, IAPs are important molecular targets for the development of cancer-specific therapeutic approaches.     

Comparison of the effects of differentially sulphated bovine kidney- and porcine intestine-derived heparan sulphate on breast carcinoma cellular behaviour

Heparan sulphate is a sulphated glycosaminoglycan and is able to bind to and regulate the activity of many growth and signalling factors. We have previously shown that its expression is correlated with tumour grade and cell proliferation in breast phyllodes tumours. In this study, we examined the use of heparan sulphate as a biomarker of invasive ductal carcinoma and the effects of differentially sulphated heparan species on breast cancer cell behaviour. Immunohistochemistry using the 10E4 monoclonal antibody was carried out on 32 paraffin-embedded breast cancer specimens and paired non-cancerous breast tissues to compare the expression patterns of heparan sulphate. Upregulated expression of the sulphated 10E4 epitope in heparan sulphate was detected in both epithelial and stromal compartments of breast cancer compared with normal mammary tissues, with a 2.8X increase in immunoreactivity score. To determine the effects of differentially sulphated heparan sulphate molecules on breast cancer behaviour, cultured breast carcinoma cells were treated with chlorate, a competitive inhibitor of glycosaminoglycan sulphation, and two different heparan sulphate species. Inhibition of glycosaminoglycan sulphation resulted in a significant increase in cancer cell adhesion and a reduction in cell migration, together with upregulated expression of focal adhesion kinase and paxillin. Both porcine intestine- and bovine kidney-derived heparan sulphate species could block the change in cell adhesion. However, the former heparan sulphate species completely abolished, while the latter exacerbated, the chlorate-induced decrease in cell migration. The results show that heparan sulphate is a useful biomarker of breast invasive ductal carcinoma. Different sulphation patterns of heparan sulphate residues have differential effects in regulating breast cancer cellular behaviour, and this may be exploited to develop heparan sulphate into a useful target for treatment of breast carcinoma.