Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Accelerated partial breast irradiation with interstitial brachytherapy as second conservative treatment for ipsilateral breast tumour recurrence: multicentric study of the GEC-ESTRO Breast Cancer Working Group

PURPOSE To analyse the clinical outcome after salvage lumpectomy and multi-catheter brachytherapy (MCB) for ipsilateral breast tumour recurrence (IBTR). MATERIAL AND METHODS Between 09/00 and 09/10, 217 patients presenting an IBTR underwent lumpectomy and MCB (low, pulsed, or high-dose rate). Survival rates without second local recurrence (2nd LR), distant metastasis (DM), and overall survival (OS) were analysed as well as late effects and cosmetic results. Univariate and multivariate analyses (MVA) based on IBTR data were performed to find prognostic factors for 2nd LR, DM, and OS. RESULTS Median follow-up after the IBTR was 3.9 years [range: 1.1-10.3]. Five and 10-year actuarial 2nd LR rates were 5.6% [range: 1.5-9.5] and 7.2% [range: 2.1-12.1], respectively. Five and 10-year actuarial DM rates were 9.6% [range: 5.7-15.2] and 19.1% [range: 7.8-28.3], respectively. Five and 10-year actuarial OS rates were 88.7% [range: 83.1-94.8] and 76.4% [range: 66.9-87.3], respectively. In MVA, histological grade was prognostic factor for 2nd LR (p=0.008) and OS (p=0.02); while tumour size was prognostic factor for DM (p=0.03). G3-4 complication rate was 11%. Excellent/good cosmetic result was achieved in 85%. CONCLUSION This study suggests that in case of IBTR, lumpectomy plus MCB is feasible and effective in preventing 2nd LR with an OS rate at least equivalent to those achieved with salvage mastectomy.

Investigating the Effects of Silencing EphA2 in Metastatic Breast Cancer Cells

EphA2, also known as ECK (epithelial cell kinase), is a transmembrane receptor tyrosine kinase that is commonly over-expressed in cancers such as those of the prostate, colon, lung, and breast. For breast cancers, EphA2 overexpression is most prominent in the ER-negative subtype, and is associated with a higher rate of lung metastasis. Studies conducted to demonstrate the role of EphA2 in a non-cancerous environment have shown that it is very important in developmental processes, but not in normal adult tissues. These results make EphA2 a prospective therapeutic target since new therapies are needed for the more aggressive ER-negative breast cancers. A panel of breast cancer cell lines was screened for expression of EphA2 by immunoblotting. Several of the overexpressing cell lines, including BT549, MDA-MB-231, and HCC 1954 were selected for experiments utilizing siRNA for transient knockdown and shRNA for stable knockdown. Targeted knockdown of EphA2 was measured using RT-PCR and immunoblotting techniques. Here, the functions of EphA2 in the process of metastasis have been elucidated using in vitro assays that indicate cancer cell metastatic potential and in vivo studies that reveal the effect of EphA2 on mammary fat pad tumor growth, vessel formation, and the effect of using EphA2-targeting siRNA on pre-established mammary fat pad tumors. A decrease in EphA2 expression both in vitro and in vivo correlated with reduced migration and experimental metastasis of breast cancer cells. Current work is being done to investigate the mechanism behind EphA2’s participation in some of these processes. These studies are important because they have contributed to understanding the role that EphA2 plays in the progression of breast cancers to a metastatic state.

Development of a Tumour Growth Inhibition Model to Elucidate the Effects of Ritonavir on Intratumoural Metabolism and Anti-tumour Effect of Docetaxel in a Mouse Model for Hereditary Breast Cancer

In a mouse tumour model for hereditary breast cancer, we previously explored the anti-cancer effects of docetaxel, ritonavir and the combination of both and studied the effect of ritonavir on the intratumoural concentration of docetaxel. The objective of the current study was to apply pharmacokinetic (PK)-pharmacodynamic (PD) modelling on this previous study to further elucidate and quantify the effects of docetaxel when co-administered with ritonavir. PK models of docetaxel and ritonavir in plasma and in tumour were developed. The effect of ritonavir on docetaxel concentration in the systemic circulation of Cyp3a knock-out mice and in the implanted tumour (with inherent Cyp3a expression) was studied, respectively. Subsequently, we designed a tumour growth inhibition model that included the inhibitory effects of both docetaxel and ritonavir. Ritonavir decreased docetaxel systemic clearance with 8% (relative standard error 0.4%) in the co-treated group compared to that in the docetaxel only-treated group. The docetaxel concentration in tumour tissues was significantly increased by ritonavir with mean area under the concentration-time curve 2.5-fold higher when combined with ritonavir. Observed tumour volume profiles in mice could be properly described by the PK/PD model. In the co-treated group, the enhanced anti-tumour effect was mainly due to increased docetaxel tumour concentration; however, we demonstrated a small but significant anti-tumour effect of ritonavir addition (p value <0.001). In conclusion, we showed that the increased anti-tumour effect observed when docetaxel is combined with ritonavir is mainly caused by enhanced docetaxel tumour concentration and to a minor extent by a direct anti-tumour effect of ritonavir.

Circulating tumor cells as trigger to hematogenous spreads and potential biomarkers to predict the prognosis in ovarian cancer.

Despite several improvements in the surgical field and in the systemic treatment, ovarian cancer (OC) is still characterized by high recurrence rates and consequently poor survival. In OC, there is still a great lack of knowledge with regard to cancer behavior and mechanisms of recurrence, progression, and drug resistance. The OC metastatization process mostly occurs via intracoelomatic spread. Recent evidences show that tumor cells generate a favorable microenvironment consisting in T regulatory cells, T infiltrating lymphocytes, and cytokines which are able to establish an "immuno-tolerance mileau" in which a tumor cell can become a resistant clone. When the disease responds to treatment, immunoediting processes and cancer progression have been stopped. A similar inhibition of the immunosuppressive microenvironment has been observed after optimal cytoreductive surgery as well. In this scenario, the early identification of circulating tumor cells could represent a precocious signal of loss of the immune balance that precedes cancer immunoediting and relapse. Supporting this hypothesis, circulating tumor cells have been demonstrated to be a prognostic factor in several solid tumors such as colorectal, pancreatic, gastric, breast, and genitourinary cancer. In OC, the role of circulating tumor cells is still to be defined. However, as opposed to healthy women, circulating tumor cells have been demonstrated in peripheral blood of OC patients, opening a new research field in OC diagnosis, treatment monitoring, and follow-up.

COX-2 expression in operable triple negative breast cancer: A hospital based cross-sectional study

Objectives. Triple Negative Breast Cancer (TNBC) lack expression of estrogen receptors (ER), progesterone receptors (PR), and absence of Her2 gene amplification. Current literature has identified TNBC and over-expression of cyclo-oxygenase-2 (COX-2) protein in primary breast cancer to be independent markers of poor prognosis in terms of overall and distant disease free survival. The purpose of this study was to compare COX-2 over-expression in TNBC patients to those patients who expressed one or more of the three tumor markers (i.e. ER, and/or PR, and/or Her2).^ Methods. Using a secondary data analysis, a cross-sectional design was implemented to examine the association of interest. Data collected from two ongoing protocols titled "LAB04-0657: a model for COX-2 mediated bone metastasis (Specific aim 3)" and "LAB04-0698: correlation of circulating tumor cells and COX-2 expression in primary breast cancer metastasis" was used for analysis. A sample of 125 female patients was analyzed using Chi-square tests and logistic regression models. ^ Results. COX-2 over-expression was present in 33% (41/125) and 28% (35/124) patients were identified as having TNBC. TNBC status was associated with elevated COX-2 expression (OR= 3.34; 95% CI= 1.40–8.22) and high tumor grade (OR= 4.09; 95% CI= 1.58–10.82). In a multivariable analysis, TNBC status was an important predictor of COX-2 expression after adjusting for age, menopausal status, BMI, and lymph node status (OR= 3.31; 95% CI: 1.26–8.67; p=0.01).^ Conclusion. TNBC is associated with COX-2 expression—a known marker of poor prognosis in patients with operable breast cancer. Replication of these results in a study with a larger sample size, or a future randomized clinical trial demonstrating an improved prognosis with COX-2 suppression in these patients would support this hypothesis.^

Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184135 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study. (c) 2005 Elsevier Inc. All rights reserved.

Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families

Genetic screening of women from multiple-case breast cancer families and other research-based endeavors have identified an extensive collection of germline variations of BRCA1 and BRCA2 that can be classified as deleterious and have clinical relevance. For some variants, such as those in the conserved intronic splice site regions which are highly likely to alter splicing, it is not possible to classify them based on the identified DNA sequence variation alone. We studied 11 multiple-case breast cancer families carrying seven distinct splice site region genetic alterations in BRCA1 or BRCA2 (BRCA1, c.IVS6-2delA, c.IVS9-2A>C, c.IVS4-1G>T, c.IVS20+1G>A and BRCA2, c.IVS17-1G>C, c.IVS20+1G>A, c.IVS7-1G>A) and applied SpliceSiteFinder to predict possible changes in efficiency of splice donor and acceptor sites, characterized the transcripts, and estimated the average age-specific cumulative risk (penetrance) using a modified segregation analysis. SpliceSiteFinder predicted and we identified transcipts that illustrated that all variants caused exon skipping, and all but two led to frameshifts. The risks of breast cancer to age 70 yrs, averaged over all variants, over BRCA1 variants alone, and over BRCA2 variants alone, were 73% (95% confidence interval 47-93), 64% (95%CI 28-96) and 79% (95%CI 48-98) respectively (all P

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.

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.

Evaluating the evidence for targeting FOXO3a in breast cancer:a systematic review

Background: Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics. Methods: Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment. Results: Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells. Discussion: FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.