Chemoresistance In Breast Cancer Cells Research Articles

Abstract 831: Increased resistance of breast cancer cells to doxorubicin by clinically relevant doses of temsirolimus

Abstract The phosphoinositide-3 kinase (PI3K) pathway is often activated in breast cancer due to frequent mutations in PIK3CA, loss of expression of PTEN or over-expression of receptor tyrosine kinases. This results in constitutive activation of this pathway and in turn resistance to anticancer treatments. One strategy to reverse this chemoresistance was to inhibit PI3K pathway by targeting key regulatory proteins such as PI3K, Akt or mTOR. Many inhibitors have been developed but clinical results obtained have been disappointing so far, probably due to the absence of information on the functional activities of the targeted kinases. We wanted to test how quantitative changes in enzymatic activities of PIK3 pathway would predict a decrease in chemoresistance to conventional therapeutic agents when combined with mTOR inhibitors. A prerequisite was to determine if the genomic mutations of PI3K pathway mentioned above were followed by quantitative changes in the corresponding enzymatic activities targeted by the specific inhibitors. We determined the enzymatic activities of the PI3K pathway in breast cancer cell lines with either mutation in PIK3CA (MCF-7 and T-47D cells), deletion of PTEN (MDA-MB-468 cells) or no alteration in the pathway (MDA-MB-231). Enzymatic activities were evaluated by quantifying the percentage of phosphorylated proteins. As expected, a genomic alteration of a PI3K pathway component resulted in activation of downstream kinases of the pathway (PDK1, mTORC2, Akt and mTORC1) but their level of activation varied depending on the cell line tested. Next, we treated those cell lines with clinically relevant, non-toxic doses (0.1nM or 1nM) of temsirolimus (mTORC1 inhibitor) and carried out a dose-response (from 0.1nM up to 10µM) of doxorubicin (anthracyclin). Temsirolimus induced the phosphorylation of Akt on both Ser473 and Thr308 in MCF-7 cells. This Akt activation remained much lower in MCF-7 than constitutive activation measured in MDA-MB-468 cells. As expected, doxorubicin, used at IC25 (66 nM) also induced the activation of Akt in MCF-7 cells. Addition of temsirolimus did not further increase doxorubicine toxicity. On the opposite, a marked increased resistance of cells to doxorubicin was measured in MCF-7 and in MDA-MB-468 cells treated with temsirolimus. Such induction of chemoresistance was not detected in MDA-MB-231 cells. Similar results were obtained with PI-103, a dual inhibitor of PI3K and mTORC1. Although PI3K pathway inhibitors have been used in clinical settings to restore the effects of conventional anti-cancer drugs, we found that some inhibitors of this pathway increased chemoresistance in breast cancer cells, depending on the altered enzymatic activities in PI3K pathway. Thus, in addition to identifying genomic alterations, kinases activities of the PI3K pathway should be quantified in breast cancer biopsies prior to contemplating the use of targeted drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 831. doi:1538-7445.AM2012-831

Abstract 3833: Ceramide/tamoxifen nanoliposomal combination - A promising strategy for treatment of chemoresistant breast cancer

Abstract Drug resistance in breast and other cancers results from multiple gene interactions. Ceramide, a key intermediate in the sphingolipid pathway, can act as a powerful tumor suppressor. The employ of ceramide-based agents as opposed to the administration of ceramide generators such as daunorubicin, could be a smart strategy for cancer therapy. Polychemotherapy remains the best option for treatment of breast cancer at the aggressive, metastatic stage. The aim of our study was to investigate the cytotoxic and antiproliferative effects of ceramide (short-chain C6-ceramide) administered in combination with tamoxifen, used here as an inhibitor of ceramide glycosylation. We previously demonstrated the cytotoxicity of this combination in MDA-MB-231 cells; however, little regarding mechanism was established. The present study employs nanoliposomal formulations of both agents, a strategy to enhance efficacy. Four triple-negative chemoresistant breast cancer cell lines, MDA-MB-231, MDA-MB-468, BT-20 and Hs578T were used. Cytotoxicity assays revealed that C6-ceramide/tamoxifen synergistically reduced viability, compared to single agents in all four cell lines. For example, in MDA-MB-468 cells, nanoliposomal C6-ceramide (2.5 µM), tamoxifen (5 µM), and the combination reduced cell viability to 70, 85, and 10% of control, respectively (96 hr). A hallmark of cancer, implicated in tumor growth as well as metastasis, is resistance to induction of apoptosis. Assessment of cellular apoptosis showed that C6-ceramide/tamoxifen treatment promoted DNA fragmentation in a dose-dependant manner, attaining a value that was 36% over control at 24 hr. Introduction of the pan-caspase inhibitor, z-VAD-fmk, completely reversed DNA fragmentation, showing that apoptosis was mediated by caspase activation. The aggressive and metastatic behavior of any tumor is dependent on the ability of the cells to proliferate. Interestingly, the lower concentrations of our drug combination (2.5 µM) induced cell cycle arrest at G1, the first growth phase. In summary, this approach has potential to circumvent limitations of ceramide-based therapies, such as rapid ceramide clearance due to metabolism, the inability to attain therapeutic levels when ceramide-generating agents are employed, and inherent insolubility that is encountered. These findings demonstrate the in vitro efficacy of combination C6-ceramide/tamoxifen nanoliposomal formulations for suppressing growth of breast cancer cells, and suggest that tamoxifen may be an effective adjuvant for enhancing ceramide-driven cell death cascades. Supported by NIGMS 77391 and ABC's, Los Angeles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3833. doi:1538-7445.AM2012-3833

A Marine Anthraquinone SZ-685C Overrides Adriamycin-Resistance in Breast Cancer Cells through Suppressing Akt Signaling

Breast cancer remains a major health problem worldwide. While chemotherapy represents an important therapeutic modality against breast cancer, limitations in the clinical use of chemotherapy remain formidable because of chemoresistance. The HER2/PI-3K/Akt pathway has been demonstrated to play a causal role in conferring a broad chemoresistance in breast cancer cells and thus justified to be a target for enhancing the effects of anti-breast cancer chemotherapies, such as adriamycin (ADR). Agents that can either enhance the effects of chemotherapeutics or overcome chemoresistance are urgently needed for the treatment of breast cancer. In this context, SZ-685C, an agent that has been previously shown, as such, to suppress Akt signaling, is expected to increase the efficacy of chemotherapy. Our current study investigated whether SZ-685C can override chemoresistance through inhibiting Akt signaling in human breast cancer cells. ADR-resistant cells derived from human breast cancer cell lines MCF-7, MCF-7/ADR and MCF-7/Akt, were used as models to test the effects of SZ-685C. We found that SZ-685C suppressed the Akt pathway and induced apoptosis in MCF-7/ADR and MCF-7/Akt cells that are resistant to ADR treatment, leading to antitumor effects both in vitro and in vivo. Our data suggest that use of SZ-685C might represent a potentially promising approach to the treatment of ADR-resistant breast cancer.

IKK-β mediates chemoresistance by sequestering FOXO3; A critical factor for cell survival and death

Chemotherapeutic drugs proved only 50% successful in breast cancer because of cell type-dependent resistance mechanisms. FOXO3 is known to be involved in the regulation of several cell death-related genes; however, the extent of FOXO3 regulation in chemoresistance is still not fully understood. Here, we show that FOXO3 critically mediates cisplatin chemosensitivity of MCF-7 breast cancer cells which express higher levels of FOXO3 compared to resistant MDA-MB-231 cells. Administration of cisplatin induces apoptosis in MCF-7 cells in a FOXO3-dependent manner as indicated by RNA interference. On the other hand, IKK-β (IκB kinase) appears to inhibit FOXO3 action after cisplatin treatment and promotes chemoresistance in MDA-MB-231 cells. IKK-β directly interacts and sequesters FOXO3 in the cytosol preventing its nuclear localization. Moreover, cisplatin treatment induces autophagosome formation through LC-3 conversion while inhibiting the cleavage of caspase 9 and caspase 3 in MDA-MB-231 cells manipulated to overexpress FOXO3. In brief, our findings demonstrate that in addition to cellular level of active FOXO3, cisplatin chemoresistance is also regulated by IKK-β sequestration of FOXO3 in cytosol.

The stem cell factor antibody enhances the chemotherapeutic effect of adriamycin on chemoresistant breast cancer cells

BackgroundThe outcome of chemotherapy in breast cancer is strongly influenced by multidrug resistance (MDR). Several surrogate markers of chemoresistance have been identified including - CD24 (cluster differentiation 24) expression, stem cell growth factor (SCF), B-cell lymphocyte protein 2 (Bcl-2) and annexin V. The present study aimed to examine the expression of CD24 in the sensitive breast cancer cell line MCF-7 (Michigan Foudation-7) and MCF-7/adriamycin resistant (MCF-7/AdrRes) cells, and, if minimal effective doses of the anthracycline drug adriamycin (0.579 μM and 88.2 μM) would be enhanced by the antibody to SCF (anti-SCF).MethodsCD24 expression was analysed by flow cytometry. Both Bcl-2 and annexin V protein expression were quantitatively assessed by the enzyme-linked immunosorbent assay (ELISA).ResultsIn MCF-7/AdrRes cells the expression of CD24 was significantly higher compared to MCF-7 cells, 86.6% and 16.3% (p < 0.001), respectively. Bcl-2 expression was significantly increased in the presence of adriamycin and SCF (p < 0.038) and decreased in the presence of adriamycin and anti-SCF. When adriamycin, anti-SCF and SCF were combined or when adriamycin was used alone the decrease in Bcl-2 expression was insignificantly altered. In the presence of both adriamycin and SCF the expression of annexin V was decreased. However, it was significantly increased in the presence of adriamycin and anti-SCF (p < 0.042), as well as adriamycin, anti-SCF and SCF combined.In MCF-7 cells the effect of adriamycin alone or with either SCF, anti-SCF or anti-SCF or SCF combined, did not significantly alter the expression of Bcl-2. However, in the presence of both adriamycin and SCF the expression of annexin V was decreased, but was significantly increased in the presence of adriamycin and anti-SCF (p < 0.001), adriamycin, anti-SCF and SCF combined and adriamycin alone. Our results demonstrate that anti-SCF with low dose of adriamycin reduces Bcl-2 expression in MCF-7/AdrRes cells and increases annexin V expression in both MCF7/AdrRes and MCF-7 cells.ConclusionAdding anti-SCF to the chemotherapeutic regime of adriamycin may strongly enhance its chemotherapeutic effect in the treatment of patients with breast cancer.

Inflammatory Breast Cancer Stem Cells: Contributors to Aggressiveness, Metastatic Spread and Dormancy

Cancer stem cell populations have been identified for several types of cancers and suggest a way for tumor cells to be resistant to therapies. Further, because of the longevity, endurance and replicative potential of cancer cells with stem-like properties, other malignant attributes such as recurrence after long periods of dormancy can also be explained. Inflammatory breast cancer (IBC) is a unique and aggressive form of breast cancer that has a clinical course unlike other forms of breast cancer. The main hallmark of IBC is prolific invasion of the dermal lymphatic vessels by tumor emboli leading to rapid metastasis of the disease. Despite an extremely aggressive treatment approach, the majority of women with IBC present with disease recurrence suggesting the presence of chemo resistant and/or dormant breast cancer cells. Current evidence suggests that IBC tumor emboli contain distinct populations of cells with stem cell-like properties. Thus, specific targeting of these stem cell-like cancer cells may be the key to effectively treating IBC.

Context-Dependent Bidirectional Regulation of the MutS Homolog 2 by Transforming Growth Factor β Contributes to Chemoresistance in Breast Cancer Cells

The TGF-β, a tumor suppressive cytokine in normal cells, is abused in cancer to promote the malignancy. In this study, we reported that TGF-β downregulated the mutS homolog 2 (MSH2), a central component of the DNA mismatch repair (MMR) system, in HER2-transformed MCF10A mammary epithelial cells and in breast cancer (BC) cells. This was mediated by a TGF-β-induced micro RNA (miRNA), miR-21, which targeted the 3' untranslated region of MSH2 mRNA and downregulated its expression. A negative correlation between the expression of TGF-β1 and MSH2 was also detected in primary breast tumors. In contrast, TGF-β upregulated MSH2 in nontransformed cells through Smad-mediated, p53-dependent promoter activation, which was absent in BC cells with impaired p53 function. Although this upregulating mechanism also existed in MCF10A/HER2 and p53-proficient BC cells, both basal and TGF-β-induced MSH2 promoter activities were significantly lower than those in MCF10A. Moreover, the basal and TGF-β-induced miR-21 levels were markedly higher in transformed cells, suggesting that the preset levels of miR-21 and MSH2 promoter activity, which is affected by the p53 status, determine the outputs of the bidirectional regulation of MSH2 by TGF-β in a certain cellular context. We further found that by downregulating MSH2, TGF-β contributed to resistance to DNA-damaging chemotherapy agents in cancer cells. Our results indicated a regulatory antagonism between promoter activation and miRNA-mediated posttranscriptional inhibition underlying a dual effect of TGF-β on the DNA repair machinery, which may influence the genomic stability in a context-dependent manner and contribute to chemoresistance in cancer.

Novel anti-viability ceramide analogs: Design, synthesis, and structure–activity relationship studies of substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides

A group of novel l-serinamides, substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides (3a–o) and substituted (S)-2-(benzylamino)-3-hydroxy-N-tetradecyl propanamides (4c, 4i, 4l, and 4o), were synthesized as potential anti-tumor lead compounds. In vitro cell viability assay results indicate treatment with 3a–o compounds resulted in significant inhibition of cell viability in the chemoresistant breast cancer cell line, MCF-7TN-R. Compounds 3i and 3l, both ortho-substituted analogs, show the greatest efficacy with IC50 values of 10.3μM and 12.5μM, respectively. The SAR analysis indicate that the imine functional group of 3a–o is critical for the cellular anti-viability effect, and the partial atomic charge (PAC) value of imine C atom is a valuable structural parameter for predicting the activity of these ceramide analogs.

Correlation of the methylation status of CpG islands in the promoter region of 10 genes with the 5-Fu chemosensitivity in 3 breast cancer cell lines

To explore the relationship between the methylation status of CpG islands in the promoter region of 10 genes in breast cancer cells and their sensitivity to 5-fluouracil (5-Fu), and to identify the genes responsible for the 5-Fu resistance in breast cancer. Three cell lines (differently resistant to chemotherapy) were used in this study: Bcap-37 (IC(50): 289.77 microg/ml), T47D (IC(50): 134.16 microg/ml) and ZR-75-30 (IC(50): 4.20 microg/ml). The methylation profile of 10 genes (BAG1, C11ORF31, CBR1, CBR4, GJA1, FOXL2, IGFBP6, P4HA1, SRI and TYMS) in the 3 breast cancer cell lines was determined by methylation specific PCR. The steady-state mRNAs of ABCC8, CHFR and IGFBP6 genes were quantified by real-time RT PCR analysis. Among the 10 genes, only genes IGFBP6 and FOXL2 displayed differential DNA methylation pattern between the 5-Fu-resistant and 5-Fu-sensitive cell lines. The mRNA expression level of genes PRSS21, LOX, IGFBP6, ABCC8 and CHFR was quantified by real-time RT-PCR analysis. Except for CHFR, the expression level of the other 4 genes was correlated with the methylation status of CpG islands, namely, a lower expression level with methylation status and a higher level with demethylation status. The results of the present study have demonstrated that there are 8 genes with differential methylation status in chemosensitive and chemoresistant breast cancer cell lines, i.e. two genes more than the six genes we reported previously. Our findings provide both mechanistic insights for the drug resistance of breast cancer and the basis for further studies on potential application of the DNA methylation in this set of genes for prediction of chemosensitivity of breast cancer.

Abstract 1031: Sphingolipid metabolism as a therapeutic target in chemotherapy resistant and hormone therapy resistant breast cancer

Abstract Endocrine- and chemo-resistance are major causes for failure of treatment in recurrent breast cancer. Normal apoptotic pathways targeted by chemotherapeutic agents become altered, diminishing their efficacy and increasing resistance mechanisms. The proapoptotic sphingolipid compound N-Octanoyl-D-erythro-sphingosine (C8-ceramide) mimics endogenous long chain ceramide and is a potent drug in endocrine-/chemo-resistant breast cancer cells. In this study, we examined basal expression levels of modifier enzymes for endogenous ceramide regulation across drug sensitive, chemoresistant and hormone therapy- resistant breast cancer cells lines. Differences were found among UDP-galactose ceramide galactosyltransferase, sphingosine kinase-1, and acid sphingomyelinase gene expression in: MCF-10A, MCF-7, MDA-MB-231 and MCF-7TN-R cell lines. Using C8-ceramide as the lead compound, 8 novel ceramide analogs were synthesized with either enol or amide backbone substitutions and tested for biological activity. The enol substitution was found to be the most potent, with (2S,3R,E)-2-((E)benzylideneamino)octadec-4-ene1,3-diol (Analog C) displaying the greatest reduction in proliferation. Analog B appeared to be the most potent cytotoxic drug in MCF-7 and MDA-MB-231 cell lines with IC50 values of 6.2uM and 3.0 uM respectively. In the cytotoxicity assay MCF-7TN-R cell line, Analog A appeared to be the most potent with an IC50 of 2.4uM. The clonogenic proliferation assay provided very different, yet promising results. Analog D had the lowest IC50 of 460nM in MDA-MB-231, Analog C had an IC50 of 100nM in MCF-7TN-R, and Analog A had an IC50 of 1.48uM in the MCF-7 breast cancer cell line. All of the IC50s for proliferation assays were at least an order of magnitude more potent than the control drug C8 (IC50 of 5uM). Due to the potency of these drugs, these results exemplify the potential of novel ceramide-targeted therapies in chemo-resistant and endocrine-resistant breast cancer cell lines. 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 1031.

Novel d-erythro N-octanoyl sphingosine analogs as chemo- and endocrine-resistant breast cancer therapeutics

Resistance to endocrine and chemotherapies remains the primary cause of breast cancer treatment failure. We have synthesized four novel D: -erythro N-octanoyl sphingosine analogs and catalogued their activity in drug-sensitive (MCF-7), endocrine-resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability; colony assay was performed to determine effects on clonogenic survival and (1)H NMR, (13)C NMR, HPLC spectra and elemental analytical data analyses were used to determine analog identity and purity. All four analogs inhibited both viability and clonogenic survival, with analog C exhibiting a log-fold improvement in anti-survival activity compared to the parent compound. With resistance to current breast cancer chemotherapies on the rise, the development of novel therapeutic targets is of growing importance. Our results show that lipid analogs have therapeutic potential in treating chemo- and endocrine-resistant breast cancer.

The Association of Cancer Stem Cells and Chemo-Resistance in Breast, Colon and Prostate Cancer Cells

The Association of Cancer Stem Cells and Chemo-Resistance in Breast, Colon and Prostate Cancer Cells

Design, Synthesis, and Biological Activity of a Family of Novel Ceramide Analogues in Chemoresistant Breast Cancer Cells

Resistance to chemotherapy and endocrine therapy is a major cause of breast cancer treatment failure. We have synthesized six novel analogues using C8-ceramide as the lead analogue and studied their effect on hormone therapy resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. Pharmacologic intervention using these ceramide analogues inhibited clonogenic survival and induced apoptosis, with one analogue being more effective than C8-ceramide. Our results show ceramide-based therapy has therapeutic potential in treating drug resistant breast cancer.

Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress

Moderate activity of the endoplasmic reticulum (ER) stress response system exerts anti-apoptotic function and supports tumor cell survival and chemoresistance, whereas its more severe aggravation may exceed the protective capacity of this system and turn on its pro-apoptotic module. In this study, we investigated whether the combination of two pharmacologic agents with known ability to trigger ER stress via different mechanisms would synergize and lead to enhanced tumor cell death. We combined the HIV protease inhibitor nelfinavir (Viracept ®) and the cyclooxygenase 2 (COX-2) inhibitor celecoxib (Celebrex ®) and investigated their combined effect on ER stress and on the viability of breast cancer cells. We found that this drug combination aggravated ER stress and caused pronounced toxicity in human breast cancer cell lines, inclusive of variants that were highly resistant to other therapeutic treatments, such as doxorubicin, paclitaxel, or trastuzumab. The anti-tumor effects of celecoxib were mimicked at increased potency by its non-coxib analog, 2,5-dimethyl-celecoxib (DMC), but were substantially weaker in the case of unmethylated-celecoxib (UMC), a derivative with superior COX-2 inhibitory efficacy. We conclude that the anti-tumor effects of nelfinavir can be enhanced by celecoxib analogs in a COX-2 independent fashion via the aggravation of ER stress, and such drug combinations should be considered as a beneficial adjunct to the treatment of drug-resistant breast cancers.

ErbB2-Mediated Src and Signal Transducer and Activator of Transcription 3 Activation Leads to Transcriptional Up-Regulation of p21Cip1 and Chemoresistance in Breast Cancer Cells

Overexpression of the ErbB2 receptor tyrosine kinase is prevalent in approximately 30% of human breast cancers and confers Taxol resistance. Our previous work has shown that ErbB2 inhibits Taxol-induced apoptosis in breast cancer cells by transcriptionally up-regulating p21(Cip1). However, the mechanism of ErbB2-mediated p21(Cip1) up-regulation is unclear. Here, we show that ErbB2 up-regulates p21(Cip1) transcription through increased Src activity in ErbB2-overexpressing cells. Src activation further activated signal transducer and activator of transcription 3 (STAT3) that recognizes a SIE binding site on the p21(Cip1) promoter required for ErbB2-mediated p21(Cip1) transcriptional up-regulation. Both Src and STAT3 inhibitors restored Taxol sensitivity in resistant ErbB2-overexpressing breast cancer cells. Our data suggest that ErbB2 overexpression can activate STAT3 through Src leading to transcriptional up-regulation of p21(Cip1) that confers Taxol resistance of breast cancer cells. Our study suggests a potential clinical application of Src and STAT3 inhibitors in Taxol sensitization of ErbB2-overexpressing breast cancers.

Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase overcomes chemoresistance of breast cancer cells

One of the mechanisms of multiple drug resistance (MDR) is inappropriate sequestration of basic chemotherapeutic agents in acidic endo-lysosomes of cells. The protonation, sequestration, and secretion (PSS) model indicates that drug distribution can be affected by intracellular pH such as lysosomal pH. The vacuolar-H +-ATPase (V-ATPase) plays an important role in regulation of intracellular pH by pumping protons into acidic endosomes via an ATP-driven process. In this study, ATP6L, the 16 kDa subunit of V-ATPase, was knocked-down by anti-ATP6L small interfering RNA (siRNA) to study the effect on chemosensitivity in the human drug-resistant breast cancer cells MCF-7/ADR. Introduction of anti-ATP6L small interfering RNA duplex into drug-resistant cancer cells significantly inhibited the expression of ATP6L mRNA and protein, as detected by qRT-PCR and Western blot. Inhibition of ATP6L expression by siRNA in MCF-7/ADR sensitized the cells to the cytotoxicity of basic chemotherapeutic agents like doxorobicin, 5-fluorourocil and vincristine. This effect was mediated by a significant increase in lysosomal pH and retention of anticancer drugs into nuclei of cells. These results support the role of tumor acidity in resistance to chemotherapy and provide a rationale for the use of tumor pH modifier agents as coadjuvants in novel anticancer therapies.

Prediction of chemotherapy response in breast cancer by using a four-gene panel including osteopontin, activating leukocyte cell adhesion molecule (ALCAM), HER2 and estrogen receptor (ER).

Abstract Abstract #2033 Background: Several prognostic factors such as TNM-stage, hormone receptor and HER2 status refer to therapeutical decisions towards adjuvant chemotherapy. Up to now it is still difficult to predict the response to chemotherapy for a single patient, which is a challenging problem in order to spare patients from cost-intensive ineffective treatment and adverse side effects. Therefore there is urgent need for additional predictive markers. Our previous findings implicated that tumor tissue expression of Activated Leucocyte Cell Adhesion Molecule (ALCAM/CD166) is associated with chemotherapy response. Osteopontin (OPN) has been described as prognostic marker in breast cancer and recently it was shown that high OPN levels in vitro mediate chemoresistance of breast cancer cells by inhibition of apoptosis. If these findings result in chemoresistance in vivo has not been investigated so far. Material and Methods: Primary breast cancer tissues from 100 patients treated with Taxane-free adjuvant standard chemotherapy regimen were collected at surgery prior to any additional therapy. The median follow-up time was 81 months. We analyzed ALCAM and OPN mRNA expression in the biological context of additional and predefined breast cancer markers, like estrogen receptor (ER), Her-2/neu (HER2), members of the urokinase plasminogen activator (uPA) system, vascular endothelial growth factor (VEGF) using oligonucleotide microarrays (Affymetrix HG-U133A). Hierarchical cluster analysis was performed to develop a gene algorithm predicting outcome after standard chemotherapy. Results: In contrast to ALCAM overexpression, which was associated with better outcome upon chemotherapy, OPN overexpression was associated with a significantly higher recurrence rate (p=0,027), but not with shorter overall survival. In addition, there were significant positive correlations of OPN with uPA, PAI-1, uPAR and VEGF-A mRNA expression. By cluster analysis based on the four markers ER, HER2, ALCAM and OPN, we identified a group of high-risk patients, which was characterized by low ER and HER2 expression, high OPN and low ALCAM levels, and with significantly shorter recurrence-free and overall survival (p&amp;lt;0,001). Discussion: In our cohort of patients the combination of ALCAM, OPN, ER and HER2 expression levels turned out as valuable predictive marker for response to adjuvant Taxane-free chemotherapy. The ratio of high OPN and low ALCAM levels was particularly associated with chemoresistance and a poor prognosis in tumors having low ER and HER2 expression levels. We hypothesize that this simple four-gene panel could help to reassign an optimized therapy to the single patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2033.

Cyclo-oxygenase 2 modulates chemoresistance in breast cancer cells involving NF-kappaB.

Background: Breast cancer cells can develop chemoresistance after prolonged exposure to cytotoxic drugs due to expression of the multi drug resistance (MDR) 1 gene. Type 2 cyclo-oxygenase (COX-2) inhibitors reverse the chemoresistance phenotype of a medullary thyroid carcinoma cell line, TT, and of a breast cancer cell line, MCF7, by inhibiting MDR1 expression and P-gp function. Aim: investigate the role of prostaglandin (PG) in modulating chemoresistance in MCF7 cells and to explore the involved intracellular mechanisms. Methods: native and chemoresistant MCF7 cells were treated with PGH2 and resistance to Doxorubicin was tested in the presence or absence of COX-2 inhibitors. Results: PGH2 restores resistance to the cytotoxic effects of Doxo, with concomitant nuclear translocation of the transcription factor NF-κB. Conclusions: COX-2 inhibitors prevent chemoresistance development in breast cancer cells by inhibiting P-gp expression and function by a mechanism that involves PGH2 generation and NF-κB activation.

P-02 Growth hormone induces chemoresistance in breast cancer cells

P-02 Growth hormone induces chemoresistance in breast cancer cells

Downregulation of Aurora-A overrides estrogen-mediated growth and chemoresistance in breast cancer cells

Estrogen is known to play a causative role in the development of sporadic breast cancers and chemoresistance. However, studies on the mechanism and proteins involved in mediating the oncogenic effects of estrogen are very limited. Recently, Aurora-A, a centrosomal protein kinase, which induces centrosome amplification and genomic instability, has been shown to be upregulated during long-term treatment of rats with estrogen and was implicated in estrogen-induced oncogenesis. Herein, we present results of the studies carried out in short-term in vitro cultures to understand the regulation of Aurora-A by estrogen and the effect of downregulation of Aurora-A on estrogen-induced breast tumorigenesis and chemoresistance. Treatment of breast cancer cells with 10 nM 17beta-estradiol (E(2)) resulted in the upregulation of Aurora-A levels in an estrogen receptor-dependent manner. However, the upregulation by E(2) was not restricted to Aurora-A alone. Following release from the tamoxifen-induced arrest, the appearance of Aurora-A in the presence of estradiol in MCF7 cells coincided with the appearance of other mitotic markers suggesting that the spike in Aurora-A levels is an indirect consequence of estrogen-mediated cell proliferation. Thus, at least in short-term in vitro studies, Aurora-A is not a specific direct target of estrogen. However, downregulation of Aurora-A by RNA interference led to a significant decrease in estrogen-induced, anchorage-dependent, and independent growth of MCF7 cells. Moreover, knockdown of Aurora-A could overcome estrogen-induced decrease in docetaxel sensitivity of MCF7 cells. Cumulatively, we propose that the upregulation of Aurora-A by estrogen in short-term in vitro cultures is an indirect consequence of estrogen-induced cell proliferation. Nevertheless, Aurora-A inhibitors could be exploited to override the effects of estrogen on breast tumorigenesis and chemoresistance.