Paclitaxel-resistant Breast Cancer Research Articles

ERα positively regulated DNMT1 expression by binding to the gene promoter region in human breast cancer MCF-7 cells

Estrogen receptors (ER) are expressed in approximately 65% of human breast cancer. Clinical trials and retrospective analyses showed that ER-positive (ER+) tumors were more vulnerable to development of chemotherapy resistance than ER-negative (ER−) tumors. The underlying mechanism is still to be elucidated. Aberrant DNA methylation has been recognized to be associated with cancer chemotherapy resistance. Recently, steroid hormone and their receptors have been found to be involved in the regulation of methyltransferases (DNMTs) and thereby contribute to chemotherapy resistance. The purpose of this study is to explore whether ERα could directly regulate the DNMTs expression. We first analyzed the methylation alterations and its correlation with the expression levels of three types of DNMTs in our established paclitaxel-resistant breast cancer lines, MCF-7(ER+)/PTX and MDA-MB-231(ER−)/PTX cell lines, using qMSP, real-time PCR and Western blot. Then we determined the function of ERα in regulation of DNMT1 using luciferase report gene systems. Our data demonstrated for the first time that ERα could upregulate DNMT1 expression by directly binding to the DNMT1 promoter region in MFC-7(ER+)/PTX cells.

Lin28 Mediates Paclitaxel Resistance by Modulating p21, Rb and Let-7a miRNA in Breast Cancer Cells

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.

Abstract 5585: Generation of novel cell lines for analyzing the role of TLR4 in breast cancer

Abstract Background: Toll like receptor 4 (TLR4) is a transmembrane receptor expressed in macrophages that plays a major role in pathogen recognition and activation of innate immunity. TLR4 signaling has been linked to tumorigenesis and promotion of metastasis in ovarian, lung, prostate and head-neck cancers through up-regulation of the expression of inflammatory cytokines (e.g., IL-6, IL-8 and TNF-alpha). These factors increase cancer cell survival and resistance to chemotherapy through up-regulation of anti-apoptotic and down-regulation of apoptotic proteins, respectively. The goal of this study was to generate new isogenic cell lines that would allow analysis of the TLR4 role in paclitaxel resistance in breast cancer. To this end, we used TLR4-positive and negative lines (MDA-MB-231 and HCC1806, respectively) to generate stable TLR4 knockdown and over-expressing line derivatives. Methods: To generate a TLR4 knockdown line, MDA-MB-231 cells were transfected with pisiRNA-TLR4 plasmid and selected with zeocin (50μg/ml). To generate a TLR4 over-expressing line, HCC1806 cells were transfected with pKT2-TLR4-PURO plasmid and selected with puromycin (1μg/ml). Ten to 20 monoclonal colonies were selected from each line and characterized for TLR4 expression and TLR4-dependent cell activities by qRT-PCR and western blot. Results: TLR4 mRNA expression was reduced by >90% as compared with parental line in five out of 8 MDA-MB-231TLR4− clones examined. One clone had 60% reduction and two clones had no effect on TLR4 mRNA determined by qRT-PCR. TLR4 mRNA was up-regulated 1,000-12,000 fold in five (out of 11) clones derived from HCC1806 line. Two clones overexpressed TLR4 mRNA by 300-500 fold and four clones had increased TLR4 expression by 1-6 fold. All selected clones were analyzed for TLR4 functional signaling by exposure to LPS (1μg/ml), which was reduced by 50-70% in MDA-MB-231TLR4− clones and increased by 15-25 folds in HCC1806TLR4+ sub-lines, as compared with cells transfected with empty vectors. Conclusions: These data show that we successfully created sub-lines of MDA-MB-231 with stable knockdown of TLR4 and sub-lines of breast carcinoma HCC1806 line with stable over-expression of TLR4. Ectopically expressed human TLR4 as well as TLR4 downregulating sequence had significant functional effects on the TLR4 signaling as demonstrated by increase and decrease of LPS-activated targets (IL-6, IL-8 and TNF-alpha) in MDA-MB-231TLR4− and HCC1806TLR4+ sub-lines, respectively. These new sub-lines will serve as excellent tools for analyzing the role of the TLR4 pathway signaling in breast cancer progression, metastasis and resistance to therapy. * These authors contributed equally in this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5585. doi:10.1158/1538-7445.AM2011-5585

Abstract 2183: High-throughput screening, identification and characterization of novel kinases involved in chemosensitization of breast cancer cells

Abstract Background - Development of drug resistance is considered to be one of the most challenging issues in chemotherapy for cancer patients. As one of the conventional chemotherapeutic agent, paclitaxel was originally extracted from Pacific yew tree and has been applied widely in ovarian cancer, breast cancer and lung cancer. Resistance to paclitaxel has posed severe problems in cancer treatment. We are therefore interested in identifying the genes that render paclitaxel-resistant breast cancer cells to become more sensitive with the drug treatment. The mechanisms and signaling pathways of these kinases in relations to paclitaxel resistance and their molecular targets have been investigated. Methods - We have screened a shRNA library containing 592 novel kinase genes with 1306 clones for the chemosensitizer loci. DNA was extracted in a high throughput manner, and cell transfection/transduction was carried out on 384 well plates using a unique solid-phase DNA delivery method. The positive hits were selected according to the alteration on their IC50 values for the drug using cell viability assays. Quantitative PCR, Western blot, and caspase assays have been employed to elucidate the signaling pathways of these kinases in response to paclitaxel. Protein purification and interaction studies have also been carried out in attempt to elucidate the putative substrates for the kinases and their interaction partners. Results and Conclusions - We have established a high throughput screening platform for identification of the chemosensitizer loci. High transfection/transduction efficiencies can be achieved using our solid-phase gene delivery method. Several novel unknown kinase genes that render drug-resistant cancer cells to become sensitive have been selected. The positive shRNA hits were further verified by quantitative real-time PCR. Among the 20 hits that showed diminished IC50 values of paclitaxel in breast cancer cells, we have selected two candidates for further studies. Knocking down of TAOK3 or VRK3 in breast cancer cells resulted in reduction of IC50 of paclitaxel to half of the original value. Furthermore, we have investigated the signaling pathways of these 2 kinases in relation to paclitaxel resistance. The different signaling pathways elucidated have revealed the complex nature of drug resistance development in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2183.

Prediction of Paclitaxel Resistance in Breast Cancer: is CYP1B1*3 a New Factor of Influence?

This article focuses on the recent findings by Marsh and colleagues, and also discusses recent findings with regards to breast cancer. Taxanes are amongst the most active agents in the treatment of breast cancer. However, many tumors are intrinsically resistant. Therefore, it would be an enormous progress, if factors could be identified that reliably differentiate between taxane-sensitive and -resistant patients. Marsh and colleagues analyzed the CYP1B1*3 (Val432Leu) polymorphism in patients with high-risk stage III and IV breast cancer, who received dose-intense paclitaxel in combination with doxorubicin and cyclophosphamide. They report for the first time that patients with two leucine alleles in codon 432 of CYP1B1 experience a longer progression-free survival compared with patients with the Val/Leu or Val/Val genotypes. If confirmed in independent cohorts CYP1B1*3 may prove to be an important factor that helps to differentiate between paclitaxel-sensitive and resistant breast cancer patients. However, the mechanism behind the association between CYP1B1*3 and prognosis of paclitaxel-treated patients remains unclear. Several studies provide strong evidence that CYP1B1 does not influence tumor progression independently from paclitaxel chemotherapy, and that CYP1B1 itself does not alter paclitaxel resistance. In addition, CYP1B1 mRNA expression does not correlate with paclitaxel sensitivity of primary tumor cells. Although still speculative, a possible explanation is an association between CYP1B1*3 with still unknown factors that, on their part, influence paclitaxel sensitivity. In the future, studies with SNP chips and studies on the transcriptome, proteome and metabolome level should be performed in order to identify signatures differentiating between paclitaxel-sensitive and -resistant patients.

Pharmacodynamics of Tubulin and Tubulin-Binding Agents: Extending Their Potential Beyond Taxanes

Chemotherapeutic agents that disrupt the assembly or disassembly of microtubules, including paclitaxel and docetaxel, are among the most commonly prescribed anticancer therapies. However, the utility of taxane-based therapy is limited principally by problems with formulation, slow administration, cumulative neurotoxicity, and resistance in part through induction of P-glycoprotein. The broad-spectrum anticancer activity of taxane therapy has encouraged investigators to identify a class of structurally novel microtubulin-stabilizing agents that could produce comparable outcomes with fewer problems. Preclinical studies indicate that epothilones have a broad spectrum activity in paclitaxel-resistant breast cancer models. Several epothilone analogues have displayed promising antitumor activity in initial clinical trials. Ixabepilone, an epothilone derivative in the later stages of clinical development, has exhibited antitumor activity in breast cancers, with or without previous taxane therapy. The most common adverse events associated with ixabepilone are reversible sensory neuropathy and neutropenia. This review briefly outlines the basic science behind microtubule-targeting agents and examines the preclinical studies of several of these agents in breast cancer models. Also discussed are results from clinical trials of epothilones alone and in combination in patients with breast cancer.

2121 POSTER Axillary recurrence after negative sentinel node procedure in breast cancer patients

Chemotherapeutic agents that disrupt the assembly or disassembly of microtubules, including paclitaxel and docetaxel, are among the most commonly prescribed anticancer therapies. However, the utility of taxane-based therapy is limited principally by problems with formulation, slow administration, cumulative neurotoxicity, and resistance in part through induction of P-glycoprotein. The broad-spectrum anticancer activity of taxane therapy has encouraged investigators to identify a class of structurally novel microtubulin-stabilizing agents that could produce comparable outcomes with fewer problems. Preclinical studies indicate that epothilones have a broad spectrum activity in paclitaxel-resistant breast cancer models. Several epothilone analogues have displayed promising antitumor activity in initial clinical trials. Ixabepilone, an epothilone derivative in the later stages of clinical development, has exhibited antitumor activity in breast cancers, with or without previous taxane therapy. The most common adverse events associated with ixabepilone are reversible sensory neuropathy and neutropenia. This review briefly outlines the basic science behind microtubule-targeting agents and examines the preclinical studies of several of these agents in breast cancer models. Also discussed are results from clinical trials of epothilones alone and in combination in patients with breast cancer.

Pharmacogenetic analysis of paclitaxel transport and metabolism genes in breast cancer

Paclitaxel is commonly used in the treatment of breast cancer. Variability in paclitaxel clearance may contribute to the unpredictability of clinical outcomes. We assessed genomic DNA from the plasma of 93 patients with high-risk primary or stage IV breast cancer, who received dose-intense paclitaxel, doxorubicin and cyclophosphamide. Eight polymorphisms in six genes associated with metabolism and transport of paclitaxel were analyzed using Pyrosequencing. We found no association between ABCB1, ABCG2, CYP1B1, CYP3A4, CYP3A5 and CYP2C8 genotypes and paclitaxel clearance. However, patients homozygous for the CYP1B1*3 allele had a significantly longer progression-free survival than patients with at least one Valine allele (P=0.037). This finding could reflect altered paclitaxel metabolism, however, the finding was independent of paclitaxel clearance. Alternatively, the role of CYP1B1 in estrogen metabolism may influence the risk of invasive or paclitaxel resistant breast cancer in patients carrying the CYP1B1*3 allele.

Head-to-head: docetaxel challenges paclitaxel

Among the novel chemotherapeutic drugs introduced in the 1990s, the taxoids have emerged as the most powerful compounds in metastatic breast cancer. For a number of years, clinical trials have investigated the efficacy of the two taxoids, docetaxel (Taxotere | and paclitaxel (Taxol| and oncologists have long asked the question whether the two agents are clinically different. However, until this time, only indirect clinical comparisons of these agents had been performed, which were imprecise owing to differences in patient populations. The TAX 311 study is the first and only head-to-head randomised phase III study to compare directly the efficacy and safety of docetaxel and paclitaxel for patients with advanced breast cancer. An early study in patients with paclitaxel-resistant breast cancer revealed differences between the taxoids and now, with the results of the newly published TAX 311 trial, it is possible to make a definitive comparison of docetaxel and paclitaxel. As previously mentioned, paclitaxel was isolated from the bark of the Pacific yew tree (Taxus brevifolia) and docetaxel was, some years later, prepared from 10-deacetylbaccatin III, isolated from the needles of the European yew tree (Taxus baccata)[1]. Despite both compounds being members of the same class of cytotoxic agent, preclinical studies demonstrated that docetaxel had a number of advantages over paclitaxel. Compared with paclitaxel, docetaxel had a wider cell-cycle activity and demonstrated a greater affinity for the (5-tubulin binding site [2]. These effects were accompanied by greater uptake and slower efflux of docetaxel from tumour cells, resulting in longer intracellular retention time and higher intracellular concentrations than those achieved with paclitaxel[3]. Compared with paclitaxel, docetaxel demonstrated more potent induction of bcl-2 phosphorylation and exhibited up to a 12-fold increase in cytotoxic activity[2,4 6]. Also, growth inhibition in human epidermal growth t%ctor receptor 2 (HER2)-positive cells was greater for docetaxel than for paclitaxel [7]. Lastly, the increased upregulation of thymidine phosphorylase observed with docetaxel and

Contribution of reactive oxygen species and caspase-3 to apoptosis and attenuated ICAM-1 expression by paclitaxel-treated MDA-MB-435 breast carcinoma cells.

Paclitaxel is a microtubule-stabilizing and apoptosis-inducing drug that is commonly used to treat metastatic breast cancer, although the mechanism of paclitaxel-induced apoptosis remains incompletely understood. Furthermore, adhesion molecule expression is attenuated on mouse mastocytoma and human leukemia cells that survive short-term culture in the presence of paclitaxel. In the present study we show that MDA-MB-435 human breast carcinoma cells that survived culture for 72 h in the presence of submaximal cytotoxic concentrations of paclitaxel (0.02 and 0.01 microg/ml) showed decreased expression of the adhesion molecule ICAM-1. Paclitaxel treatment of MDA-MB-435 cells was associated with the generation of reactive oxygen species (ROS), dissipation of mitochondrial transmembrane potential, and the activation of caspase-3. The antioxidant glutathione protected MDA-MB-435 cells from paclitaxel-induced cytotoxicity and reduced ICAM-1 expression. In addition, a selective inhibitor of caspase-3 (Z-DEVD-FMK), as well as a pan-caspase inhibitor (Z-VAD-FMK), partially prevented the decrease in ICAM-1 expression observed following paclitaxel treatment, but did not protect against paclitaxel-induced cytotoxicity. We conclude that the paclitaxel-induced reduction in ICAM-1 expression by MDA-MB-435 breast carcinoma cells is both ROS- and caspase-dependent, whereas paclitaxel-induced cytotoxicity is ROS-dependent and does not involve caspases. Decreased ICAM-1 expression by breast carcinoma cells that survive paclitaxel treatment may negatively impact on cytotoxic lymphocyte-mediated destruction of paclitaxel-resistant breast cancer cells in the context of chemo-immunotherapy or chemo-adoptive immunotherapy.

A phase II study of docetaxel in paclitaxel-resistant ovarian and peritoneal carcinoma: a gynecologic oncology group study

Objectives Docetaxel is an inhibitor of microtubule depolymerization and has demonstrated activity in paclitaxel-resistant breast cancer and gynecologic cancer. The Gynecologic Oncology Group (GOG) conducted a study of docetaxel in paclitaxel-resistant ovarian and peritoneal carcinoma to determine its activity, and nature and degree of toxicity, in this cohort of patients. Methods Patients with platinum- and paclitaxel-resistant ovarian or peritoneal carcinoma, defined as progression while on or within 6 months of therapy, were eligible if they had measurable disease and had not received more than one chemotherapy regimen. Docetaxel at a dose of 100 mg/m 2 was administered iv over 1 h every 21 days. A prophylactic regimen of oral dexamethasone 8 mg bid was begun 24 h before docetaxel administration and continued for 48 h thereafter. Hepatic function was strictly monitored. Results Sixty patients were entered and treated with a total of 256 courses, with all 60 evaluable for toxicity and 58 evaluable for response. Responses were observed in 22.4% of patients, with 5.2% achieving complete response and 17.2% achieving partial response (95% CI, 12.5–35.3%). The median duration of response was 2.5 months. The likelihood of observing a response did not appear to be related to the length of the prior paclitaxel-free interval or duration of prior paclitaxel infusions. The principal adverse effect of grade 4 neutropenia occurred in 75% of patients. There was one treatment-related death. Dose reductions were required in 36% of patients. Conclusions Docetaxel is active in paclitaxel-resistant ovarian and peritoneal cancer but, in view of significant hematologic toxicity, further study is warranted to ascertain its optimal dose and schedule.