Mapping loss of heterozygosity in normal human breast cells from BRCA1/2 carriers

We have studied loss of heterozygosity at the BRCA1 and BRCA2 loci in 992 normal cell clones derived from topographically defined areas of normal tissue in four samples from BRCA1/BRCA2 mutation carriers. The frequency of loss of heterozygosity in the clones was low ( 1.01%), but it was found in all four samples, whether or not a tumour was present. Topographical mapping revealed that the genetic changes were clustered in some breast samples. Our study confirms the previous finding that a field of genetic instability can exist around a tumour, suggesting that sufficient tissue must be removed at surgery to avoid local recurrence. We also demonstrate that such a field of genetic change can exist in morphologically normal tissue before a tumour develops and, for the first time, we demonstrate that the field is of a size greater than one terminal duct-lobular unit. The genetic changes are not identical, however, which suggests that genetic instability in these regions may play an early role in tumour development. We also confirm and extend our original observation of loss of the wild-type BRCA1 allele in some clones, and loss of the mutant allele in others, demonstrating that loss of either allele is a stochastic event.

FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas

Purpose: Classic lobular carcinomas (CLC) account for 10% to 15% of all breast cancers. At the genetic level, CLCs show recurrent physical loss of chromosome16q coupled with the lack of E-cadherin (CDH1 gene) expression. However, little is known about the putative therapeutic targets for these tumors. The aim of this study was to characterize CLCs at the molecular genetic level and identify putative therapeutic targets. Experimental Design: We subjected 13 cases of CLC to a comprehensive molecular analysis including immunohistochemistry for E-cadherin, estrogen and progesterone receptors, HER2/ neu and p53; high-resolution comparative genomic hybridization (HR-CGH); microarray-based CGH (aCGH); and fluorescent and chromogenic in situ hybridization for CCND1 and FGFR1. Results: All cases lacked the expression of E-cadherin, p53, and HER2, and all but one case was positive for estrogen receptors. HR-CGH revealed recurrent gains on 1q and losses on 16q (both, 85%). aCGH showed a good agreement with but higher resolution and sensitivity than HR-CGH. Recurrent, high level gains at 11q13 (CCND1) and 8p12-p11.2 were identified in seven and six cases, respectively, and were validated with in situ hybridization. Examination of aCGH and the gene expression profile data of the cell lines, MDA-MB-134 and ZR-75-1, which harbor distinct gains of 8p12-p11.2, identified FGFR1 as a putative amplicon driver of 8p12-p11.2 amplification in MDA-MB-134. Inhibition of FGFR1 expression using small interfering RNA or a small-molecule chemical inhibitor showed that FGFR1 signaling contributes to the survival of MDA-MB-134 cells. Conclusions: Our findings suggest that receptor FGFR1 inhibitors may be useful as therapeutics in a subset of CLCs.

A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells

Phytochemicals have provided an abundant and effective source of therapeutics for the treatment of cancer. Here we describe the characterization of a novel plant toxin, persin, with in vivo activity in the mammary gland and a p53-, estrogen receptor-, and Bcl-2-independent mode of action. Persin was previously identified from avocado leaves as the toxic principle responsible for mammary gland-specific necrosis and apoptosis in lactating livestock. Here we used a lactating mouse model to confirm that persin has a similar cytotoxicity for the lactating mammary epithelium. Further in vitro studies in a panel of human breast cancer cell lines show that persin selectively induces a G(2)-M cell cycle arrest and caspase-dependent apoptosis in sensitive cells. The latter is dependent on expression of the BH3-only protein Bim. Bim is a sensor of cytoskeletal integrity, and there is evidence that unique structure of the compound, persin could represent a novel class of microtubule-targeting agent with potential specificity for breast cancers.

Disruption of BRCA I function results in telomere lengthening and increased anaphase bridge formation in immortalized cell lines

BRCA1 is a tumor suppressor that functions in controlling cell growth and maintaining genomic stability. BRCA1 has also been implicated in telomere maintenance through its ability to regulate the transcription of hTERT, the catalytic subunit of telomerase, resulting in telomere shortening, and to colocalize with the telomere-binding protein TRF1. The high incidence of nonreciprocal translocations in tumors arising from BRCA1 mutation carriers and Brca1-null mice also raises the possibility that BRCA1 plays a role in telomere protection. To date, however, the consequences for telomere status of disrupting BRCA1 have not been reported. To examine the role of BRCA1 in telomere regulation, we have expressed a dominant-negative mutant of BRCA1 (trBRCA1), known to disrupt multiple functions of BRCA1, in telomerase-positive mammary epithelial cells (SVCT) and telomerase-negative ALT cells (GM847). In SVCT cells, expression of trBRCA1 resulted in an increased incidence of anaphase bridges and in an increase in telomere length, but no change in telomerase activity. In GM847 cells, trBRCA1 also increased anaphase bridge formation but did not induce any change in telomere length. BRCA1 colocalized with TRF2 in telomerase-positive cells and with a small subset of ALT-associated PML bodies (APBs) in ALT cells. Together, these results raise the possibility that BRCA1 could play a role in telomere protection and suggest a potential mechanism for one of the phenotypes of BRCA1 deficient cells. (c) 2005 Wiley-Liss, Inc.

The key role of CD40 ligand in overcoming tumor-induced dendritic cell dysfunction

Overcoming dendritic cell (DC) dysfunction is a prerequisite for successful active immunotherapy against breast cancer. CD40 ligand (CD40L), a key molecule in the interface between T-lymphocytes and DCs, seems to be instrumental in achieving that goal. Commenting on our data that CD40L protects circulating DCs from apoptosis induced by breast tumor products, Lenahan and Avigan highlighted the potential of CD40L for immunotherapy. We expand on that argument by pointing to additional findings that CD40L not only rescues genuine DCs but also functionally improves populations of immature antigen-presenting cells that fill the DC compartment in patients with breast cancer.

Anti-proliferative effects of novel Glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)2 group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 μM and 10 μM compound (11) and 10 μM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/ or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness. © 2006 Bentham Science Publishers Ltd.

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation ( FISH). Participating laboratories were blinded to these test results. Oestrogen receptor ( ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH ( kappa coefficient=1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good ( kappa coefficient=0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade, A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not, Thus, tyrosine phosphorylation of cyclin D2 may be a hey regulatory target for FGF-2 signaling. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

The role of TG2 in regulating S100A4-mediated mammary tumour cell migration

The importance of S100A4, a Ca2+-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca2+-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and a5ß1 integrin co-signalling pathways linked by activation of PKCa in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

Tissue Transglutaminase (TG2) is a potential therapeutic target in the treatment of chemoresistant breast cancer

Tissue transglutaminase (TG2) has been suggested to be a key player in the progression and metastasis of chemoresistant breast cancer. One of the foremost survival signalling pathways implicated in causing drug resistance in breast cancer is the constitutive activation of NFκB (Nuclear Factor -kappa B) induced by TG2. This study provides a mechanism by which TG2 constitutively activates NFκB which in turn confers chemoresistance to breast cancer cells against doxorubicin. Breast cancer cell lines with varying expression levels of TG2 as well as TG2 null breast cancer cells transfected with TG2 were used as the major cell models for this study. This study made use of cell permeable and impermeable TG2 inhibitors, specific TG2 and Rel A/ p65 targeting siRNA, TG2 functional blocking antibodies, IKK inhibitors and a specific targeting peptide against Rel A/p65 to investigate the pathway of activation involved in the constitutive activation of NFκB by TG2 leading to drug resistance. Crucial to the activation of Rel A/p65 and drug resistance in the breast cancer cells is the interaction between the complex of IκBα and Rel A/p65 with TG2 which results in the dimerization of Rel A/p65 and polymerization of IκBα. The association of TG2 with the IκBα-NFκB complex was determined to be independent of IKKα/β function. The polymerized IκBα is degraded in the cytoplasm by the μ-calpain pathway which allows the cross linked Rel A/ p65 dimers to translocate into the nucleus. Using R283 and ZDON (cell permeable TG2 activity inhibitors) and specific TG2 targeting siRNA, the Rel A/ p65 dimer formation could be inhibited. Co-immunoprecipitation studies confirmed that the phosphorylation of the Rel A/p65 dimers at the Ser536 residue by IKKε took place in the cell nucleus. Importantly, this study also investigated the transcriptional regulation of the TGM2 gene by the pSer536 Rel A/ p65 dimer and the importance of this TG2-NFκB feedback loop in conferring drug resistance to breast cancer cells. This data provides evidence that TG2 could be a key therapeutic target in the treatment of chemoresistant breast cancer.