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.

Calcium transport and signaling in the mammary gland: Targets for breast cancer

The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of reaulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer. (c) 2005 Elsevier B.V. All rights reserved.

Expression profiling correlates with treatment response in women with advanced serous epithelial ovarian cancer

The majority of epithelial ovarian carcinomas are of serous subtype, with most women presenting at an advanced stage. Approximately 70% respond to initial chemotherapy but eventually relapse. We aimed to find markers of treatment response that might be suitable for routine use, using the gene expression profile of tumor tissue. Thirty one women with histologically-confirmed late-stage serous ovarian cancer were classified into 3 groups based on response to treatment (nonresponders, responders with relapse less than 12 months and responders with no relapse within 12 months). Gene expression profiles of these specimens were analyzed with respect to treatment response and survival (minimum 36 months follow-up). Patients' clinical features did not correlate with prognosis, or with specific gene expression patterns of their tumors. However women who did not respond to treatment could be distinguished from those who responded with no relapse within 12 months based on 34 gene transcripts (p < 0.02). Poor prognosis was associated with high expression of inhibitor of differentiation-2 (ID2) (p = 0.001). High expression of decorin (DCN) and ID2 together was strongly associated with reduced survival (p = 0.003), with an estimated 7-fold increased risk of dying (95% CI 1.9-29.6; 14 months survival) compared with low expression (44 months). Immunohistochemical analysis revealed both nuclear and cytoplasmic distribution of ID2 in ovarian tumors. High percentage of nuclear staining vas associated with poor survival, although not statistically significantly. In conclusion, elevated expression of ID2 and DCN was significantly associated with poor prognosis in a homogeneous group of ovarian cancer patients for whom survival could not be predicted from clinical factors. (c) 2006 Wiley-Liss, Inc.

Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL- 1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSC LC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 and DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2$-deoxy-cytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC.

Evaluation of oestrogen and progesterone receptor status in HER-2 positive breast carcinomas and correlation with outcome

Aim: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients. Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/ PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed > 80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen. Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

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.

On the simultaneous use of clinical and microarray expression data in the cluster analysis of tissue samples

This paper considers a model-based approach to the clustering of tissue samples of a very large number of genes from microarray experiments. It is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. Frequently in practice, there are also clinical data available on those cases on which the tissue samples have been obtained. Here we investigate how to use the clinical data in conjunction with the microarray gene expression data to cluster the tissue samples. We propose two mixture model-based approaches in which the number of components in the mixture model corresponds to the number of clusters to be imposed on the tissue samples. One approach specifies the components of the mixture model to be the conditional distributions of the microarray data given the clinical data with the mixing proportions also conditioned on the latter data. Another takes the components of the mixture model to represent the joint distributions of the clinical and microarray data. The approaches are demonstrated on some breast cancer data, as studied recently in van't Veer et al. (2002).

Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA

The effects of ethanol on PPARS in MCF-7 human breast cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in various metabolic diseases. In the liver, PPARα is involved in alcohol metabolism and may lead to the development of alcoholic fatty liver and other alcohol mediated liver injuries. PPARβ modulation by ethanol induces abnormal myelin production by oligodendrocytes. PPARα and PPARβ are PPAR isoforms expressed in the human breast cell lines. Epidemiological studies show a positive correlation between alcohol intake and breast cancer risk, however, the molecular mechanisms involved are unclear. We hypothesized that ethanol would affect the expression and transactivation of human PPAR isoforms in estrogen receptor (ER) positive and ER negative breast cancer cells. Using real time RT-PCR we looked at the transcription of PPAR isoforms in the presence of increasing concentrations of ethanol and saw isoform and time dependent specific effects. Gene reporter assays enabled us to ascertain the effects of ethanol on ligand-mediated activation of human PPARα and PPARβ at concentrations equivalent to both moderate and chronic alcohol consumption. Ethanol differentially blocked the ligand-mediated activation of both PPARα and PPARβ. Since PPARα and PPARβ are involved in the differentiation and proliferation of breast cancer cells, PPARs may be a possible mechanism involved in the effect of ethanol in breast cancer.