Breast Cancer Research - Latest articles

Serum biomarker profiles and response to neoadjuvant chemotherapy for locally advanced breast cancer

2008-05-12

IntroductionNeoadjuvant chemotherapy has become the standard of care for the diverse population of women diagnosed with locally advanced breast cancer. Serum biomarker levels are increasingly being investigated for their ability to predict therapy response and aid in the development of individualized treatment regimens. Multianalyte profiles may offer greater predictive power for neoadjuvant treatment response than the individual biomarkers currently in use. Materials and Methods: Serum samples were collected from 44 patients enrolled in a phase I-II, open-label study of liposomal doxorubicin and paclitaxel in combination with whole breast hyperthermia for the neoadjuvant treatment of locally advanced breast cancer (stage IIB or III). Samples were collected prior to each of four rounds of treatment and prior to definitive surgery. Samples were assayed by Luminex for 55 serum biomarkers including: cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipokines, cytokines, chemokines, hormones, and other proteins. Results: Biomarker levels were compared retrospectively with clinical and pathologic treatment response. Univariate analysis of the data identified several groups of biomarkers that differed significantly among treatment outcome groups early in the course of neoadjuvant chemotherapy. Multivariate statistical analysis revealed multi-biomarker panels that could differentiate between treatment response groups with high sensitivity and specificity. Conclusions: We demonstrate here that serum biomarker profiles may offer predictive power concerning treatment response and outcome in the neoadjuvant setting. The continued development of these findings will be of considerable clinical utility in the design of treatment regimens for individual breast cancer patients. Trial Registration: #NCT00346229.

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells

2008-05-12

IntroductionTumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer. Methods: This study was designed to determine the effects of the NO-releasing prodrug JS-K on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion was determined using Matrigel invasion assays. The expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) were determined using an MMP array kit and enzyme-linked immunosorbent assays. The activity and expression of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) were determined using Western blot. Results: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (p < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (p < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (p < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of ERK1/2 and JNK were unaffected. Conclusions: We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Estrogen and progesterone induce persistent increases in p53-dependent apoptosis and suppress mammary tumors in BALB/c-Trp53+/- mice

2008-05-09

IntroductionTreatment with estrogen and progesterone mimics the protective effect of parity on mammary tumors in rodents and depends upon the activity of p53. The following experiments tested whether exogenous estrogen and progesterone prime p53 to be more responsive to DNA damage and if these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome. Methods: Mice that differ in p53 status (Trp53+/+, Trp53+/-, Trp53-/-) were treated with estrogen and progesterone (E+P) for 14 days, then were tested for p53-dependent responses to ionizing radiation. Responses were also examined in parous and age-matched virgins. The effects of hormonal exposures on tumor incidence were examined in BALB/c-Trp53+/- mammary tissues. Results: Nuclear accumulation of p53 and apoptotic responses were increased similarly in the mammary epithelium from E+P-treated and parous mice compared to placebo and age-matched virgins. This effect is sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone-stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c-Trp53+/- mice, but were intermediate compared to Trp53+/+ and Trp53-/- tissues indicating haploinsufficiency. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. The majority of tumors lacked estrogen receptor (ER), but ER-positive tumors were observed in both nulliparous and parous mice. However, apoptotic responses to ionizing radiation and tumor incidence did not differ among outgrowths of epithelial transplants from E+P-treated donors and nulliparous donors. Conclusions: Therefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppresses mammary tumorigenesis, but this effect was not retained in epithelial outgrowths.

Intact and total insulin-like growth factor-binding protein-3 (IGFBP-3) levels in relation to breast cancer risk factors: a cross-sectional study

Caroline Diorio, Jacques Brisson, Sylvie Berube and Michael Pollak — 2008-05-09

IntroductionLevels of insulin-like growth factor (IGF)-I and its main binding protein (IGFBP-3) have been associated with breast cancer risk among premenopausal women. However, associations of IGFBP-3 levels with breast cancer risk have been inconsistent, possibly due to the different predominant forms of circulating IGFBP-3 (intact versus fragmented) that were measured in these studies. Here, we examine the association of breast cancer risk factors with intact and total IGFBP-3 levels. Methods: This cross-sectional study includes 737 premenopausal women recruited at screening mammography. Plasma IGF-I, intact and total IGFBP-3 levels were measured by ELISA methods. Percent and absolute breast density were evaluated using a computer-assisted method. The associations were evaluated using generalized linear models and Pearson's (r) or Spearman's (rs) partial correlation coefficients. Results: Means +/- standard deviations of intact and total IGFBP-3 levels (ng/mL) were 1044 +/- 234 and 4806 +/- 910, respectively. Intact and total IGFBP-3 levels were correlated with age and smoking. Levels of intact IGFBP-3 were negatively correlated with WHR (r = -0.128; P = 0.0005), parity (rs = -0.078; P = 0.04), alcohol intake (r = -0.137; P = 0.0002), and positively correlated with energy intake (r = 0.075; P = 0.04). In contrast, total IGFBP-3 levels were positively correlated with WHR (r = 0.115; P = 0.002), parity (rs = 0.089; P = 0.02), BMI (r = 0.115; P = 0.002), physical activity (r = 0.118; P = 0.002), IGF-I levels (r = 0.588; P < 0.0001), and negatively correlated with percent or absolute breast density (r = -0.095; P = 0.01 and r = -0.075; P = 0.04, respectively). Conclusions: Our data show that associations of some breast cancer risk factors with intact levels of IGFBP-3 are different from those with total (intact and fragmented) IGFBP-3 levels. These findings suggest that different molecular forms of IGFBP-3 may bear different relations to premenopausal breast cancer risk.

Breast cancer tumor growth estimated through mammography screening data

2008-05-08

IntroductionKnowledge of tumor growth is important in the planning and evaluation of screening programs, clinical trials, and epidemiological studies. Studies of tumor growth rates in humans are usually based on small and selected samples. In the present study based on the Norwegian Breast Cancer Screening Program, tumor growth was estimated from a large population using a new estimating procedure/model. Methods: A likelihood based estimating procedure was used, where both tumor growth and screen test sensitivity [STS] were modeled as continuously increasing functions of tumor size. The method was applied to cancer incidence and tumor measurement data from 395 188 women aged 50-69 years. Results: Tumor growth varied considerably between subjects, with 5 % of tumors using less than 1.2 months to grow from 10 to 20 mm in diameter, and another 5 % using more than 6.3 years. The mean time a tumor needed to grow from 10 to 20 mm in diameter was estimated to 1.7 years, increasing with age. STS was estimated to increase sharply with tumor size, going from 26 % at 5 mm to 91 % at 10 mm. Compared to previously used Markov models for tumor progression, the applied model gave considerably higher model fit (85 % increased predictive power) and provided estimates directly linked to tumor size. Conclusion: Screening data with tumor measurements can provide populationbased estimates of tumor growth and STS directly linked to tumor size. There is a large variation in breast cancer tumor growth, with faster growth among younger women.

Autoantibodies as potential biomarkers for breast cancer

2008-05-06

IntroductionCurrently only a limited number of tumor markers for breast cancer are available. Antibodies to tumor-associated proteins may expand the number of available tumor markers for breast cancer and be used together in a serum profile to enhance sensitivity and specificity. Methods: In this study, we interrogated a breast cancer cDNA T7 phage library for tumor-associated proteins using biopan enrichment techniques with sera from normal and breast cancer patients. The enrichment of tumor-associated proteins after biopanning was tested using plaque-lift assay and immunochemical detection. The putative tumor-associated phage clones were collected for PCR and sequencing analysis. Unique and ORF phage-expressed proteins were then used to develop phage protein enzyme-linked immunosorbent assays (ELISAs) to measure corresponding autoantibodies using 87 breast cancer patients and 87 normal serum samples. Logistic regression model and leave-one-out validation were used to evaluate predictive accuracies with single marker as well as combined markers. Identities of those selected proteins were revealed through sequence BLAST. Results: We harvested 100 putative tumor-associated phage clones after biopan enrichment. Sequencing analysis revealed that six phage proteins were in-frame and unique. Antibodies to these six phage-expressed proteins were measured by ELISAs and results showed that three of the phage clones had statistical significance in discriminating patients from normals. BLAST results of the three proteins showed great matches to ASB-9, SERAC1, and RELT. Measurements of the three predictive phage proteins were combined in a logistic regression model that achieved 80% sensitivity and 100% specificity in prediction of sample status, whereas leave-one-out validation achieved 77.0% sensitivity and 82.8% specificity among 87 patient and 87 control samples. ROC curve analysis and leave-one-out method both showed that combined measurements of the three antibodies were more predictive of disease than any of the single antibodies studied, underscoring the importance of identifying multiple potential markers. Conclusions: For breast cancer, serum autoantibody profiling is a promising approach for early detection and diagnosis. Rather than one, a panel of autoantibodies appears preferable to achieve superior accuracy. Further refinements will need to be made to further improve the accuracy. Once refined the assay must be applied to a prospective patient population to demonstrate applicability.

Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1 mediated metabolism

2008-05-02

Background: The natural product eupatorin has been reported to have antiproliferative activity in tumor cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1 and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumor cells. In this study we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family mediated metabolism. Methods: The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468, and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined. Results: Eupatorin showed a dose dependent inhibitory effect of cell growth on MDA-MB-468 cells with a sub-micromolar IC50 whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect was mainly attributed to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells, as measured by EROD assay and western immunoblotting. Moreover, CYP1 family enzymes were shown to metabolize eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was noticed in MCF-10A cells which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1-inhibitor acacetin. Conclusion: The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumor cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate, which should be further examined in an in vivo study.

Nonsteroidal anti-inflammatory drugs and breast cancer risk in the National Institutes of Health–AARP Diet and Health Study

2008-04-30

IntroductionBy inhibiting cyclooxygenase-2, nonsteroidal anti-inflammatory drugs (NSAIDs) decrease aromatase activity and might reduce breast cancer risk by suppressing estrogen synthesis. Epidemiologic evidence for a protective role of NSAIDs in breast cancer, however, is equivocal. Methods: We tested NSAID use for its association with breast cancer incidence in the National Institutes of Health–AARP Diet and Health Study, where 127,383 female AARP (formerly known as the American Association of Retired Persons) members with no history of cancer, aged 51 to 72 years, completed a mailed questionnaire (1996 to 1997). We estimated relative risks of breast cancer for NSAID exposures using multivariate Cox proportional hazards regression models. The state cancer registry and mortality index linkage identified 4,501 primary incident breast cancers through 31 December 2003, including 1,439 estrogen receptor (ER)-positive cancers and 280 ER-negative cancers. Results: Proportional hazards models revealed no statistically significant association between overall NSAIDs and total breast cancer. As cyclooxygenase inhibition by aspirin (but not other NSAIDs) is irreversible, we tested associations by NSAID type. Although we observed no significant differences in risk for daily use (versus nonuse) of aspirin (relative risk = 0.93, 95% confidence interval = 0.85 to 1.01) or nonaspirin NSAIDS (relative risk = 0.96, 95% confidence interval = 0.87 to 1.05), risk of ER-positive breast cancer was significantly reduced with daily aspirin use (relative risk = 0.84, 95% confidence interval = 0.71 to 0.98) – a relationship not observed for nonaspirin NSAIDS. Neither aspirin nor nonaspirin NSAIDs were associated with risk of ER-negative breast cancer. Conclusion: Breast cancer risk was not significantly associated with NSAID use, but daily aspirin use was associated with a modest reduction in ER-positive breast cancer. Our results provide support for further evaluating relationships by NSAID type and breast cancer subtype.

Cancer and fertility preservation: fertility preservation in breast cancer patients

2008-04-29

Aggressive chemotherapy has improved the life expectancy for reproductive-age women with breast cancer, but it often causes infertility or premature ovarian failure due to destruction of the ovarian reserve. Many questions concerning fertility preservation in breast cancer patients remain unanswered – for example, whether fertility preservation methods interfere with chemotherapy, and whether subsequent pregnancy has negative effects on the prognosis. Fertility preservation is a critical factor in decision-making for younger breast cancer patients, however, and clinicians should address this. The present article reviews the incidence of chemotherapy-induced amenorrhea, and discusses fertility-preservation options and the prognosis for patients who become pregnant after breast cancer.

Evaluation of biological pathways involved in chemotherapy response in breast cancer

2008-04-29

IntroductionOur goal was to examine the association between biological pathways and response to chemotherapy in estrogen receptor (ER)-positive and ER-negative breast cancers separately. Methods: Gene set enrichment analysis including 852 predefined gene sets, was applied to gene expression data from 51 ER-negative and 82 ER-positive breast cancers that were all treated with a preoperative paclitaxel, 5-fluoruracil, doxorubicin and cyclophosphamide chemotherapy. Results: Twenty-seven (53%) ER-negative and 7 (9%) ER-positive patients had pathologic complete response (pCR) to therapy. Among the ER-negative cancers, a proliferation gene signature (FDR q=0.1), the Genomic Grade Index (FDR q =0.044) and the E2F3 pathway (FDR q =0.22, p=0.07) signature were enriched in the pCR group. Among the ER-positive cancers, the proliferation signature (FDR q =0.001) and the Genomic Grade Index (FDR q =0.015) were also significantly enriched in cases with pCR. Ki67 expression, as single gene marker of proliferation, did not provide the same information as the entire proliferation signature. An ER-associated gene set (FDR q = 0.03) and a mutant p53-gene signature (FDR q = 0.0019) were enriched in ER-positive cancers with residual cancer. Conclusion: Proliferation- and genomic grade-related gene signatures are associated with chemotherapy sensitivity in both ER-negative and -positive breast cancers. Genes involved in the E2F3 pathway are associated with chemotherapy sensitivity among ER-negative cancers. The mutant p53-signature and expression of ER-related genes were associated with lower sensitivity to chemotherapy in ER-positive breast cancers only.

TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells

Jillian Howlin, Jeanette Rosenkvist and Tommy Andersson — 2008-04-24

Background: Amplification of the TNK2 gene in primary tumours was shown to correlate with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42 driven migration by activation of BCAR1, however, distinct from this effect, is evidence for a role of TNK2 in the regulation of EGFR endocytosis and degradation. In this study, we sought to investigate if negative targeting of TNK2 by siRNA could be used to inhibit cancer cell invasion, establish the contribution of its effect on the EGFR and consequently attempt to resolve the issue of TNK2's mechanism of action. Methods: We used siRNA to knockdown expression of TNK2 and its proposed effector BCAR1 in order to analyse the effect of this knockdown on cancer cell behaviour in vitro. We examined morphological changes using phase-contrast microscopy and immuohistochemistry. Functional parameters examined included apoptosis, proliferation, migration and invasion. We also performed flow cytometry analysis to examine EGFR cell surface expression and Western blot to examine total EGFR levels. Results: We observed that targeting of TNK2 by siRNA in breast cancer cells resulted in distinct morphological changes characterised by a stellate appearance and absence of protrusions at membrane edges. These changes were not recapitulated upon siRNA targeting of BCAR1. We thus hypothesised that a component of the effects induced by TNK2 may be independent of BCAR1. Consistent with the idea of an alternative mechanism for TNK2, we observed that TNK2 associates with activated EGFR in breast cancer cells in a TNK2-kinase independent manner. Furthermore, we demonstrated that TNK2 functions to maintain EGFRs on the cell surface. We could demonstrate that the main functional effect of activating these surface EGFRs in breast cancer cells is stimulation of migration. In accordance, TNK2 silencing by siRNA, led to a significant reduction in cell surface EGFR and a concomitant decrease in the migratory and invasive capacity of breast cancer cells. Conclusions: Our data suggest that TNK2 can enhance migration and invasion of breast cancer cells via preservation of EGFR expression, notwithstanding its previously reported signalling via BCAR1, explaining its oncogenic behaviour in vitro and correlation with metastatic human breast cancer in vivo.