No abstract

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an ''inverse genomics'' approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

No abstract

The expression of microRNAs is altered in various cancer types, leading to their definition as onco-and tumor-suppressor microRNAs. In our study, we investigated the role of miR-335 in the formation of sporadic human breast cancer and its involvement in the regulatory network of the breast cancer susceptibility gene BRCA1. To validate single components of the BRCA1 cascade, microRNA overexpression was performed in a cell culture model with subsequent protein analysis and luciferase reporter assays. Here, we were able to identify miR-335 as simultaneously regulating the known BRCA1 activators ERa, IGF1R, SP1 and the repressor ID4, including a feedback regulation of miR-335 expression by estrogens. Overexpression of miR-335 resulted in an upregulation of BRCA1 mRNA expression, suggesting a functional dominance of ID4 signaling. The relevance of the miR-335 regulation for human breast cancer was confirmed in primary sporadic breast cancer specimens with significantly decreased miR-335 levels (p < 0.05) in comparison to normal controls. Interestingly, the microRNA expression level correlated positively to the BRCA1 transcript level, supporting the hypothesis of a miR-335-mediated regulation of the tumor suppressor gene. Functionally, overexpression of miR-335 led to decreased cell viability and an increase in apoptosis, supporting its tumor-suppressive function. In summary, our data indicate that miR-335 affects different targets in the upstream BRCA1-regulatory cascade with impact on key cellular functions such as proliferation and apoptosis. Deregulation of the microRNA during breast cancer development and progression may thereby lead to an increased tumorigenic potential by inactivating crucial tumor-suppressive signals.In Western countries, breast cancer is the most frequent tumor found in women, with a low incidence in men. The tumor develops in a multistep process from ductal epithelial cells by triggers as yet poorly defined. Human breast cancers can be divided into two major subtypes, the inherited (5-10%) and the sporadic form, representing the greatest part. 1 The inherited subtype is predominantly characterized by germline mutations in one of the cancer susceptibility genes, known as BRCA1 and BRCA2, favoring a high predisposition to develop breast and ovarian cancer. 2 Although BRCA1 mutations have been found only rarely in the sporadic form, decreased expression levels of the gene were identified in these tumors. 3,4 As BRCA1 expression is embedded in a tightly controlled network, involving various activating and repressing factors, an altered transcriptional regulation may cause deregulation of the gene. Here, the expression of BRCA1 was determined to be hormone-dependent via direct activation through the estrogen (ERa), the aryl hydrocarbon (AhR) and the insulinlike growth factor 1 (IGF1R) receptors. [5][6][7] These are supported by hormone-independent factors such as the specific protein 1 (SP1) and inhibited via the inhibitor of DNA binding 4 (ID4). 8,9 These factors were identified as potent regula...