Herceptin-resistant Breast Cancer Research Articles

Anti–MUC1 nanobody can synergize the Tamoxifen and Herceptin effects on breast cancer cells by inducing ER, PR and HER2 overexpression

IntroductionOne of the most pressing concerns associated with breast cancer-targeted therapies is resistance to Tamoxifen and Herceptin. Such drug resistance is usually characterized by reduced expression of certain cell surface receptors. Some biological regimens can induce perceptible overexpression of these receptors in favor of drug responsiveness. Material and MethodsIn this research, drug-responsive MCF-7 and SKBR-3, along with drug-resistant MCF-7R (Tamoxifen resistant) and JIMT-1 (Herceptin resistant) breast cancer cell lines in 2D and 3D cultures were exposed to anti-MUC1 nanobody and then assessed for their ER, PR, and HER2 gene and protein expression using qRT-PCR and immunofluorescent staining analyses. Cell viability and the synergistic relationships of combination treatments were determined with MTT assay followed by CompuSyn software. Apoptotic cells were evaluated with Annexin V/propidium Iodide (PI) and acridine orange/ethidium bromide (AO/EB) staining. ResultsAnti-MUC1 exposure elevated the expression levels of ER (42 folds), PR (18.5 folds), and HER2 (4.7 folds). As a result of co-treatment, the IC50 levels for Tamoxifen and Herceptin were reduced by up to 10 and 3 folds, respectively. MCF-7R cells responded positively to Tamoxifen, as evidenced by a 5-fold reduction in the IC50 and enhanced apoptosis. ConclusionThe ER, PR, and HER2 overexpression after MUC1 blocking could signal drug hypersensitization and facilitate drug resistance management.

Coupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks

The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate complexes during lysis and enrichment procedures. Advantages of this new approach are illustrated through application to PTP1B interaction networks in models of HER2-positive and Herceptin-resistant breast cancer. We have demonstrated that inhibitors of PTP1B significantly reduced proliferation and viability in cell-based models of acquired and de novo Herceptin resistance in HER2-positive breast cancer. Using differential analysis, comparing substrate-trapping to wild-type PTP1B, we have identified multiple unreported protein targets of PTP1B with established links to HER2-induced signaling and provided internal validation of method specificity through overlap with previously identified substrate candidates. Overall, this versatile approach can be readily integrated with evolving proximity-labeling platforms (TurboID, BioID2, etc.), and is broadly applicable across all PTP family members for the identification of conditional substrate specificities and signaling nodes in models of human disease.

Abstract 1790: Herceptin-resistant breast cancer cells exhibits distinct sensitivity to lapatinib in in vivo tumor xenograft models

Abstract Introduction: HER2-targeted therapies, including the monoclonal antibody (Ab) herceptin and the tyrosine kinase inhibitor (TKI) lapatinib, have been commonly used to treat HER2-positive breast cancer (BC). However, resistance to herceptin and/or lapatinib frequently occurs and currently represents a significant clinical problem. We previously demonstrated that both HER3- and IGF-1 receptor (IGF-1R)-initiated signaling played crucial roles in the development of herceptin resistance. Further studies showed that our herceptin-resistant SKBR3-pool2 and BT474-HR20 sublines, as compared their parental SKBR3 and BT474 cells, respectively, also exhibited refractoriness to lapatinib in vitro. However, the in vivo efficacy of lapatinib against the herceptin-resistant BC models (SKBR3-pool2 and BT474-HR20) remains unclear. Methods: Tumor xenograft models were established via subcutaneous inoculation of SKBR3-pool2 or BT474-HR20 cells into athymic nude mice. Tumor formation was assessed by palpation and measured with fine calipers twice a week. The tumor-bearing mice were treated with vehicle (DMSO) or lapatinib (80 mg/kg) via intraperitoneal injection daily for three weeks. Immunohistochemistry (IHC) assays were performed to assess the expression and activation of proteins. Two individuals independently read and quantified all IHC slides by considering both the intensity of immunostaining and the percentage of positive-staining tumor cells. Results: Treatment with lapatinib profoundly inhibited growth of the tumors established with SKBR3-pool2 cells. In contrast, lapatinib slightly, but not significantly, reduced tumor growth in BT474-HR20 cells-derived xenograft models. IHC analyses of the tumors obtained from both SKBR3-pool2 and BT474-HR20 xenografts showed that lapatinib treatment had little effect on the expression of HER3 and the protein phosphatase PPP3CB. Lapatinib also did not alter the levels of phosphorylated Akt (p-Akt) and FOXO3a (p-FOXO3a) in vivo. Interestingly, in BT474-HR20-derived tumors, lapatinib treatment dramatically enhanced expression of insulin receptor substrate-1 (IRS1) and increased the levels of phosphorylated HER3 (p-HER3). However, such phenomena were not observed in the tumors established with SKBR3-pool2 cells. Conclusion: While the tumors derived from BT474-HR20 cells retain their resistant phenotype to lapatinib, SKBR3-pool2-tumors are highly sensitive to lapatinib treatment. It seemed that lapatinib-induced upregulation of IRS1 and activation of HER3, evidenced by increased p-HER3, contributed to the inefficacy of lapatinib against BT474-HR20-tumors in vivo. Keywords: HER3, IRS1, treatment resistance, HER2-targeted therapy, breast cancer Citation Format: Sanbao Ruan, Hao Liu, Hui Lyu, Congcong Tan, Bolin Liu. Herceptin-resistant breast cancer cells exhibits distinct sensitivity to lapatinib in in vivo tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1790.

Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

Resistance to Herceptin represents a significant challenge for successful treatment of HER2-positive breast cancer. Here, we show that in Herceptin-sensitive cells, FOXO3a regulates specific miRNAs to control IGF2 and IRS1 expression, retaining basic IGF2/IGF-1R/IRS1 signaling. The basic activity maintains expression of PPP3CB, a subunit of the serine/threonine-protein phosphatase 2B, to restrict FOXO3a phosphorylation (p-FOXO3a), inducing IGF2- and IRS1-targeting miRNAs. However, in Herceptin-resistant cells, p-FOXO3a levels are elevated due to transcriptional suppression of PPP3CB, disrupting the negative feedback inhibition loop formed by FOXO3a and the miRNAs, thereby upregulating IGF2 and IRS1. Moreover, we detect significantly increased IGF2 in blood and IRS1 in the tumors of breast cancer patients with poor response to Herceptin-containing regimens. Collectively, we demonstrate that the IGF2/IGF-1R/IRS1 signaling is aberrantly activated in Herceptin-resistant breast cancer via disruption of the FOXO3a-miRNA negative feedback inhibition. Such insights provide avenues to identify predictive biomarkers and effective strategies overcoming Herceptin resistance.

Kinetic Modeling of DUSP Regulation in Herceptin-Resistant HER2-Positive Breast Cancer.

Background: HER2 (human epidermal growth factor 2)-positive breast cancer is an aggressive type of breast cancer characterized by the overexpression of the receptor-type protein tyrosine kinase HER2 or amplification of the HER2 gene. It is commonly treated by the drug trastuzumab (Herceptin), but resistance to its action frequently develops and limits its therapeutic benefit. Dual-specificity phosphatases (DUSPs) were previously highlighted as central regulators of HER2 signaling; therefore, understanding their role is crucial to designing new strategies to improve the efficacy of Herceptin treatment. We investigated whether inhibiting certain DUSPs re-sensitized Herceptin-resistant breast cancer cells to the drug. We built a series of kinetic models incorporating the key players of HER2 signaling pathways and simulating a range of inhibition intensities. The simulation results were compared to live tumor cells in culture, and showed good agreement with the experimental analyses. In particular, we observed that Herceptin-resistant DUSP16-silenced breast cancer cells became more responsive to the drug when treated for 72 h with Herceptin, showing a decrease in resistance, in agreement with the model predictions. Overall, we showed that the kinetic modeling of signaling pathways is able to generate predictions that assist experimental research in the identification of potential targets for cancer treatment.

Modelling the role of dual specificity phosphatases in herceptin resistant breast cancer cell lines

BackgroundBreast cancer remains the most lethal type of cancer for women. A significant proportion of breast cancer cases are characterised by overexpression of the human epidermal growth factor receptor 2 protein (HER2). These cancers are commonly treated by Herceptin (Trastuzumab), but resistance to drug treatment frequently develops in tumour cells. Dual-specificity phosphatases (DUSPs) are thought to play a role in the mechanism of resistance, since some of them were reported to be overexpressed in tumours resistant to Herceptin. ResultsWe used a systems biology approach to investigate how DUSP overexpression could favour cell proliferation and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. We measured the expression of 20 DUSP genes in two breast cancer cell lines following long-term (6 months) exposure to Herceptin, after confirming that these cells had become resistant to the drug. We constructed several Boolean models including specific substrates of each DUSP, and showed that our models correctly account for resistance when overexpressed DUSPs were kept activated. We then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed. ConclusionsThese results show how a combination of experimental analysis and modelling help to understand cell survival mechanisms in breast cancer tumours, and crucially enable us to generate testable predictions potentially leading to new treatments of resistant tumours.

Abstract 5888: Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells

Abstract Resistance to HER2-target therapy remains a clinical challenge for HER2+ breast cancer subtype. The molecular mechanisms for Herceptin resistance remain elusive. Here, we demonstrated that the IGF2 and IRS1 protein levels were increased in Herceptin resistant breast cancer cells and that the IGF2/IGF1R/IRS1/Akt/mTOR signaling was involved in Herceptin resistance. In Herceptin sensitive HER2+ breast cancer cells, IRS1 protein level resulted from mTOR2 mediated FOXO3a status determined the feedback regulation of IGF2. Upon high dose rhIGF2 treatment, the mTOR1 translationally upregulated PPP3CB protein level that recovered the function of FOXO3a inactivated by mTOR2. FOXO3a positively transcriptionally regulated miR-128-3p and miR-30-5p expression that targeted IRS1 mRNA to control the feedback regulation of IGF2 signaling. In agreement, in Herceptin resistant cells, the decrease of miR-128-3p and miR-30-5p resulted from constitutively inactivation of FOXO3a by mTOR2, led to increase of IRS1 protein level, even in response to high endogenous IGF2 protein level in culture medium. High IGF2 protein level was resulted from decrease of miR-193a-5p that was also regulated by FOXO3a. Unexpectedly, in Herceptin resistant cells, Src/p-STAT5 signaling cooperating with HDAC1 transcriptionally repressed PPP3CB expression resulting into FOXO3a inactivation. Moreover, we confirmed that IRS1 knockdown reversed the Herceptin resistance in vivo using a xenograft mouse model. Serums form patients with poor response to Herceptin, had higher IGF2 protein level. Tissues from patients with poor response had higher IRS1, p-Akts473, p-FOXO3a and p-Src levels, but had lower PPP3CB, miR-128-3p, miR-30-5p and miR-193a-5p levels. In conclusions, disruption of PPP3CB-FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling plays pivotal role in Herceptin resistance of HER2+ breast cancer. Related molecules in this signaling may represent potential biomarkers and therapeutic targets for HER2+ breast cancer treatment. Keywords: Breast cancer, Herceptin resistance, IGF2, IRS1, FOXO3a This study was supported by grants from the National Natural Science Foundation of China: No.81672616, No.81402196 and No.81272450, supported by grants from Guangdong Natural Science Funds for Distinguished Young Scholars No.2016A030306003 (G Zheng). Citation Format: Zhimin He. Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5888.

The function and mechanism of ErbB3 and IGF1R in breast cancer with Herceptin resistance

Objective To investigate the role of epidermal growth factor receptor 3 (ErbB3) and insulin-like growth factor-1 receptor (IGF1R) in enhancing the resistance of Herceptin in human breast cancer. Methods HRG (Heregulin, the ligand of ErbB3) or IGF2 (insulin-like growth factor2, the ligand of IGF1R) was correspondingly added into breast cancer cells SKBR3 and BT474, and then 3-(4, 5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and were performed in these cells to evaluate the sensitivity of these cells to Herceptin. Furthermore, we used HRG or IGF2 antibodies to inhibit their joint receptors in Herceptin-resistant breast cancer cells SKBR3/POOL2 and BT474/HR20. Finally, the sensitivity of these treated cells to Herceptin was detected via MTS assay. HRG or IGF2 was added into breast cancer cell BT474, and co-IP assay was used to detect the expressions of ErbB3 and IGF1R which combined with ErbB2. Results The treatment groups used HRG or IGF2 enhanced the resistance of Herceptin in Herceptin-sensitive breast cancer cells. On the other hand, we used antibodies of HRG and IGF2 to block their combining with their receptors in Herceptin-resistant breast cancer cells, the cells became more sensitive to Herceptin. BT474 cell was treated with HRG or IGF2. The expressions of ErbB3 and IGF1R which combined with ErbB2 were increased. Conclusions The formation of heterodimers ErbB2/ErbB3 and ErbB2/IGF1R might enhance the resistance of Herceptin in ErbB2-overexpression human breast cancers. Key words: Receptor, epidermal growth factor; Receptor, IGF type 1; Breast neoplasms/DT/ME; Drug tolerance; Antibodies, monoclonal/TU

Abstract 347: Developing a novel immunotoxin that targets cells overexpressing ErbB2

Abstract Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. The use of a membrane lysin as the toxin domain distinguishes our immunotoxins from numerous others in that internalization of the toxin is not required to induce cell death. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics. While Herceptin treatment has been effective in patients with ErbB2 positive breast cancer, our novel immunotoxin may ultimately prove to be a novel and efficacious addition to the arsenal of approaches used to eliminate Herceptin-resistant breast cancer cells. Citation Format: Luqun Shen, Kelsey Weigel, Clayton Thomas, Lauren Drapalik, Zachary T. Schafer, Shaun Lee. Developing a novel immunotoxin that targets cells overexpressing ErbB2. [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 347.

Abstract 1677: Annexin A2 antibody inhibits the progression of triple-negative and herceptin-resistant breast cancer by blocking epidermal growth factor receptor functions

Abstract Short Description: Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown. Methods: Triple-negative (MDA-MB-231) and Herceptin-resistant (JIMT-1) breast cancer cell lines were grown in complete DMEM and DMEM/F12 medium respectively, in a humidified incubator at 37°C with 5% CO2. The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study Results: We demonstrate that in triple-negative and Herceptin-resistant breast cancer cells a large amount of AnxA2 is specifically translocated to the outer membrane domain. In addition, AnxA2 interacts with extracellular domain of epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalization by blocking its homodimerization. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration. Conclusions: These studies indicate that association of AnxA2 with EGFR in the membrane domain might play a positive regulatory role in keeping EGFR signaling events in an activated state in triple negative and Herceptin-resistant breast cancer thus making AnxA2 an important therapeutic target. Citation Format: Pankaj Chaudhary, Lee Daniel Gibbs, Jamboor K. Vishwanatha. Annexin A2 antibody inhibits the progression of triple-negative and herceptin-resistant breast cancer by blocking epidermal growth factor receptor functions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2015-1677

87P - Inhibition of triple negative and herceptin-resistant breast cancer proliferation and migration by annexin A2 antibodies

87P - Inhibition of triple negative and herceptin-resistant breast cancer proliferation and migration by annexin A2 antibodies

Inhibition of triple-negative and Herceptin-resistant breast cancer cell proliferation and migration by Annexin A2 antibodies.

Background:Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown.Methods:The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study.Results:We demonstrate that AnxA2 interacts with epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalisation by blocking its homodimerisation. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration.Conclusions:These findings suggest that cell-surface AnxA2 has an important regulatory role in EGFR-mediated oncogenic processes by keeping EGFR signalling events in an activated state. Therefore, AnxA2 could potentially be used as a therapeutic target in triple-negative and Herceptin-resistant breast cancers.

Correction: Reciprocal Regulation of Annexin A2 and EGFR with Her-2 in Her-2 Negative and Herceptin-Resistant Breast Cancer

Correction: Reciprocal Regulation of Annexin A2 and EGFR with Her-2 in Her-2 Negative and Herceptin-Resistant Breast Cancer

Reciprocal Regulation of Annexin A2 and EGFR with Her-2 in Her-2 Negative and Herceptin-Resistant Breast Cancer

Alternative survival pathways are commonly seen to be upregulated upon inhibition of receptor tyrosine kinases (RTK), including Her-2. It is established that treatment with Herceptin leads to selective overexpression and activation of epidermal growth factor receptor (EGFR) and Src which further contributes to oncogenesis in Herceptin resistant and triple negative breast cancer (TNBC) patients. Here, we show a co-regulated upregulation in the expression of Annexin A2 (AnxA2), a known substrate of Src and one of the regulators of EGFR receptor endocytosis, in Herceptin resistant and Her-2 negative breast cancer. Immunohistochemical expression analysis revealed a reciprocal regulation between Her-2 and AnxA2 in breast cancer clinical samples as well as in cell lines as confirmed by protein and RNA analysis. The siRNA and Herceptin mediated downregulation/inhibition of Her-2 in Her-2 amplified cells induced AnxA2 expression and membrane translocation. In this study we report a possible involvement of AnxA2 in maintaining constitutively activated EGFR downstream signaling intermediates and hence in cell proliferation, migration and viability. This effect was consistent in Herceptin resistant JIMT-1 cells as well as in Her-2 negative breast cancer. The siRNA mediated AnxA2 downregulation leads to increased apoptosis, decreased cell viability and migration. Our studies further indicate the role of AnxA2 in EGFR-Src membrane bound signaling complex and ligand induced activation of downstream signaling pathways. Targeting this AnxA2 dependent positive regulation of EGFR signaling cascade may be of therapeutic value in Her-2 negative breast cancer.

Abstract 3812: Deguelin as a novel therapeutic agent for Herceptin resistant breast cancers

Abstract Human breast cancers overexpressing HER-2 are generally highly aggressive. Current therapeutic strategies to target HER-2 signaling pathway with antibodies or small molecule tyrosine kinase inhibitors have shown impressive success in the management of these tumor types, although most women exhibit short term response to these therapies. Acquired resistance to HER-2 target drugs, especially Herceptin is a major challenge to clinicians. Patients receiving Herceptin therapy invariably recur and most often with highly aggressive tumors. Thus identifying new drugs which target multiple molecular pathways is desired to treat Herceptin resistant breast cancer. Using paired HER-2 overexpressing parental Herceptin responsive (BT-474) and its Herceptin resistant variant cells (BT-474HR) we showed that acquired resistance to Herceptin is associated with enhanced expression of HER-2, EGFR, CyclinD1, P-AKT (Ser-473), PI-3K, JAB1 proteins and decreased expression of nuclear pten and p27 proteins. These results are similar to those observed in recurrent breast tumors in patients following Herceptin treatment. Deguelin, a natural product derivative of rotenoids and has been shown to inhibit growth of diverse cancer types. Deguelin is a known AKT inhibitor, however its efficacy is never tested in breast cancers resistant to Herceptin which express high levels of p-AKT. We examined effect of Deguelin on HER-2 overexpressing BT-474 BT-474-HRcells. Deguelin inhibited growth of both, BT-474 and BT-474HR cells in concentration (0-250nM) and time dependent (24-72h) manner. There was a 40-50%- inhibition of both BT474and BT-474HR cells at 25nM Deguelin treatment at 72h. Deguelin decreased cell surface expression of HER-2 in both these cell lines. In parental BT-474 cells both Herceptin 5-10ug/ml and Deguelin down regulated HER-2 expression, but only Deguelin reduced HER-2 (125-250nM) over expression in both BT-474 and BT-474HR cells. Down regulation of HER-2 following Deguelin treatment was associated with simultaneous down regulation of nuclear accumulation of cyclin D1, and JAB1, and p-AKT (Ser-473) proteins and up regulation of nuclear p27 and p53in both BT-474 and BT474 HR cells. These results suggest that Deguelin inhibits growth of both HER-2 overexpressing and those resistant to Herceptin therapy breast cancer cells. Effect of Deguelin appears to be mediated through down regulation of HER-2, PI3K AKT, JAB1, Cyclin D1, and p27 pathway. Our preliminary results warrant further investigation into Deguelin as a potential therapeutic agent for Herceptin resistant breast cancers. (This work was supported by NCI CA140321.) 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 3812. doi:1538-7445.AM2012-3812

Targeting Bcl-2 in Herceptin-Resistant Breast Cancer Cell Lines

Acquired resistance to Herceptin is a major clinical problem in the treatment of HER2-overexpressing breast cancer. Understanding the molecular mechanisms leading to resistance will allow identification of novel therapeutic targets and predictors of therapeutic response. To this end, up-regulation of anti-apoptotic proteins has been associated with resistance to the HER2-targeted drug lapatinib, but has not yet been linked to Herceptin resistance. The aim of the current study was to determine if the Bcl-2 anti-apoptotic protein is a potential therapeutic target in cells with acquired Herceptin resistance. The BT474 HER2-overexpressing breast cancer cell line and BT474-derived acquired Herceptin-resistant clones were used as models in this study. Bcl-2 and Bax expression were assessed by Western blotting. Proliferation assays were performed on cells treated with the Bcl-2 inhibitor ABT-737 in the absence or presence of Herceptin. Finally, the effect of PI3K inhibition or IKK inhibition on Bcl-2 expression and Herceptin sensitivity was examined by Western blotting and established proliferation assays. We show that cells with acquired resistance to Herceptin have an increased Bcl-2:Bax ratio. Resistant cells have increased sensitivity to ABT-737. Further, pharmacologic inhibition of Bcl-2 improved sensitivity to Herceptin in acquired resistant cells. Finally, PI3K and IKK inhibition down-regulated Bcl-2 expression and increased sensitivity to Herceptin in resistant cells. Taken together, these new observations support further study of Bcl-2-targeted therapies in Herceptin-resistant breast cancers, and importantly, future investigation of Bcl-2 expression as a potential predictor of Herceptin response in patients with HER2-overexpressing breast cancer.

Abstract 1759: Design optimization and characterization of novel herceptin-based immunotoxins for cancer therapy. Enhanced antitumor activity in vitro and in vivo

Abstract The human epidermal growth factor receptor (Her) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of Her2/neu is associated with aggressive disease and poor prognosis. Herceptin, the only anti-Her2/neu, humanized monoclonal antibody approved by FDA for therapy of mammary carcinoma is currently and successfully prescribed for treatment of Her2/neu positive cancer. However, in 40-60% of all patients with Her2/neu-overexpressing tumors, Herceptin has little or no effect. The goal of this study was to demonstrate the construction and characterization of various Herceptin-based immunotoxins, loaded with a highly cytotoxic recombinant gelonin (rGel) molecule. Utilizing a humanized anti-Her2/neu scFv (4D5), we generated 2 orientation constructs: 4D5/rGel and rGel/4D5. We also generated a conventional chemical conjugate with full-length Herceptin (Herceptin/rGel conjugate), and we generated a unique fusion of anti-Her2/neu IgG containing 2 rGel molecules (Aglyco-Herceptin/rGel). This is the first report of the preparation of active full-length antibody-toxin construct from E.coli. In vitro studies showed that all immunotoxins retained the affinity and specificity of the original antibody, as well as the biological activity of rGel toxin. Against the Her2/neu overexpressing cancer cell lines, all immunotoxins demonstrated specific cytotoxic effects, even against Herceptin-resistant breast cancer cells. Against ovarian (SKOV3) cells, the Herceptin/rGel conjugate, Aglyco-Herceptin/rGel and rGel/4D5 fusion constructs demonstrated the best in vitro efficacy (IC50 of 0.28nM, 12.19nM and 1.57nM for each construct respectively vs 70.86 nM for 4D5/rGel). rGel/4D5 outperformed 4D5/rGel demonstrating improved internalization efficiency and increased intracellular delivery of rGel. All the immunotoxins displayed similar minimal exposure times to target cells for optimal cytotoxic activity. All constructs demonstrated a distinct cell-killing efficiency time course. Functional stability studies of the constructs showed that all remained biologically active after incubation in human plasma. Efficacy studies of the various constructs against SKOV3 tumor xenografts are ongoing and will be reported. Based on our in vitro results, the bivalent full-length antibody-toxin immunotoxins were superior to the corresponding recombinant monovalent constructs. Among monovalent constructs, the rGel/4D5 fusion was superior in vitro overall to 4D5/rGel. Different orientations of antibody and toxin can promote optimal cell internalization and toxicity of the immunotoxin, and may be valuable considerations for further construct optimization. Research conducted, in part, by the Clayton Foundation for Research. 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 1759. doi:10.1158/1538-7445.AM2011-1759

Abstract 611: Etanercept as a new tool for treatment of Herceptin-resistant breast cancer induced by tumor necrosis factor alpha

Abstract Breast cancer overexpressing ErbB2 is associated with high aggressiveness and elevated metastatic potential. Several therapies have been designed targeting receptor tyrosine kinase ErbB2, such as the low molecular weight inhibitor Lapatinib and the monoclonal antibody Herceptin. We have already demonstrated that tumor necrosis factor alpha (TNF) induces proliferation of BT-474 and SKBR-3 human and C4HD murine breast cancer cells which overexpress ErbB2, through the transactivation of ErbB2 and subsequent activation of transcription factor NF-κB, and through increase of Cyclin D1 protein expression. In the present study we evaluated the effectiveness of clinical drugs which block ErbB2, in order to inhibit NF-κB transcriptional activity and TNF-induced proliferation. We transfected BT-474 and SKBR-3 breast cancer cells with a luciferase reporter gene under control of a NF-κB response element and observed that the pharmacological ErbB2 inhibitor AG825 (100 μM)and the Lapatinib analog GW2974 (1 μM) blocked NF-κB activity induced by TNF. However, when we used 10 μg/ml Herceptin, TNF still activated NF-κB. We also monitored NF-κB activation through Western blot of phosphorylated IκBα, the protein which inhibits NF-κB traslocation to the nucleus, with same results. By reporter gene assays, we then explored activation of Cyclin D1 promoter, a target gene of NF-κB. While both AG825 and GW2974 blocked TNF-induced Cyclin D1 promoter activation, Herceptin did not. The endogenous protein Cyclin D1 mirrored the profile obtained with reporter gene assays. BT-474 proliferation increased in the presence of TNF (75 ± 12% vs control cells) measured by 3H-thimidine incorporation, cell count and flow cytometry of cells stained with propidium iodide. AG825 and GW2974 completely blocked TNF-induced proliferation, but Herceptin failed to do so. Bearing in mind that TNF is frequently expressed in invasive breast tumors, our results suggest that TNF may be one of the factors which confer Herceptin resistance. We therefore hypothesized that blocking TNF through the TNFR2-FcIgG fusion protein Etanercept may overcome Herceptin resistance. We observed that Etanercept (5 mg/kg, i.p. twice wk) inhibited TNF-induced in vitro proliferation of C4HD cells, a murine breast adenocarcinoma that produces TNF. When this tumor was growth in nude mice and treated with Etanercept the tumor size was reduced by 37.6 ± 1.1 % (P<0.05). Interestingly, histological examination of said tumors revealed that there were fewer mitotic figures and a lower percentage of necrotic and fibrotic areas in the tumors from mice treated with Etanercept, as compared to the group treated with an irrelevant IgG. Our results propose Etanercept as a new agent to overcome clinically observed Herceptin resistance. 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 611.

Improvement of sensitivity to tamoxifen in estrogen receptor-positive and Herceptin-resistant breast cancer cells

ERBB2 overexpression in estrogen receptor (ER)-positive breast cancer cells such as BT474 (BT) cells has been found to confer resistance to tamoxifen, and suppression of ERBB2 improves the antiproliferative effects of tamoxifen. In this study, the responsiveness to tamoxifen in the BT/HerR, Herceptin-resistant BT cell lines established through constant Herceptin exposure, was evaluated. Compared with BT cells, improvement of sensitivity to tamoxifen in BT/HerR was demonstrated by ER functional analysis and cell proliferation assay. Tamoxifen in the resistant cell line was found to inhibit 17beta-estradiol-stimulating estrogen-responsive gene pS2 expression more effectively than in BT cells in real-time PCR assay. Western blot analysis showed that cross-phosphorylation between ER and downstream components of ERBB2 was attenuated in BT/HerR cells. ER redistribution from cytoplasm to nucleus could be found in these cells through immunofluorescence and confocal studies, and importantly, chromatin immunoprecipitation studies demonstrated that tamoxifen induced occupancy of the pS2 promoter by ER and nuclear receptor corepressor (NCOR1) instead of coactivator NCOA3 in these cells. Finally, combination of tamoxifen and Herceptin was found to improve the sensitivity of BT/HerR cells to Herceptin. Our results suggest that the ER genomic pathway in the ER-positive and Herceptin-resistant breast cancer cells may be reactivated, allowing tamoxifen therapy to be effective again, and a combination of tamoxifen and Herceptin can be a potential therapeutic strategy for ER-positive and Herceptin-resistant human breast cancer.

The role of insulin-like growth factor-I receptor in the development of Herceptin resistance

To the Editor: We read with great interest the article by Tanner et al. regarding development of a novel cell line established from patient with Herceptin-resistant breast cancer ([1][1]). They showed that lack of growth inhibition was rationalized by the unaltered Akt phosphorylation in these