Taxol-resistant Breast Cancer Research Articles

71P Breast cancer cells’ taxol-resistance is associated with cytoplasmic actin isoforms balance shift and acquisition of more malignant phenotype

Breast cancer is one of the most common and aggressive types of malignancy which is associated with an unfavourable prognosis. Conventional chemotherapy for breast cancer is currently based on the use of classical cytotoxic drugs including taxol. But such therapeutic strategies unfortunately often lead to acquisition of drug resistance which is one of the main reasons of relapse and metastasis. In the present study we investigated potential molecular mechanisms of more malignant phenotype acquisition by taxol-resistant breast cancer cells.

LncRNA NEAT1 promotes the Taxol resistance of breast cancer via sponging the miR-23a-3p-FOXA1 axis.

Breast cancer is the most prevalent malignancy among women worldwide. Paclitaxel (Taxol) is a widely applied chemotherapeutic agent against breast cancer. Although Taxol therapy has achieved improvements recently, development of chemoresistance of breast cancer patients is a major obstacle, leading to therapeutic failure. Long non-coding RNAs (lncRNAs) play pivotal roles in tumorigenesis and progresses of breast cancer. However, the biological roles and molecular targets of lncRNA NEAT1 in Taxol-resistant breast cancer remain unclear. Here, we report that NEAT1 is significantly upregulated in breast tumors and cell lines. In addition, silencing NEAT1 effectively sensitizes breast cancer cells to Taxol. Bioinformatical analysis and luciferase assay demonstrated that miR-23a-3p could be sponged and downregulated by NEAT1. We demonstrated that miR-23a-3p was downregulated and functioned as a tumor suppressor in breast cancer. Furthermore, in the established Taxol-resistant MDA-MB-231 breast cancer cell line, we detected significantly increased NEAT1 expression and downregulated miR-23a-3p expression. Importantly, FOXA1 was identified and validated as a direct target of miR-23a-3p in breast cancer cells. Rescue experiments demonstrated that the restoration of miR-23a-3p in NEAT1-overexpressing Taxol-resistant breast cancer cells successfully overcame the NEAT1-promoted Taxol resistance. Taken together, our results revealed the clinical roles and molecular mechanisms for the NEAT1-mediated chemoresistance, providing new insights into the development of non-coding RNA-based therapeutic strategies for enhancing the anti-cancer effects of traditional chemotherapeutic drugs.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Interfering cellular lactate homeostasis overcomes Taxol resistance of breast cancer cells through the microRNA-124-mediated lactate transporter (MCT1) inhibition

BackgroundBreast cancer, the most common invasive cancer of women, is a malignant neoplasm and the second main cause of cancer death. Resistance to paclitaxel (Taxol), one of the frequently used chemotherapy agents for breast cancer, presents a major clinical challenge. Recent studies revealed that metabolic alterations of cancer cells play important roles in chemo-resistance.Materials and methodsIn this study, Human breast cancer cells, BT474, SKBR3 and MCF7 were used to study the causal relationship between the lactate exporter, MCT1 (SLC16A1)-modulated glucose metabolism and Taxol resistance of breast cancer cells. Taxol resistant breast cancer cells were established. The intracellular lactate and extracellular lactate levels as well glucose uptake and oxygen consumption were measured. MicroRNA-124 expressions were detected by qRT-PCR from both breast cancer patient samples and breast cancer cells. Target of miR-124 was predicted and verified by Western blot and luciferase assay. An xenograft mice model was established and evaluated for the in vivo tumor therapeutic effects of MCT1 inhibitor plus microRNA-124 treatments.ResultsLow toxic Taxol treatments promoted cellular glucose metabolism and intracellular lactate accumulation with upregulated lactate dehydrogenase-A (LDHA) and MCT1 expressions. By establishing Taxol resistant breast cancer cell line, we found Taxol resistant cells exhibit upregulated LDHA and MCT1 expressions. Furthermore, glucose consumption, lactate production and intracellular ATP were elevated in Taxol resistant MCF7 cells compared with their parental cells. The miR-124, a tumor suppressive miRNA, was significantly downregulated in Taxol resistant cells. Luciferase assay and q-RT-PCR showed MCT1 is a direct target of miR-124 in both breast cancer cell lines and patient specimens. Moreover, co-treatment of breast cancer cells with either MCT1 inhibitor or miR-124 plus Taxol led to synergistically cytotoxic effects. Importantly, based on in vitro and in vivo results, inhibition of MCT1 significantly sensitized Taxol resistant cells. Finally, rescue experiments showed restoration of MCT1 in miR-124 overexpressing cells promoted Taxol resistance.ConclusionsThis study reveals a possible role of miRNA-214-mediated Taxol resistance, contributing to identify novel therapeutic targets against chemoresistant breast cancers.

Taxol Induces Brk-dependent Prosurvival Phenotypes in TNBC Cells through an AhR/GR/HIF-driven Signaling Axis.

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF-dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors (n = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread.Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761-72. ©2018 AACR.

Abstract 1084: Combination of eribulin and AURKA inhibitor prevents metastatic colonization and eradicates established metastases in breast cancer

Abstract The majority of cancer-related deaths (90%) are happened due to metastasis from the primary tumor site to distant organs. MLN8237 is a small, highly selective molecule inhibitor of Aurora A kinase (AURKA), which results in disruption of the mitotic spindle, chromosome segregation collapse, and inhibition of cell proliferation. Numerous studies have showed that levels of AURKA are elevated in many types of cancer, including breast cancer. Our previous studies indicated that MLN8237 is extremely potent against pulmonary metastasis, but not the primary tumor in orthotropic xenograft model. To further enhance MLN8237 based regiment, its combination with other therapeutic compounds can be used. As a potential partner we chose eribulin - fully synthetic macrocyclic analogue of the marine natural product halichondrin B. Eribulin belong to the class of non-taxane microtubule destabilizing molecules, currently used in clinic to treat taxol resistant metastatic breast cancer. In present study we have investigated the effect of MLN8237 and eribulin against breast cancer in vivo and in vitro. The results showed that combination of drugs possess a synergistic effect on both primary tumor and metastases, through inducing cytotoxic autophagy and apoptosis. This data clearly indicate great potential behind the MLN8237 based therapies and introduce a new hope for the eradication of metastatic breast cancer. Citation Format: Elena N. Pugacheva, Varvara Kozyreva, Anna Kiseleva, Ryan Ice, Brandon Jones, Yuriy Loskutov. Combination of eribulin and AURKA inhibitor prevents metastatic colonization and eradicates established metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2017-1084

WX-132-18B, a novel microtubule inhibitor, exhibits promising anti-tumor effects

Cancer drug researchers have been seeking microtubule-inhibiting agents (MIAs) with higher bioactivity and lower toxicity than currently marketed drugs. WX-132-18B, a novel structural compound synthesized at our institute, specifically bound to the colchicine-binding site on tubulin rather than the vinblastine site, and concentration-dependently reduced microtubule content via depolymerization. It exhibited the same cellular phenotypic profiles as the classic MIAs (colchicine, vincristine, and taxol), including inducing cell cycle arrest at the G2/M phase, triggering tumor cell apoptosis, promoting nuclear membrane permeability, reducing mitochondrial membrane potential, and disrupting the redox system balance. Importantly, WX-132-18B displayed more potent in vitro bioactivity (IC50 0.45–0.99 nM) than did the classic MIAs; it inhibited the proliferation of human umbilical vein endothelial cells and seven types of human tumor cells, especially the taxol-resistant breast cancer cells MX-1/T. WX-132-18B also dose-dependently inhibited mice sarcoma, human lung, and gastric cancer xenograft tumors and the formation of tumor blood vessels in mice. In conclusion, WX-132-18B is a novel microtubule-depolymerizing agent that selectively acts on the colchicine-binding site of tubulin and exerts potent in vitro and in vivo anti-tumor effects. These characteristics, along with its anti-angiogenesis and anti-drug resistance properties, make WX-132-18B a promising anti-tumor drug candidate.

Silencing Aurora A leads to re-sensitization of breast cancer cells to Taxol through downregulation of SRC-mediated ERK and mTOR pathways.

While Taxol has been reported to improve the clinical survival of breast cancer patients, subsequently developed drug-resistance of the cancer cells limits its final efficacy and applications. Previous studies suggested that Aurora A is involved in the development of the Taxol-resistance of breast cancer. We established Taxol-resistant breast cancer MCF-7/T cells and xenograft models to explore the role of Aurora A in Taxol resistant ER-positive breast cancer. Compared with their parental MCF-7/C cells, the Taxol-resistant MCF-7/T cells exhibited enhanced colony formation, less cell death and higher invasive ability. The resistant cells presented overexpressed Aurora A, elevated phosphorylated SRC and upregulated Ras/Raf/ERK and Akt/mTOR pathways. Silencing of Aurora A reduced the activity of SRC and downregulated the ERK and Akt/mTOR pathways, which led to re-sensitization of the resistant MCF-7/T cells to Taxol in vitro. These results suggested that the activation of Aurora A and the subsequent upregulation of ERK and Akt through SRC induced Taxol-resistance in breast cancer cells, and inhibiting Aurora A and the related SRC/EKT/Akt pathway could restore the sensitivity of breast cancer cells to Taxol. These results might shed light on the development of strategies to circumvent Taxol-related chemoresistance in breast cancer clinical practice.

Se/Ru-Decorated Porous Metal-Organic Framework Nanoparticles for The Delivery of Pooled siRNAs to Reversing Multidrug Resistance in Taxol-Resistant Breast Cancer Cells.

We report here a novel and personalized strategy of selenium/ruthenium nanoparticles modified metal organic frameworks MIL-101(Fe) for delivering pooled small interfering RNAs (siRNAs) to enhance therapy efficacy by silencing multidrug resistance (MDR) genes and interfere with microtubule (MT) dynamics in MCF-7/T (Taxol-resistance) cell. The existence of coordinatively unsaturated metal sites in MIL-101(Fe) can strongly interact with the electron-rich functional groups of cysteine, which can be regarded as the linkage between selenium/ruthenium nanoparticles and MIL-101(Fe). Se@MIL-101 and Ru@MIL-101 loaded with MDR gene-silencing siRNAs via surface coordination can significantly enhance protection of siRNAs against nuclease degradation, increase siRNA cellular uptake, and promote siRNA escape from endosomes/lysosome to silence MDR genes in MCF-7/T cell, resulting in enhanced cytotoxicity through the induction of apoptosis with the signaling pathways of phosphorylation of p53, MAPK, and PI3K/Akt and the dynamic instability of MTs and disrupting normal mitotic spindle formation. Furthermore, in vivo investigation of the nanoparticles on nude mice bearing MCF-7/T cancer xenografts confirmed that Se@MIL-101-(P+V)siRNA nanoparticles can significantly enhance cancer therapeutic efficacy and decrease systemic toxicity in vivo.

Decreased expression of microRNA-17 and microRNA-20b promotes breast cancer resistance to taxol therapy by upregulation of NCOA3.

Chemoresistance is a major obstacle to effective breast cancer chemotherapy. However, the underlying molecular mechanisms remain unclear. In this study, nuclear receptor coactivator 3 (NCOA3) was found to be significantly increased in taxol-resistant breast cancer tissues and cells. Moreover, overexpression of NCOA3 enhanced breast cancer cell resistance to taxol, whereas depletion of NCOA3 decreased taxol resistance. Subsequently, we investigated whether NCOA3 expression was regulated by miRNAs in breast cancer. By bioinformatics prediction in combination with the data of previous report, miR-17 and miR-20b were selected as the potential miRNAs targeting NCOA3. By real-time PCR analysis, we found that miR-17 and miR-20b were significantly reduced in taxol-resistant breast cancer tissues and cells. In addition, we provided some experimental evidences that miR-17 and miR-20b attenuated breast cancer resistance to taxol in vitro and in vivo models. Furthermore, by luciferase reporter assays, we further validated that both miR-17 and miR-20b directly binded the 3′-untranslated region of NCOA3 mRNA and inhibited its expression in breast cancer cells. Finally, both miR-17 and miR-20b levels were found to be significantly negatively correlated with NCOA3 mRNA levels in breast cancer tissues. Together, our results indicated that loss of miR-17 and miR-20b enhanced breast cancer resistance to taxol by upregulating NCOA3 levels. Our study suggested miR-17, miR-20b and NCOA3 may serve as some predictive biomarkers and potential therapeutic targets in taxol-resistant breast cancer treatment.

Abstract 3325: Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance

Abstract In recent years, impressive technical advancements have been made in the isolation and validation of the mammary stem cells (MaSCs) and cancer stem cells (CSCs). However, the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to resistance to chemotherapy and radiation therapy. However, an effective therapeutic strategy to overcome this resistance is yet to be identified. We have recently discovered that the DNA methyltransferases, especially DNMT1, play critical role in MaSCs and CSCs self-renewal and targeted deletion of this gene impaired mammary tumor formation by inhibiting CSCs formation. However, the molecular mechanism(s) by which DNMTs control CSCs and the therapeutic relevance of DNMTs inhibitors in regulation of CSCs and overcome drug resistance are also largely unknown. In this study, using MMTV-Neu-Tg mouse mammary tumor model, we found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which leads to activation of un-activated Neu-Tg into transformed tumor forming phenotype. Combination of 5-Azacytidine, a DNMT inhibitor, and butyrate, a HDAC inhibitor, markedly reduces CSCs and consequently increases the overall survival of the animal. RNA-seq analysis of the CSCs treated with 5-AzaC+butyrate provides evidence that combined inhibition of DNMTs and HDACs reduces CSCs pool in the mammary gland by blocking growth-promoting signaling molecules like RAD51AP1 and SPC25. RAD51AP1 and SPC25, which are known to play a key role in DNA damage repair and kinetochore assembly, are significantly overexpressed in breast tumor tissues and associated with decreased overall patients’ survival. Further, these two genes are overexpressed in Tamoxifen and Taxol resistant human breast cancer cell lines. Functional inactivation of these genes in breast caner cells facilitates chemotherapy-induced apoptosis and reduces tumor growth. Overall, our studies provide strong evidence that breast CSCs (both basal stem cells and luminal progenitor cells) are susceptible for genetic and epigenetic modifications and associated with resistance to chemo- and radiotherapy. Thus, combination of DNMT and HDAC inhibitors can serve as an effective therapeutic strategy to block mammary tumor growth and to overcome drug resistance by inhibiting CSCs. Citation Format: Rajneesh Pathania, Ravindra B. Kolhe, Sabarish Ramachandran, Gurusamy Mariappan, Priyanka Thakur, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3325.

Synergistic effects of negative-charged nanoparticles assisted by ultrasound on the reversal multidrug resistance phenotype in breast cancer cells

We have fabricated a negative-charged nanoparticle (Heparin-Folate-Tat-Taxol NP, H-F-Tat-T NP) with dual ligands, tumor targeting ligand folate and cell-penetrating peptide Tat, to deliver taxol presenting great anticancer activity for sensitive cancer cells, while it fails to overcome multidrug resistance (MDR) in MCF-7/T cells (taxol-resistant breast cancer cells). Ultrasound (US) can increase the sensitivity of positive-charged NPs thereby making it possible to reverse MDR through inducing NPs’ drug release. However, compared with the negative-charged NPs, positive-charged NPs may cause higher toxic effect. Hence, the combination of negative-charged NPs and US may be an efficient strategy for overcoming MDR. The conventional procedure to treat with NPs followed by US exposure possibly destruct multifunctional NPs resulting in its bioactivity inhibition. Herein, we have further improved the operating approach to eliminate US mechanical damage and keep the integrity of negative-charged NPs: cells are exposed to US with microbubbles (MBs) prior to the treatment of H-F-Tat-T NPs. Superior to the conventional method, US sonoporation affects the physiological property of cancer cells while preventing direct promotion of drug release from NPs. The results of the present study displayed that US in condition (1MHz, 10% duty cycle, duration of 80s, US intensity of 0.6W/cm2 and volume ratio of medium to MBs 20:1) combined with H-F-T-Tat-T NPs can achieve optimal reversal MDR effect in MCF-7/T cells. Mechanism study further disclosed that the individual effect of US was responsible for the enhancement of cell membrane permeability, inhibition of cell proliferation rate and down-regulation of MDR-related genes and proteins. Simultaneously, US sonoporation on resistant cancer cells indirectly increased the accumulation of NPs by inducing endosomal escape of negative-charged NPs. Taken together, the overcoming MDR ability for the combined strategy was achieved by the synergistic effect from individual function of NPs, physiological changes of resistant cancer cells and behavior changes of NPs caused by US.

High Bak Expression Is Associated with a Favorable Prognosis in Breast Cancer and Sensitizes Breast Cancer Cells to Paclitaxel.

Breast cancer has become the leading cause of cancer-related death among women. A large number of patients become resistant to drug chemotherapy. Paclitaxel (Taxol) is an effective chemotherapeutic agent used to treat cancer patients. Taxol has been widely used in human malignancies including breast cancer because it can stabilize microtubules resulting in cell death by causing an arrest during the G2/M phase of the cell cycle. Pro-apoptotic Bcl-2 antagonist killer 1 (Bak) plays an important role in Taxol-induced apoptosis in breast cancer. In our present study, we investigated the expression of the Bak protein and clinicopathological correlations in a large sample of breast cancer tissues by immunohistochemistry. We found that the percentage of high scores of Bak expression in breast cancer was significantly lower than that of the non-cancerous breast control tissue. In addition, lower Bak expression was positively associated with the clinical TNM stage of breast cancer with a significant decrease in overall survival compared with those with higher Bak expression especially in the Luminal and HER2 subtypes. Importantly, higher Bak expression predicted a favorable clinical outcome in the cases treated with Taxol indicated by a higher overall survival than that of patients with lower Bak expression especially in Luminal and HER2 subtypes. Furthermore, these results were confirmed in vitro since overexpression of Bak sensitized breast cancer cells to Taxol by inhibiting proliferation and promoting apoptosis; in contrast, downregulation of Bak through siRNA transfection inhibited Taxol induced-apoptosis. Therefore, our results demonstrate that Bak acts as a sensitive biomarker and favorable prognostic factor for Taxol treatment in breast cancer. The restoration of Bak expression would be therapeutically beneficial for Taxol resistant breast cancer patients.

Silencing of glutaminase 1 resensitizes Taxol-resistant breast cancer cells to Taxol

Taxol is a front‑line chemotherapeutic agent for the treatment of patients with multiple types of tumor. However, resistance to Taxol remains one of the principal causes of cancer‑associated mortality. Glutamine, which is metabolized via a glutaminase (GLS)‑dependent process, termed glutaminolysis, is important in cell growth and metabolism. The present study reported a novel mechanism underlying Taxol resistance in breast cancer cells. By investigating the glutamine metabolism of breast cancer cells in response to treatment with Taxol invitro, it was observed that Taxol induced the uptake of glutamine and the expression of GLS1. Notably, Taxol‑resistant cancer cells exhibited upregulation in the metabolism of glutamine and expression of GLS1. In addition, overexpression of GLS1 rendered cancer cells resistant to Taxol, indicating that GLS1 may be the therapeutic target for overcoming Taxol resistance in clinical therapeutics. The results also demonstrated that knock‑down of GLS1 using small interfering RNA, resensitized the Taxol‑resistant breast cancer cells to Taxol.

Sensitization of breast cancer cells to taxol by inhibition of taxol resistance gene 1

The purpose of this study was to determine the role of taxol resistance gene 1 (TXR1) in a taxol-resistant breast cancer cell line. Expression levels of TXR1 and thrombospondin-1 (TSP1) were detected by RT-PCR and Western blot analysis. In MCF-7 cells transfected with TXR1 plasmids, the expression of a number of drug resistance genes was monitored, as well as cell proliferation. MCF-7 cells were also transiently transfected with a chemically synthesized small interfering RNA (siRNA) targeting TXR1. Taxol concentrations varying from 0.6 to 6 μg/ml were shown to inhibit the proliferation of MCF-7 cells in a time- and dose-dependent manner by arresting cells in the G2/M phase. Additionally, 0.06 μg/ml taxol was used to establish a taxol-resistant MCF-7 cell line, MCF-7/T. When TXR1 was exogenously expressed, a taxol-resistant phenotype was further induced and the expression levels of BCRP, GSTP1 and MVP increased. Transient transfection of TXR1-targeting siRNAs was shown to restore the chemosensitivity of MCF-7 cells. These results suggest that an increased expression of TXR1 is a novel mechanism for reversing the taxol resistance of breast cancer cells.

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol‐resistant human breast cancer cell line MDA‐MB‐231

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and β-tubulins (more specifically the βIII and βIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.

Function of Aurora kinase A in Taxol-resistant breast cancer and its correlation with P-gp

Breast cancer is one of the most common malignant diseases among women. In early and metastatic breast cancer, Taxane (Taxol) is widely used as an adjuvant and neoadjuvant therapies. Although breast cancer is initially responsive to Taxol, inherent or developed resistance to Taxol often limits the efficacy of the drug. The oncogene Aurora kinaseA is frequently up-regulated in human cancer, and is associated with sensitivity to chemotherapy in certain types of cancer. In the present study, we aimed to clarify the functional role of Aurora kinaseA in breast cancer resistance to Taxol, and to determine the means to overcome this resistance. The correlation between the expression levels of Aurora kinaseA and chemoresistance to Taxol in breast cancer cells, and resistance to Taxol in a xenograft model were demonstrated. MTT assay was performed to determine cell viability. Subsequently, the relationship of Aurora kinaseA with the expression and functional role of P-gp was clarified, as well as its relationship with p-ERK2, which regulates the expression of P-gp. The expression of AuroraA was determined to be capable of enhancing the sensitivity of cells resistant to Taxol in vitro and in vivo using stable knockdown Aurora kinaseA cells. We propose that this kinase may be used as a target for overcoming chemoresistance and enhancing the chemosensitivity of breast cancer to Taxol.

Abstract 2130: Overcoming chemotherapy resistance by targeting LDHA mediated glycolysis

Abstract Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer. 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 2130. doi:10.1158/1538-7445.AM2011-2130

Abstract 5441: Preclinical assessment of the HDAC inhibitor JNJ-26481585: potent in vivo activity across a broad spectrum of human tumor xenografts

Abstract JNJ-26481585 is a promising “second generation” oral pan-HDAC inhibitor. Recently, we have published data describing the identification and characterization of the molecule as a potent inhibitor of HDAC1 with a favorable pharmacodynamic profile. Subsequent analysis has shown that the compound has pan-HDAC inhibitory activity and demonstrates effective inhibition of tumor cell growth across a broad spectrum of solid and hematological tumor cell lines. Recent data has also shown that in vivo JNJ-26481585 alone, or in combination with bortezomib, is an effective agent in myeloma. Herein we describe the activity of JNJ-26481585 against a broad range of preclinical solid tumor xenograft models, at doses substantially below MTD and with different schedules of administration in order to address the clinical utility of this agent. Significant growth inhibition was achieved when dosing JNJ-26481585 at MTD with T/C in tumors of; A2780 ovarian tumor xenograft (3%), ras-mutant colon (HCT-116), (7%), MDA-MB-231 breast (38%), DU-145 prostate (11%) and lung H460, A549 and ras mutant NCI-H1373 (29%, 15% and 32% respectively). In the A2780 model, activity of JNJ-26481585 was superior to that of five other HDAC inhibitors tested. This class leading activity was observed at MTD and optimal route of administration of all compounds and importantly, we also observed significant activity of JNJ-26481585 at concentrations as low as 12.5% of MTD (T/C = 35%). Both class leading activity and significant tumor growth inhibition at concentrations substantially below MTD was also observed in A549, DU145 and HCT116 tumor xenografts. Furthermore, in HCT116, in line with taxotere in prostate models, JNJ-26481585 was shown to be more effective than the SOC, 5-FU. The HCT116 model was used further to address the potential of the compound for clinical utility by exploring alternative dosing schedules to that of QD IP dosing. 3 days on/4 days off, 1 week on/ 1 week off, Q2D and Q7D were all investigated alongside QD dosing and observed to inhibit tumor growth in all cases; Q7D was the only alternative schedule not to achieve efficacy equivalent to that with QD dosing. Finally, data indicating greater activity or equivalence with the SOC suggested that JNJ-26481585 could be used in those indications, thus potency was investigated in drug resistant patient material. In both taxol resistant NSCLC and doxorubicin resistant breast cancer cultures, JNJ-26481585 dramatically inhibited proliferation. We conclude that in preclinical models, JNJ-26481585 was effective at reducing the tumor size to the greatest extent of all HDAC agents studied. Potent antitumoral activity was also observed at doses substantially below MTD and from alternative dosing schedules, suggesting that this could be a promising agent for clinical development in such indications. JNJ-26481585 is currently in Phase I clinical trials. 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 5441.

Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol

BackgroundTaxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.ResultsTaxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.ConclusionLDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.