Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer

BACKGROUND: FOXM1 regulates expression of cell cycle related genes that are essential for progression into DNA replication and mitosis. Consistent with its role in proliferation, elevated expression of FOXM1 has been reported in a variety of human tumour entities. FOXM1 is a gene of interest because recently chemical inhibitors of FOXM1 were described to limit proliferation and induce apoptosis in cancer cells in vitro, indicating that FOXM1 inhibitors could represent useful anticancer therapeutics. METHODS: Using immunohistochemistry (IHC) we systematically analysed FOXM1 expression in human invasive breast carcinomas (n = 204) and normal breast tissues (n = 46) on a tissue microarray. Additionally, using semiquantitative realtime PCR, a collection of paraffin embedded normal (n = 12) and cancerous (n = 25) breast tissue specimens as well as benign (n = 3) and malignant mammary cell lines (n = 8) were investigated for FOXM1 expression. SPSS version 14.0 was used for statistical analysis. RESULTS: FOXM1 was found to be overexpressed in breast cancer in comparison to normal breast tissue both on the RNA and protein level (e.g. 8.7 fold as measured by realtime PCR). We found a significant correlation between FOXM1 expression and the HER2 status determined by HER2 immunohistochemistry (P < 0.05). Univariate survival analysis showed a tendency between FOXM1 protein expression and unfavourable prognosis (P = 0.110). CONCLUSION: FOXM1 may represent a novel breast tumour marker with prognostic significance that could be included into multi-marker panels for breast cancer. Interestingly, we found a positive correlation between FOXM1 expression and HER2 status, pointing to a potential role of FOXM1 as a new drug target in HER2 resistant breast tumour, as FOXM1 inhibitors for cancer treatment were described recently. Further studies are underway to analyse the potential interaction between FOXM1 and HER2, especially whether FOXM1 directly activates the HER2 promoter

Expressionsanalyse und funktionelle Charakterisierung potentieller Biomarker des humanen Mammakarzinoms in vitro und in einem SFRP1-Knockout Mausmodell

Every year around one million new incidences for breast cancer are registered worldwide. Analyses of the molecular origins of breast cancer as well as the identification of new distinctive biomarkers which indicate tumour development and allow to predict the tumour outcome are of increasing interest for modern medicine. Suitable biomarkers can help with an earlier diagnosis of tumours, a better prediction of disease progression and with improved prognostics for the selection of optimized treatment strategies. Results from recent studies point towards a possible association between the deregulated activity of HH and WNT signalling and the development of human breast tumours. SFRP1 is an important inhibitor of WNT signalling to which a remarkable role as a tumour suppressor in human breast tumourigenesis has been assigned. In line with these studies, the quality of the three HH signalling members SHH, GLI1 and FOXM1 as molecular biomarkers of the human mammary carcinoma was evaluated. The results from protein and mRNA expression analyses showed an overexpression of all three molecules within the analysed set of breast tumours (n = 204) as well as in a set of breast tumour cell lines (n = 9), which correlated to certain clinic pathological characteristics of the tumours. A significant association was detected between increased levels of cytoplasmic SHH expression and the tumour characteristics size (P < 0.01), histological grade (P < 0.01) and HER2 receptor status (P = 0.049). Expression analyses on SHH further showed that the transcription of this gene is regulated through combined mechanisms of promoter methylation and the use of alternative transcription start sites. For the transcription factor GLI1, we found that increased levels of nuclear GLI1 expression correlated significantly with tumour stage (P < 0.001), and also showed a significant association with the nodal status of the analysed tumours (P = 0.027). An abundant nuclear appearance of GLI1 was further associated with a significantly reduced overall survival (P = 0.019) of breast cancer patients. For FOXM1 increased levels of nuclear expression were also detected in the analysed tumourous tissues and showed a significant correlation to the HER2 receptor status (P = 0.045), as well as to reduced overall survival tendencies of the analysed patients (P = 0.019). First functional analyses on this molecule also showed that an inhibition of FOXM1 expression leads to reduced cell proliferation, migration and invasiveness of human breast tumour cell lines in vitro. Furthermore Cyclin B1 and Aurora B Kinase could be identified as potential target genes of FOXM1 mediated transactivation. Taken together these data point towards a potential role of the HH signalling pathway in human breast tumourigenesis and further characterizes this signalling pathway as a possible new therapeutic target in HER2 positive breast cancers. As an additional project the functional consequences of a Sfrp1 loss in vivo were analysed on the basis of a Sfrp1-knockout mouse model. Results from the phenotypical analyses of homozygous knockout mice showed a functional influence of Sfrp1 on fertility characteristics of these animals. Reduced numbers of homozygous litters, as well as histological abnormalities in uterine and breast tissues of homozygous Sfrp1-knockout mice were observed shortly after birth. Also Oxytocin receptor mRNA levels in breast tissues of homozygous Sfrp1-knockout mice were increased during pregnancy and shortly after birth. Analyses of Sfrp1 expression in breast tissues of wildtype mice from different age and developmental stages showed that Sfrp1 expression is increased during pregnancy, lactation, involution, as well as in breast tissues of elder mice. These processes are associated with the morphological reorganisation of breast tissues and as all growth and developmental processes they underlie strict cellular control mechanisms, which inhibit non-directed and abnormal proliferation of the cells. An increased expression of Sfrp1 within these important stages therefore underlines the putative tumour suppressive function of this gene. In summary, the presented study successfully identified the three HH signalling members SHH, GLI1 and FOXM1 as novel and potentially useful biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies. Also for the first time histological differences in uterine and breast tissues of homozygous Sfrp1 knockout mice from certain developmental stages were detected that may contribute to the definition of Sfrp1 associated functions in vivo

Expressionsanalyse und funktionelle Charakterisierung potentieller Biomarker des humanen Mammakarzinoms in vitro und in einem SFRP1-Knockout Mausmodell

Every year around one million new incidences for breast cancer are registered worldwide. Analyses of the molecular origins of breast cancer as well as the identification of new distinctive biomarkers which indicate tumour development and allow to predict the tumour outcome are of increasing interest for modern medicine. Suitable biomarkers can help with an earlier diagnosis of tumours, a better prediction of disease progression and with improved prognostics for the selection of optimized treatment strategies. Results from recent studies point towards a possible association between the deregulated activity of HH and WNT signalling and the development of human breast tumours. SFRP1 is an important inhibitor of WNT signalling to which a remarkable role as a tumour suppressor in human breast tumourigenesis has been assigned. In line with these studies, the quality of the three HH signalling members SHH, GLI1 and FOXM1 as molecular biomarkers of the human mammary carcinoma was evaluated. The results from protein and mRNA expression analyses showed an overexpression of all three molecules within the analysed set of breast tumours (n = 204) as well as in a set of breast tumour cell lines (n = 9), which correlated to certain clinic pathological characteristics of the tumours. A significant association was detected between increased levels of cytoplasmic SHH expression and the tumour characteristics size (P < 0.01), histological grade (P < 0.01) and HER2 receptor status (P = 0.049). Expression analyses on SHH further showed that the transcription of this gene is regulated through combined mechanisms of promoter methylation and the use of alternative transcription start sites. For the transcription factor GLI1, we found that increased levels of nuclear GLI1 expression correlated significantly with tumour stage (P < 0.001), and also showed a significant association with the nodal status of the analysed tumours (P = 0.027). An abundant nuclear appearance of GLI1 was further associated with a significantly reduced overall survival (P = 0.019) of breast cancer patients. For FOXM1 increased levels of nuclear expression were also detected in the analysed tumourous tissues and showed a significant correlation to the HER2 receptor status (P = 0.045), as well as to reduced overall survival tendencies of the analysed patients (P = 0.019). First functional analyses on this molecule also showed that an inhibition of FOXM1 expression leads to reduced cell proliferation, migration and invasiveness of human breast tumour cell lines in vitro. Furthermore Cyclin B1 and Aurora B Kinase could be identified as potential target genes of FOXM1 mediated transactivation. Taken together these data point towards a potential role of the HH signalling pathway in human breast tumourigenesis and further characterizes this signalling pathway as a possible new therapeutic target in HER2 positive breast cancers. As an additional project the functional consequences of a Sfrp1 loss in vivo were analysed on the basis of a Sfrp1-knockout mouse model. Results from the phenotypical analyses of homozygous knockout mice showed a functional influence of Sfrp1 on fertility characteristics of these animals. Reduced numbers of homozygous litters, as well as histological abnormalities in uterine and breast tissues of homozygous Sfrp1-knockout mice were observed shortly after birth. Also Oxytocin receptor mRNA levels in breast tissues of homozygous Sfrp1-knockout mice were increased during pregnancy and shortly after birth. Analyses of Sfrp1 expression in breast tissues of wildtype mice from different age and developmental stages showed that Sfrp1 expression is increased during pregnancy, lactation, involution, as well as in breast tissues of elder mice. These processes are associated with the morphological reorganisation of breast tissues and as all growth and developmental processes they underlie strict cellular control mechanisms, which inhibit non-directed and abnormal proliferation of the cells. An increased expression of Sfrp1 within these important stages therefore underlines the putative tumour suppressive function of this gene. In summary, the presented study successfully identified the three HH signalling members SHH, GLI1 and FOXM1 as novel and potentially useful biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies. Also for the first time histological differences in uterine and breast tissues of homozygous Sfrp1 knockout mice from certain developmental stages were detected that may contribute to the definition of Sfrp1 associated functions in vivo

Use of expression data and the CGEMS genome-wide breast cancer association study to identify genes that may modify risk in BRCA1/2 mutation carriers

Germline mutations in BRCA1 or BRCA2 confer an increased lifetime risk of developing breast or ovarian cancer, but variable penetrance suggests that cancer susceptibility is influenced in part by modifier genes

Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

Abstract Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.</p