Prolactin-dependent up-regulation of BRCA1 expression in human breast cancer cell lines.

We report experimental evidence that BRCA1, a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of the BRCA1 gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase in BRCA1 mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase in BRCA1 mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.     

Heregulin induces phosphorylation of BRCA1 through phosphatidylinositol 3-Kinase/AKT in breast cancer cells.

The breast cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein that acts as a tumor suppressor. Phosphorylation of BRCA1 has been implicated in altering its function, however, the pathway(s) that leads to the phosphorylation of BRCA1 has not been described. Here, a signaling pathway by which heregulin induces cell cycle-independent phosphorylation of BRCA1 was delineated. We showed that heregulin stimulation induced the phosphorylation of BRCA1 and concomitant activation of the serine/threonine kinase AKT in T47D human breast cancer cells. Heregulin-induced phosphorylation of BRCA1 was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors and by a dominant-negative AKT. In the absence of heregulin, the ectopic expression of the constitutively active p110 subunit of PI3K was sufficient to induce BRCA1 phosphorylation. Furthermore, the purified glutathione S-transferase/AKT kinase phosphorylated BRCA1 in vitro. We have also shown that the phosphorylation of BRCA1 by AKT occurs on the residue Thr-509, which is located in the nuclear localization signal. These results reveal a novel signaling pathway that links extracellular signals to the phosphorylation of BRCA1 in breast cancer cells.     

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.

Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.     

Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines

Germline alterations of the BRCA1 tumor suppressor gene have been implicated at least in half of familial breast cancers. Nevertheless, in sporadic breast cancer no mutation of this gene has been characterized to date. In sporadic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to better understand the relationship between BRCA1 expression and malignant transformation, we have adapted the new real-time quantitative PCR method based on a 5' nuclease assay and the use of doubly labeled fluorescent TaqMan probes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA with or without exon 11 in the normal breast epithelial cell line MCF10a and in three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two methods of quantification: the comparative C(T) and the standard curve. We found that the full length BRCA1 mRNA, which encodes the functional nuclear protein, was down-regulated in tumor cells when compared with MCF10a cells.     

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.     

BRCA1 accumulates in the nucleus in response to hypoxia and TRAIL and enhances TRAIL-induced apoptosis in breast cancer cells

A major contributing factor to the development of breast cancer is decreased functional expression of breast cancer susceptibility gene 1, BRCA1. Another key contributor to tumorigenesis is hypoxia. Here we show that hypoxia increased the nuclear localization of BRCA1 in MCF-7 and MDA-MB-468 human breast cancer cell lines without changing its steady-state expression level. Nuclear accumulation of BRCA1 was not evident in MCF-12A or HMEC (human mammary epithelial cell) nonmalignant mammary epithelial cells under the same conditions. Hypoxia also increased the cell surface expression of TRAIL on MDA-MB-468 cells. Neutralization of TRAIL precluded the hypoxia-induced accumulation of BRCA1 in the nucleus, whereas exogenously administered TRAIL mimicked the effect. Treatment of MDA-MB-468 cells with TRAIL resulted in a dose- and time-dependent increase in apoptosis. Furthermore, TRAIL-induced apoptosis in HCC1937 cells, which harbor a BRCA1 mutation, increased synergistically when wild-type BRCA1 was reconstituted in the cells, and downregulation of BRCA1 expression in MDA-MB-468 cells reduced the apoptotic response to TRAIL. These data provide a novel link between hypoxia, TRAIL and BRCA1, and suggest that this relationship may be especially relevant to the potential use of TRAIL as a chemotherapeutic agent.     

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.     

Cell cycle differences in DNA damage-induced BRCA1 phosphorylation affect its subcellular localization

Phosphorylation of BRCA1 tumor suppressor protein is regulated during the cell cycle and in response to DNA damage. Several Ser/Thr kinases have been implicated in BRCA1 phosphorylation, including ATM/ATR, cdk2, and hChk2 kinases. In this study, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were employed to study BRCA1 phosphorylation during the S and G2/M phases under conditions of DNA damage. We observed that IR (ionizing radiation) treatment induced phosphorylation of Ser-988/Ser-1524 during the S phase and of Ser-988/Ser-1423 during the G2/M phase. UV treatment induced phosphorylation of Ser-988 during the S phase and of Ser-1423 during the G2/M phase. Phosphorylation of serines 1423 and -1524 was not induced in HCC1937 breast cancer cells, which contain mutant BRCA1 protein. Confocal microscopy revealed that unphosphorylated BRCA1 localizes on chromosomes from metaphase through telophase, whereas Ser-988-phosphorylated BRCA1 resides in the inner chromosomal structure, centrosome, and the cleavage furrow during prophase through telophase. We also found that Ser-988-phosphorylated BRCA1 relocalizes to the perinuclear region when cells are subjected to IR or UV radiation in the S phase. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

BRCA1 gene expression in breast cancer: a correlative study between real-time RT-PCR and immunohistochemistry.

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.     

Induction or suppression of expression of cytochrome C oxidase subunit II by heregulin beta 1 in human mammary epithelial cells is dependent on the levels of ErbB2 expression

The ErbB family of receptor kinases is composed of four members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Amplification of the ErbB2/neu is found in about 30% of breast cancer patients and is associated with a poor prognosis. Heregulin (HRG) activates the ErbB2 via induction of heterodimerization with ErbB3 and ErbB4 receptors. With suppression subtractive hybridization, we demonstrated that the expression of cytochrome c oxidase subunit II (COXII) is HRG-responsive. Two nontransformed human mammary epithelial cell lines, the HB2 and the HB2(ErbB2) (the HB2 engineered to overexpress ErbB2), displayed an opposite response to HRG-mediated regulation. HRG upregulated mRNA expression of COXII in the HB2 cells, but suppressed COXII expression in the HB2(ErbB2) cells. A human breast cancer cell line (T47D), which expresses ErbB2 at a level similar to that of the HB2 cells, also responded to HRG by increasing COXII mRNA levels. Therefore, HRG regulation of COXII expression depends on the levels of ErbB2 expression. Furthermore, the expression of COXII was inversely correlated to the levels of ErbB2, i.e., the cells overexpressing ErbB2 exhibited lower COXII levels. HRG-evoked signal transduction differed between the cells with normal ErbB expression and the cells overexpressing ErbB2. The activation of both ERK and PI3-K was essential for HRG regulation of COXII, i.e., blockage of either pathway eliminated HRG-mediated alteration. This is the first report demonstrating that the expression of mitochondria-encoded COXII is HRG-responsive. The levels of ErbB2 expression are decisive for the diverse biological activities of HRG.