Role In Breast Carcinogenesis Research Articles

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

The interaction between aromatase, metalloproteinase 2,9 and cd44 in breast cancer

This study intends to verify the expression levels and correlation of aromatase, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and CD44 in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) when both are found in the same breast. One hundred and ten cases were evaluated by tissue microarray (TMA) and immunohistochemically screened with anti-aromatase polyclonal antibodies, anti-MMP-2 monoclonal antibodies, anti-MMP-9 polyclonal antibodies and anti-CD44 monoclonal antibodies. Aromatase was expressed in IDC and DCIS in 63 (57.3%) and 60 (67%) of the cases respectively; MMP-2 was similarly expressed in IDC and DCIS in 15 (13.60%) cases; MMP-9 was positively expressed in IDC and DCIS in 83 (75.50%) and 82 (74.50%) cases, respectively; CD44 was positively expressed in IDC and DCIS in 49 (44.50%) and 48 (42.60%) of the cases, respectively; all of them were highly correlated (p<0,001). The correlation analysis found positive, statistically significant correlation, in IDC between aromatase and MMP-2 (p<0.001) and between aromatase and MMP-9 (p=0.034). Positive correlation between aromatase and MMP-2 (p<0.001) and between MMP-9 and CD44 (p=0.030) were found in DCIS. These results allow us to conclude that aromatase through local estrogen synthesis in breast tissue plays an important role in breast carcinogenesis, mainly influencing MMP-2 and MMP-9 which are important participants in tumor cell invasion and dependence of their connection to CD44 for action.

Do alterations in mitochondrial DNA play a role in breast carcinogenesis?

A considerable body of evidence supports a role for oxidative stress in breast carcinogenesis. Due to their role in producing energy via oxidative phosphorylation, the mitochondria are a major source of production of reactive oxygen species, which may damage DNA. The mitochondrial genome may be particularly susceptible to oxidative damage leading to mitochondrial dysfunction. Genetic variants in mtDNA and nuclear DNA may also contribute to mitochondrial dysfunction. In this review, we address the role of alterations in mtDNA in the etiology of breast cancer. Several studies have shown a relatively high frequency of mtDNA mutations in breast tumor tissue in comparison with mutations in normal breast tissue. To date, several studies have examined the association of genetic variants in mtDNA and breast cancer risk. The G10398A mtDNA polymorphism has received the most attention and has been shown to be associated with increased risk in some studies. Other variants have generally been examined in only one or two studies. Genome-wide association studies may help identify new mtDNA variants which modify breast cancer risk. In addition to assessing the main effects of specific variants, gene-gene and gene-environment interactions are likely to explain a greater proportion of the variability in breast cancer risk.

Gene Promoter Hypermethylation in Tumors and Matched Affected Lymph Nodes of Breast Cancer Patients.

Abstract Gene promoter hypermethylation plays a pivotal role in breast carcinogenesis and breast cancer progression. In this study, we investigated 6 gene promoters (RASSF1A, HIN-1, RARβ2, TWIST, CCND2 and APC) for aberrant DNA methylation by real-time methylation-specific PCR in primary tumors and matched affected lymph nodes from 30 patients with breast cancer. These data were used to explore associations between gene promoter hypermethylation and clinicopathological features, including tumor cell proliferation, angiogenesis and hypoxia marker expression.Overall, gene promoter methylation levels (ratios of DNA copy numbers for methylated and reference genes) were higher in primary tumors than in matched lymph nodes. Notably, we found significant correlations between methylation levels in primary tumors and matched lymph nodes for 5 of 6 genes (P&amp;lt;0.05). An identical gene promoter methylation status (high or low according to the median methylation level) in primary tumors and matched lymph nodes was observed in 67% to 83% of cases. No significant associations between risk factors and gene promoter hypermethylation of the 6 genes was observed, except for the degree of spreading into nearby lymph nodes, which associated with high RASSF1A methylation levels in tumors (P=0.020) and high CCDN2 methylation levels in lymph nodes, and for HER2 expression, which was most frequently observed in tumors with low HIN-1 methylation levels (P=0.039). HIN-1 methylation levels in tumors negatively correlated with the fractions of proliferating tumor and endothelial cells determined by CD34/Ki-67 immunohistochemistry in both primary tumors (r=-0.594, P=0.001 and r=-0.619, P&amp;lt;0.001) and in lymph nodes (r=-0.412, P=0.024 and r=-0.400, P=0.029). Furthermore, high HIN-1 methylation levels in primary tumors tended to associate with breast tumors lacking expression of the hypoxia marker carbonic anhydrase IX in tumor cells (P=0.063) and associated stroma (P=0.086). CCND2 methylation levels in affected lymph nodes positively correlated with the ratios of the fractions of proliferating tumor cells to the fractions of proliferating endothelial cells observed in both primary tumors (r=0.418, P=0.002) and in lymph nodes (r=0.563, P=0.002).In summary, gene promoter hypermethylation in primary breast tumors and matched lymph nodes are highly correlated. Further studies with larger numbers of samples are necessary to investigate the prognostic or predictive potential of these observations. For two genes, HIN-1 and CCDN2, we observed correlations between gene promoter methylation levels and tumor cell proliferation. HIN-1 and CCDN2 are frequently lost in breast cancer and are both involved in cell cycle regulation. The biological significance of these findings needs to be further explored. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1149.

DNA Promoter Methylation in Breast Tumors Is Not Associated with Polymorphism in the GSTP1 Gene.

Abstract The gluthathione S-transferases (GSTs) are a family of enzymes that play an important role in detoxification of a variety of potential carcinogenic, hydrophobic and electrophilic compounds. Glutathione-S-transferase P1 (GSTP1) is involved in thiol-mediated detoxification and breakdown of reactive oxygen species created by anticancer drug exposure. It is also an inhibitor of c-Jun N-terminal kinase 1 which is involved in stress response, apoptosis and cellular proliferation. It has been postulated that a decrease in GST enzyme activity could result in higher frequency and a specific pattern of mutations in cancer. Loss of GSTP1 expression is observed in approximately two thirds of breast cancer cases, which indicates its potential role in breast carcinogenesis. The GSTP1 gene exhibits deletion polymorphisms and homozygous deletion of the gene may result in lack of enzyme activity. The polymorphism in the GSTP1 gene results from a base substitution and leads to an amino acid change at codon 105. This amino acid resides at the substrate binding site of the enzyme and may modify the catalytic activity of the enzyme several-fold.DNA methylation is an important epigenetic modification of the genome. A specific methylation pattern has been observed in various types of cancers. Hypermethylation of the GSTP1 promoter has also been associated with gene silencing in different forms of cancer. In a previous study we have found that the GSTP1 gene was the second most frequently methylated gene in breast cancer. However, a possible relationship between the methylation status of the gene promoter and GSTP1 polymorphic variants has not been investigated.In this study we investigated the role of the different allelic variants in the methylation status of the GSTP1 gene in patients with breast cancer. A total of 48 patients with breast cancer and 32 healthy women were analyzed. The GSTP1 polymorphism was determined by PCR-RFLP. Methylation of the GSTP1 gene was evaluated by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) using probes targeting the GSTP1 gene and metylation-sensitive enzyme digestion of the probe-DNA hybrids. The amplified products were analyzed by sequence-type capillary electrophoresis. Subsequent quantification of the methylation status was performed by comparing the relative signal peaks from the digested and undigested samples.The null allele was observed in % 56,2 of the patients and % 46.8 of the control group, respectively. No significant difference was observed between the distribution of the genotypes in the patients and the controls. The GSTP1 gene promoter was methylated in % 39.6 of the patients. However, the distribution of the genotypes and allele frequencies did not reveal any preferential methylation of a specific allele. The genotypes were not associated with increased likelihood of promoter methylation. Our data indicate that the deletion allele of the GSTP1 gene does affect the methylation status of the gene in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1150.

IMP3 is a novel biomarker for triple negative invasive mammary carcinoma associated with a more aggressive phenotype

IMP3, an oncofetal protein, is a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein family. Its relevance as a novel biomarker in lung, pancreatic, renal, and cervical adenocarcinoma was recently revealed. However, its role in breast carcinogenesis and tumor progression is not yet established. Basal-like carcinoma was initially identified by gene expression profiling. It accounts for 15% to 30% of all breast cancers. These tumors express basal epithelial markers including cytokeratin 5 but lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), therefore, are often referred to as triple negative breast cancer. They have been found to be associated with a worse overall and disease-free survival. In this retrospective study, we examined the IMP3 expression in invasive ductal carcinoma of the breast and correlated its expression with morphological and biologic prognostic factors. The study group comprised 138 cases of invasive ductal carcinoma retrieved from the surgical pathologic files for a 10-year period from 1997 to 2006. Survival data and clinical stage were available on all 138 patients. Tumor characteristics including size, grade, lymphovascular invasion, necrosis, lymph node metastasis, estrogen receptor, progesterone receptor, and HER2 status were obtained from pathologic reports. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue using mouse monoclonal antibody against IMP3 and CK5/6. Of the 138 breast cancer cases, IMP3 expression was seen in 45 (33%). Twenty-five of the IMP3+ cases were triple negative. We found significant correlation between IMP3 expression and higher grade (P = .001), necrosis (P< .0001) triple negative, and CK5/6 expression (P < .0001 for each). Cox multivariate analysis showed a hazard ratio of IMP3 expression at 3.14 (P = .05). IMP3 is a novel biomarker for triple negative (basal-like) invasive mammary carcinoma, and its expression is associated with a more aggressive phenotype and decreased overall survival.

Postmenopausal estrogen monotherapy–associated breast cancer risk is modified by CYP17A1_-34_T&gt;C polymorphism

Long-term hormone therapy (HT) is a recognized risk factor for postmenopausal breast cancer. Elevated steroid hormone levels play a critical role in breast carcinogenesis and this may be contributed by the efficiency of hormone biosynthesis. Within this context, genetic polymorphisms related to steroid hormone biosynthesis may modify HT-associated postmenopausal breast cancer risk. CYP17 is a key player of this pathway and the CYP17A1_-34_T > C polymorphism has been suggested to affect breast cancer risk in women using long-term HT. We genotyped 13 polymorphisms of seven genes of the steroid hormone biosynthesis pathway in 3,149 postmenopausal breast cancer patients and 5,489 age-matched controls from Germany. We observed a significant interaction of CYP17A1_-34_T > C and HT use on breast cancer risk in a co-dominant model (P (interaction) = 0.007). Current users of estrogen monotherapy showed a significantly increased risk for duration of use per 5-year increment when they were carriers of the CYP17A1_-34_TC genotype (OR 1.13, 95% CI: 1.04-1.23 per 5 years of use). We conclude that CYP17A1_-34_T > C may be part of the genetic background to contribute to postmenopausal breast cancer risk in women using estrogen monotherapy.

Analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis

Several independent studies have presented evidence for the involvement of human papillomaviruses (HPV) in the aetiology of human breast cancer, while others have reported the opposite findings. Here, we have analysed by a high sensitive multiplex PCR-based method the prevalence of alpha mucosal and beta cutaneous HPV DNA in 90 ductal lavages, colostrum and milk. Ten of the 70 DLs analyzed (14%) contained a single or multiple beta HPV types, while DNA from mucosal high-risk HPV types was detected in only one sample (1/70). A strong reduction of HPV positivity in DL fluids was observed in 45 specimens collected after removal of the superficial layers of the nipple epidermis. All DLs were negative for the mucosal low-risk HPV types 6 and 11. Finally, HPV positivity was low in colostrum and milk. Our data show that DNA of alpha mucosa and beta cutaneous HPV types are rarely present in the breast fluids and suggest that a direct role of HPV in breast carcinogenesis is unlikely.

Repeated measures of serum glucose and insulin in relation to postmenopausal breast cancer

Experimental and epidemiological evidence suggests that circulating glucose and insulin may play a role in breast carcinogenesis. However, few cohort studies have examined breast cancer risk in association with glucose and insulin levels, and studies to date have had only baseline measurements of exposure. We conducted a longitudinal study of postmenopausal breast cancer risk using the 6% random sample of women in the Women's Health Initiative clinical trials whose fasting blood samples, provided at baseline and at years 1, 3 and 6, were analyzed for glucose and insulin. In addition, a 1% sample of women in the observational study, who had glucose and insulin measured in fasting blood samples drawn at baseline and in year 3, were included in the analysis. We used Cox proportional hazards models to estimate hazard ratios and 95% confidence intervals for the association of baseline and follow-up measurements of serum glucose and insulin with breast cancer risk. All statistical tests were 2-sided. Among 5,450 women with baseline serum glucose and insulin values, 190 incident cases of breast cancer were ascertained over a median of 8.0 years of follow-up. The highest tertile of baseline insulin, relative to the lowest, was associated with a 2-fold increase in risk in the total population (multivariable hazard ratio 2.22, 95% confidence interval 1.39-3.53) and with a 3-fold increase in risk in women who were not enrolled in the intervention arm of any clinical trial (multivariable hazard ratio 3.15, 95% confidence interval 1.61-6.17). Glucose levels showed no association with risk. Analysis of the repeated measurements supported the results of the baseline analysis. These data suggest that elevated serum insulin levels may be a risk factor for postmenopausal breast cancer.

Serum oestrogen receptor α and β bioactivity are independently associated with breast cancer: a proof of principle study

Background:Oestrogens play a crucial role in breast carcinogenesis. Earlier studies have analysed the serum levels of endogenous hormones measured by conventional assays. In this study, we analysed the capacity of serum from breast cancer cases and controls to transactivate the oestrogen receptor α (ER-α) and β (ER-β).Methods:We used a receptor oestrogen-responsive element (ERE) – the green fluorescent protein (GFP)-reporter test system in Saccharomyces cerevisiae. Oestrogen receptor-α or ER-β bioactivity was determined in serum from 182 randomly chosen postmenopausal women with breast cancer and from 188 age-matched controls.Results:High serum ER-α and ER-β bioactivity were independently associated with the presence of breast cancer. Women whose levels of serum ER-α and ER-β bioactivity were in the highest quintile among controls had a 7.57-(95% confidence interval (CI): 2.46–23.32; P=0.0004) and a 10.14 (95% CI: 3.19–32.23; P<0.0001)-fold risk for general and oestrogen receptor-positive breast cancer, respectively.Conclusion:The use of serum ER-α and ER-β bioactivity assays as clinical tools in the management of breast cancer warrants further research. Future studies will dictate whether surrogate markers of ER-α and ER-β bioactivity will provide a means to monitor the efficacy of anti-endocrine, adjuvant and chemopreventive strategies.

Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

To identify genes involved in breast tumorigenesis, we applied the retroviral LoxP-Cre system to a nontumorigenic mouse mammary epithelial cell line NOG8 to create random chromosome deletion/translocation. We found that the disruption of one allele of Smyd4 (SET and MYND domain containing 4) gene through chromosome translocation led to tumorigenesis. The expression of Smyd4 was markedly decreased in tumor cells. Re-expression of Smyd4 resulted in growth suppression of tumor cells and inhibition of tumor formation in nude mice. Furthermore, the RNA interference-mediated suppression of Smyd4 expression in human MCF10A mammary epithelial cells caused their growth in soft agar. Microarray studies revealed that platelet-derived growth factor receptor alpha polypeptide (Pdgfr-alpha) was highly expressed in tumor cells compared with NOG8 cells. Re-expression of Smyd4 significantly reduced the expression of Pdgfr-alpha in tumor cells. In human breast cancers, reverse transcription-PCR results revealed that Smyd4 expression was totally silenced in 2 of 10 specimens. These findings indicate that Smyd4, as a potential tumor suppressor, plays a critical role in breast carcinogenesis at least partly through inhibiting the expression of Pdgfr-alpha, and could be a novel target for improving treatment of breast cancer.

The Macrophage Colony-Stimulating Factor 1 Response Signature in Breast Carcinoma

Macrophages play an important role in breast carcinogenesis. The pathways that mediate the macrophage contribution to breast cancer and the heterogeneity that exists within macrophages are incompletely understood. Macrophage colony-stimulating factor 1 (CSF1) is the primary regulator of tissue macrophages. The purpose of this study was to define a novel CSF1 response signature and to evaluate its clinical and biological significance in breast cancer. We defined the CSF1 response signature by identifying genes overexpressed in tenosynovial giant cell tumor and pigmented villonodular synovitis (tumors composed predominantly of macrophages recruited in response to the overexpression of CSF1) compared with desmoid-type fibromatosis and solitary fibrous tumor. To characterize the CSF1 response signature in breast cancer, we analyzed the expression of CSF1 response signature genes in eight published breast cancer gene expression data sets (n = 982) and did immunohistochemistry and in situ hybridization for CSF1 response genes on a breast cancer tissue microarray (n = 283). In both the gene microarray and tissue microarray analyses, a consistent subset (17-25%) of breast cancers shows the CSF1 response signature. The signature is associated with higher tumor grade, decreased expression of estrogen receptor, decreased expression of progesterone receptor, and increased TP53 mutations (P < 0.001). Our data show that the CSF1 response signature is consistently seen in a subset of breast carcinomas and correlates with biological features of the tumor. Our findings provide insight into macrophage biology and may facilitate the development of personalized therapy for patients most likely to benefit from CSF1-targeted treatments.

Protein levels of NAD(P)H:quinone oxidoreductase 1 and p53 correlate in breast tumors.

Abstract Abstract #4157 Background: NAD(P)H:quinone oxidoreductase 1 (NQO1) is integral flavoprotein responsible for detoxification and metabolism of quinoid compounds. NQO1 protects cells against accumulation of genotoxic quinoids, oxidative stress and stabilizes p53 from proteasomal degradation. The aim of this study was to follow the NQO1 role in breast carcinogenesis.&amp;#x2028; Methods: Levels of NQO1 transcript were monitored in frozen tumor samples by real-time PCR with absolute quantification. NQO1 protein levels were evaluated by immunohistochemistry in paraffin-embedded tumor samples. NQO1-Pro187Ser polymorphism was assessed in lymphocyte DNA by PCR with restriction fragment analysis.&amp;#x2028; Results: High interindividual variability in NQO1 expression among patients was observed. Wild-type genotype samples expressed NQO1 protein more often than heterozygotes (p=0.004; Fisher's exact test). We found strong correlation between NQO1 transcript and protein levels (p=0.004; T-test). Moreover, samples with negative NQO1 protein expression were often also negative for p53 expression (p=0.037; Fisher's exact test). No significant association of NQO1 levels and/or polymorphism with clinico-pathological characteristics of patients was found.&amp;#x2028; Conclusions: One of major findings of this study is the observed correlation of protein levels of NQO1 and p53 in breast tumors. Ability of NQO1 to modulate the degradation of p53 in the 20S proteasome was previously reported by use of cell lines and animal models [1]. Our results obtained by the use of real tumor samples further support this concept and suggest that NQO1 may play important role in carcinogenesis.&amp;#x2028; This study was supported by grants of Internal Grant Agency of Ministry of Health of the Czech Republic, no.: 9426-3, Grant Agency of the Czech Republic 305/07/P347 and the Grant Agency of Charles University, no.: 94507.1. Asher G, Lotem J, Kama R, Sachs L, Shaul Y: Proc Natl Acad Sci USA 98(3): 1188-1193, 2001. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4157.

Minireview: C2- and C4-position 17β-estradiol metabolites and their relation to breast cancer

C2- and C4-position 17beta-estradiol metabolites play an important role in breast carcinogenesis. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via two pathways. These pathways entail increased cell proliferation and the formation of reactive oxygen species that trigger an increase in the likelihood of deoxyribonucleic acid mutations. 2-Methoxyestradiol, a 17beta-estradiol metabolite, however, causes induction of apoptosis in transformed and tumor cells; thus exhibiting an antiproliferative effect on tumor growth. The 4-hydroxyestradiol:2-methoxyestradiol and 2-hydroxyestradiol:2-methoxyestradiol ratios therefore ought to be taken into account as possible indicators of carcinogenesis.

Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

We have previously demonstrated the tumor suppressor characteristics of protein tyrosine phosphatase receptor-type O (PTPRO) in leukemia and lung cancer, including its suppression by promoter methylation. Here, we show tumor-specific methylation of the PTPRO CpG island in primary human breast cancer. PTPRO expression was significantly reduced in established breast cancer cell lines MCF-7 and MDA-MB-231 due to promoter methylation compared with its expression in normal human mammary epithelial cells (48R and 184). Further, the silenced gene could be demethylated and reactivated in MCF-7 and MDA-MB-231 cells upon treatment with 5-Azacytidine, a DNA hypomethylating agent. Because PTPRO promoter harbors estrogen-responsive elements and 17beta-estradiol (E2) plays a role in breast carcinogenesis, we examined the effect of E2 and its antagonist tamoxifen on PTPRO expression in human mammary epithelial cells and PTPRO-expressing breast cancer cell line Hs578t. Treatment with E2 significantly curtailed PTPRO expression in 48R and Hs578t cells, which was facilitated by ectopic expression of estrogen receptor (ER)beta but not ERalpha. On the contrary, treatment with tamoxifen increased PTPRO expression. Further, knockdown of ERbeta by small interfering RNA abolished these effects of E2 and tamoxifen. Chromatin immunoprecipitation assay showed association of c-Fos and c-Jun with PTPRO promoter in untreated cells, which was augmented by tamoxifen-mediated recruitment of ERbeta to the promoter. Estradiol treatment resulted in dissociation of c-Fos and c-Jun from the promoter. Ectopic expression of PTPRO in the nonexpressing MCF-7 cells sensitized them to growth-suppressive effects of tamoxifen. These data suggest that estrogen-mediated suppression of PTPRO is probably one of the early events in estrogen-induced tumorigenesis and that expression of PTPRO could facilitate endocrine therapy of breast cancer.

Interaction Between Estrogen Receptor Alpha and Insulin/IGF Signaling in Breast Cancer

Estrogens and insulin/Insulin like growth factor 1 (IGF-I) have potent positive effects on the proliferation of mammary epithelial cells and estrogen-dependent breast cancer cells. A cooperative crosstalk between estrogens and insulin/IGF-I signaling pathways exists and it plays a critical role in breast carcinogenesis, tumor cell proliferation, differentiation and survival through the modulation of multiple biological events. The biological effects of estrogens are mainly mediated by the activation of estrogen receptor (ERalpha) whose activity is deeply influenced by the insulin/IGF-I signaling pathway. On the other hand, estrogens enhance insulin signaling by increasing the expression and/or the functional activity of some proteins involved in the insulin/IGF-I pathway. This review will focus on the critical node of the IGF-I network involved in the crosstalk with ERalpha and implicated in breast cancer development and progression.

Down‐regulation of claudin‐2 in breast carcinomas is associated with advanced disease

Claudin 2 (CLDN2) is a family of integral membrane tight junctions. The aim was to determine the influence of CLDN2 expression on tumour behaviour and its role in breast carcinogenesis. Thirty-seven invasive breast carcinomas and corresponding normal breast tissues were examined for CLDN2 protein and mRNA expression using Western blotting and semiquantitative reverse transcriptase-polymerase chain reaction. The expression of CLDN2 protein in 118 cases of breast carcinoma was further studied with immunohistochemistry and related to various clinicopathological parameters. CLDN2 protein expression was significantly down-regulated (0.4-fold) in tumours compared with corresponding normal breast tissue (P < 0.0001). Down-regulation of CLDN2 was significantly associated with lymph node metastasis (P = 0.047) by Western blot analysis, and with high clinical stage (P = 0.040) by immunohistochemistry. The expression levels of CLDN2 mRNA in high clinical stages (stages II and III) were lower than those in low clinical stage (stage I) and normal tissue, but not statistically significantly so. These results suggest that CLDN2 is implicated in the progression as well as the development of breast carcinoma, indicating that CLDN2 is a possible tumour suppressor gene product.

Analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis

Several independent studies have presented evidence for the involvement of human papillomaviruses (HPV) in the aetiology of human breast cancer, while others have reported the opposite findings. Here, we have analysed by a high sensitive multiplex PCR-based method the prevalence of alpha mucosal and beta cutaneous HPV DNA in 90 ductal lavages, colostrum and milk. Ten of the 70 DLs analyzed (14%) contained a single or multiple beta HPV types, while DNA from mucosal high-risk HPV types was detected in only one sample (1/70). A strong reduction of HPV positivity in DL fluids was observed in 45 specimens collected after removal of the superficial layers of the nipple epidermis. All DLs were negative for the mucosal low-risk HPV types 6 and 11. Finally, HPV positivity was low in colostrum and milk. Our data show that DNA of alpha mucosa and beta cutaneous HPV types are rarely present in the breast fluids and suggest that a direct role of HPV in breast carcinogenesis is unlikely.

Expression of TRPC6 channels in human epithelial breast cancer cells

BackgroundTRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.MethodsMolecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).ResultsA non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.ConclusionOur results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

Promoter hypomethylation of the N-acetyltransferase 1 gene in breast cancer

Arylamine N-acetyltransferase type 1 (NAT1) is reported to be involved in the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs or carcinogens. Gene-specific hypomethylation frequently occurs in a range of cancers and hypomethylation of the genes often correlates well with increased transcription levels. This study was conducted in order to evaluate the methylation status and the transcriptional activity of NAT1 in breast cancer tissues (n=72), benign breast tissues (n=31) and morphologically normal breast tissues (n=30). Our findings showed that the methylation of the NAT1 gene was identified in 39 of the breast carcinomas (54.2%), 23 normal (76.7%) and 25 benign breast tissue samples (80.6%). The breast cancer tissues showed significantly lower methylation rates of the NAT1 promoters than the normal and benign tissues (P=0.012). Furthermore, cancer tissues showed lower methylation density rates than normal and benign breast tissues (P=0.012). The tissues that showed aberrant methylation of NAT1 showed significantly less mRNA expression compared with the unmethylated cases by a thousand fold (P<0.001). Twenty cancers from the methylated group showed positive staining for the estrogen receptor (ER) (51.3%), while 72.7% from the unmethylated group stained positive (P=0.063). Our results suggest that DNA hypomethylation in the NAT1 gene appears to be present in cancerous breast tissues thus indicating that this type of methylation may significantly influence the transcriptional activation of the gene. Therefore, hypomethylation of the NAT1 gene plays a significant role in breast carcinogenesis.