HER2-positive Breast Cancer Cell Lines Research Articles

Erratum to: Evaluation of IGF1R and phosphorylated IGF1R as targets in HER2-positive breast cancer cell lines and tumours

Erratum to: Evaluation of IGF1R and phosphorylated IGF1R as targets in HER2-positive breast cancer cell lines and tumours

Abstract 443: Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib

Abstract HER2-positive breast cancers are poorly differentiated and have a higher potential for metastasis. Although trastuzumab and lapatinib have been used for the treatment of HER2-positive breast cancer, resistance to both drugs has dampened the long-term benefit of those drugs. As cancer has multiple genetic aberrations and can readily develop resistance to individual drugs with specific targets, we employed synergistic drug combination as a strategy to overcome drug resistance and to enhance the efficacy of existing drugs. From a screen of the Johns Hopkins Drug Library, we identified triptolide as a strong synergistic hit with lapatinib in HCC1954, a drug-resistant HER2-positive breast cancer cell line. The combination of lapatinib and nontoxic doses of triptolide synergistically inhibited the HER2 signaling pathway. We found that lapatinib increased both mRNA expression and the half-life of HER2 protein, whereas triptolide countered the effects of lapatinib by downregulating the expression of HER2, leading to synergistic inhibition of the HER2 signaling pathway. The synergy in cell proliferation was observed in all HER2-positive breast cancer cell lines regardless of drug resistance status, but was absent in HER2-negative ones. In mouse xenograft models, the combination of lapatinib and triptolide at their minimum effective doses almost completely inhibited the growth of the drug-resistant HER2-positive breast cancer cells. Together, these findings suggest that the combination of triptolide and lapatinib could be a promising new therapeutic regimen for drug-resistant HER2-positive breast cancer. Citation Format: Paweena Chalugun, Joong Sup Shim, Preethi C. Korangath, Saraswati Sukumar, Jun O. Liu. Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2014-443

Abstract 3914: Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer

Abstract TFAP2C and mouse homolog tcfap2c are transcription factors that play a critical role in mammary gland development and in maintaining the luminal breast cancer phenotype. Interestingly, several human HER2-positive breast cancer cell lines display genetic dependency on TFAP2C. However, the role of TFAP2C in carcinogenesis and progression in neu-activated breast cancer remains unknown. We examined the role of tcfap2c in mammary gland carcinogenesis by examining tumor formation in mice expressing an activated rat neu gene using MMTV-neu with and without mammary gland conditional knockout (KO) of tcfap2c using MMTV-Cre. The time until spontaneous palpable tumor formation was measured in mice harboring the neu oncogene with and without tcfap2c. Mice with homozygous conditional deletion of tcfap2c demonstrated a significant delay in tumor formation compared to littermate controls that retained tcfap2c expression (42 vs. 25 weeks, p<0.004). Immunohistochemistry of the tumors demonstrated a luminal phenotype; cytokeratin 8 (CK8), a luminal marker, was more strongly expressed compared to cytokeratin 5 (CK5), a basal marker, in tumors from both control (883 vs. 0.1 cells/HPF, p<0.0005) and KO animals (845 vs. 0, p<0.0005). Cleaved caspase-3 (CC3) showed that apoptosis was increased in tumors from KO compared to control animals (5 vs. 0.3 CC3-positive cells/HPF, p<0.0008). Ki-67 showed that proliferation was decreased in KO compared to control (348 vs. 142 cells/HPF, p<0.009). To validate the effects of tcfap2c, we established a cell line from a tumor obtained from an animal expressing activated neu with homozygous floxed tcfap2c lacking Cre expression. Paired cell lines with differential expression of tcfap2c were established by infecting the cells with Adenovirus-Cre (Ad-Cre) or Adenovirus-Empty (Ad-Empty). Proliferation was assessed by MTT and demonstrated that loss of tcfap2c expression resulted in a 30% decrease in proliferation rate (p<0.006). The difference in proliferation was confirmed in vivo through xenograft experiments of these paired cell lines inoculated into athymic (nude) mice. Mice xenografted with the paired cell lines demonstrated that KO of tcfap2c resulted in a significant delay in tumors reaching a 2 cm threshold requiring euthanasia (27.8 vs. 19.8 days post-inoculation, p<0.003). In order to identify a potential mechanism accounting for the differences in proliferation and delay in carcinogenesis, we identified tcfap2c target genes by comparing the binding sites of tcfap2c by chromatin-immunoprecipitation-sequencing (ChIP-seq) and differential expression by microarray/RT-PCR. We identified erbb3 and egfr as two tcfap2c target genes by ChIP-seq. KO of tcfap2c resulted in a 3-fold and 12-fold reduction in erbb3 and egfr expression, respectively (p<0.05). Taken together, these findings indicate that tcfap2c plays critical roles in oncogenesis and progression of HER2-positive mammary cancer. Citation Format: Jung M. Park, Tong Wu, George Woodfield, Anthony Cyr, James De Andrade, Weizhou Zhang, Frederick Domann, Ronald J. Weigel. Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2014-3914

Taspase1 cleaves MLL1 to activate cyclin E for HER2/neu breast tumorigenesis.

Taspase1, a highly conserved threonine protease, cleaves nuclear transcriptional regulators mixed-lineage leukemia (MLL, MLL1), MLL2, TFIIA, and ALF to orchestrate a wide variety of biological processes. In vitro studies thus far demonstrated that Taspase1 plays important roles in the proliferation of various cancer cell lines, including HER2-positive breast cancer cells. To investigate the role of Taspase1 in breast tumorigenesis in vivo, we deleted Taspase1 from mouse mammary glands by generating MMTV-neu;MMTV-cre;Tasp1F/− mice. We demonstrate that initiation of MMTV-neu- but not MMTV-wnt-driven breast cancer is blocked in the absence of Taspase1. Importantly, Taspase1 loss alone neither impacts normal development nor pregnancy physiology of the mammary gland. In mammary glands Taspase1 deficiency abrogates MMTV-neu-induced cyclins E and A expression, thereby preventing tumorigenesis. The mechanisms were explored in HER2-positive breast cancer cell line BT474 and HER2-transformed MCF10A cells and validated using knockdown-resistant Taspase1. As Taspase1 was shown to cleave MLL which forms complexes with E2F transcription factors to regulate Cyclins E, A, and B expression in mouse embryonic fibroblasts (MEFs), we investigated whether the cleavage of MLL by Taspase1 constitutes an essential in vivo axis for HER2/neu-induced mammary tumorigenesis. To this end, we generated MMTV-neu;MLLnc/nc transgenic mice that carry homozygous non-cleavable MLL alleles. Remarkably, these mice are also protected from HER2/neu-driven breast tumorigenesis. Hence, MLL is the primary Taspase1 substrate whose cleavage is required for MMTV-neu-induced tumor formation. As Taspase1 plays critical roles in breast cancer pathology, it may serve as a therapeutic target for HER2-positive human breast cancer.

Abstract 4338: The lipogenic phenotype of HER2/neu-positive breast cancer cells

Abstract 15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. We have shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis of palmitate-treated HER2/neu-positive SKBR3 cells and HER2-normal MCF7 cells suggests that palmitate induces an ER-stress response in the HER2-positive cells. This is congruent with protein markers for stress-related translational stalling, which ultimately result in CHOP-dependent cell death. Palmitate distinctly reduces the expression of the HER3 protein and combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. In addition to these translational changes, palmitate treatment of HER2/neu-positive SKBR3 cells results in AMPK activation and subsequent inhibition of fatty acid synthesis. Transcriptome and global metabolite profiling analyses of the effect of increased palmitate on these cells indicate that palmitate alters the metabolic phenotype by re-routing the glucose carbon flux in the cells. Supported by CDMRP Award W81XWH-11-1-0094 to Jan Baumann and NCI Award 5R01CA136658-03 to Douglas Conklin. Citation Format: Jan M. Baumann, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2014-4338

Abstract 153: High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line

Abstract Purpose Neuropilin-1 (Nrp1) was initially characterized as a guide for migrating cells and axons in developing nervous systems and is essential for the precise formation of neurons and vasculature. However, it was recently reported to also play an important role in the immune system. This study aimed to investigate the role of Nrp1 on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cancer cell line SKBR3. In addition, we evaluated the gene expression profile of monocytes expressing high and low levels of Nrp1. Methods Peripheral blood mononuclear cells from healthy volunteers were prepared using BD Vacutainer CPT Cell Preparation Tubes. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD14 and allophycocyanin-conjugated anti-Nrp1 antibodies, and Nrp1high and Nrp1low monocyte subsets were sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay with SKBR3 as target cells and trastuzumab as the antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. The effector cell to target cell ratios of 20:1 and 10:1 were used. Gene expression analysis was performed by Affymetrix GeneChip microarrays. All study protocols were approved by the Ethics Committee for Clinical Research, Kyoto University Hospital, Kyoto, Japan (authorization number G424). Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted Nrp1high monocytes than in Nrp1low monocytes (75% vs. 45%, p<0.01). Trastuzumab-mediated ADCC was significantly suppressed by Nrp1 neutralization antibodies in a dose-dependent manner (Control arm 14.3%, Treatment arm 6%; p<0.01). Gene expression analysis revealed that 308 genes were up-regulated in the Nrp1high population, including monocyte-specific chemokine receptor and hematopoietic transcriptional factor. Conclusion These results suggest that determination of Nrp1 expression can be used to classify monocytes as populations with and without cellular cytotoxic activity. Analysis of Nrp1 expression levels in monocytes can therefore be used to identify HER2-positive breast cancer patients for whom trastuzumab therapy will be less effective. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Masao Kawashima, Masakazu Toi. High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2014-153

Effect of Her2 Overexpression on Cancer Stem-Like Characteristics of Triple-Negative Breast Cancer Cells

ABSTRACT Aim: Cancer stem cells (CSCs) have been proposed to constitute a subpopulation of undifferentiated tumor cells that are resistant to both radio- and chemotherapy. The population exhibiting CD44+/CD24- is generally accepted to be human breast CSCs. However the overexpression of CD24 in breast cancer is associated with a poor patient prognosis. HER2-positive breast cancers are predominantly positive for CD24, which has been identified as a marker of differentiated normal mammary epithelial cells. We aimed to investigate the relationship between HER2 and CD24. Methods: We established stable HER2-expressing cell lines by introducing a wild type HER2 gene into 231-Luc triple-negative (ER- PgR- HER2-) breast cancer cells, having stem-like characteristics. Then, we determined the functional relevance of CD24 expression in HER2-positive breast cancer cells. We also investigated the expression of CD44, CD24 and HER2 by flow cytometry analysis. Results: Overexpression of HER2 decreased representative CSC properties, such as the sphere-forming ability and ALDH1 activity, and did not affect the tumorigenicity of 231-Luc triple-negative breast cancer cells. Flow cytometry analysis revealed that stable overexpression of HER2 up-regulates CD24 in triple-negative breast cancer. Positive relationship between HER2 and CD24 expression was also confirmed in HER2-positive breast cancer cell lines: BT-474, HCC202, SKBR3, and HCC1569. Given that overexpression of HER2 increases the level of Akt phosphorylation, we examined if CD24 depletion might suppress Akt phosphorylation in HER2-expressing cells. Successfully, the phosphorylation of Akt was down-regulated by CD24 depletion in the HER2-expressing cells. Moreover, knockdown of CD24 increased the sensitivity of HER2-expressing cells to lapatinib, a dual tyrosine kinase inhibitor for both EGFR and HER2. Conclusions: HER2 may have some contribution to stem-like characteristics of breast cancer cells but other factors are required to regulate CSC functions. CD24 supports both the expression of HER2 and the consequent activation of the PIK3/AKT signaling pathway so as to promote cell survival. CD24 appears to contribute to the resistance to HER2-targeted therapy and is itself a potential therapeutic target in HER2-positive breast cancer. Disclosure: All authors have declared no conflicts of interest.

Discovery of hapten-specific scFv from a phage display library and applications for HER2-positive tumor imaging.

In this study, an anti-hapten antibody (single chain Fv, scFv) against a hapten probe was developed as a unique reporter system for molecular imaging or therapy. The hapten peptide (histamine-succinyl-GSYK, Him) was synthesized for phage displayed scFv affinity selection and for conjugation with cypate (Cy-Him) for in vivo near-infrared (NIR) optical imaging. Hapten-specific scFvs were affinity selected from the human single fold phage display scFv libraries (Tomlinson I + J) with high specificity and affinity. Utilizing HER2 targeting as a model system, the highest affinity scFv (clone J42) was recombinantly fused to an anti-HER2 affibody (scFv-L-Aff) with no loss of affinity of either protein. The functionality of the hapten-scFv reporter system was tested in vitro with a HER2-positive human breast cancer cell line, SK-BR3, and in vivo with SK-BR3 xenografts. ScFv-L-Aff mediated the binding of the hapten to HER2 on SK-BR3 cells and from tissue from the SK-BR3 xenograft; however, scFv-L-Aff did not mediate uptake of the hapten in the SK-BR3 xenografted tumors, presumably due to rapid internalization of the HER2/scFv-L-Aff complex. Our results suggest that this hapten-peptide and anti-hapten scFv can be a universal reporter system in a wide range of imaging and therapeutic applications.

Role of CD24 in HER2-positive breast cancer.

e11572 Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane kinase receptor, which promotes the RAS/RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. The standard treatment for HER2-positive breast cancer is a combination of chemotherapy plus an anti-HER2 agent, such as trastuzumab and lapatinib. HER2-positive breast cancers are predominantly positive for CD24, which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein. Overexpression of CD24 in breast cancer is associated with a poor prognosis. We hypothesize that the co-expression of HER2 with CD24 is associated with the therapeutic resistance of HER2-positive breast cancer cells. Methods: We established HER2-expressing cell lines by introducing a wild type HER2 gene into MDA-MB-231 triple-negative (ER-/PgR-/HER2-) breast cancer cells. We used the HER2-positive breast cancer cell lines BT-474, HCC202, SKBR3, and HCC1569. To investigate the relevance of CD24 expression in HER2-positive breast cancer, we analyzed HER2 and...

Effect of trastuzumab/AT-101 combination on apoptosis and cytotoxicity in HER2-positive breast cancer cells: A novel combination to effect resistance to anti-HER2 therapy through inhibition of PI3K signaling.

e13512 Background: Trastuzumab is a humanized monoclonal antibody approved for the treatment of breast cancer with HER2 amplification and/or overexpression. AT-101, the (-/-) enantiomer of gossypol...

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1)

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44highCD24low breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44highCD24lowHER2low stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44highCD24low cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.

Enhanced PI3K p110α Signaling Confers Acquired Lapatinib Resistance That Can Be Effectively Reversed by a p110α-Selective PI3K Inhibitor

Although the HER2-targeting agents trastuzumab and lapatinib have improved the survival of patients with HER2-positive breast cancer, resistance to these targeted therapies is a major challenge. To investigate mechanisms of acquired lapatinib resistance, we generated acquired lapatinib resistance cell models by extended exposure of two HER2-positive breast cancer cell lines to lapatinib. Genomic and proteomic analyses revealed that lapatinib-resistant breast cancer cells gained additional phosphoinositide 3-kinase (PI3K) activation through activating mutation in PI3K p110α and/or increasing protein expression of existing mutant p110α. p110α protein upregulation in lapatinib-resistant cells occurred through gene amplification or posttranscriptional upregulation. Knockdown of p110α, but not p110β, the other PI3K catalytic subunit present in epithelial cells, inhibited proliferation of lapatinib-resistant cells, especially when combined with lapatinib. Lapatinib-resistant xenograft growth was inhibited persistently by combination treatment with the p110α-selective PI3K inhibitor BYL719 and lapatinib; the drug combination was also well tolerated in mice. Mechanistically, the combination of lapatinib plus BYL719 more effectively inhibited Akt phosphorylation and, surprisingly, Erk phosphorylation, than either drug alone in the resistance model. These findings indicate that lapatinib resistance can occur through p110α protein upregulation-mediated, and/or mutation-induced, PI3K activation. Moreover, a combinatorial targeted therapy, lapatinib plus BYL719, effectively overcame lapatinib resistance in vivo and could be further tested in clinical trials. Finally, our findings indicate that p110β may be dispensable for lapatinib resistance in some cases. This allows the usage of p110α-specific PI3K inhibitors and thus may spare patients the toxicities of pan-PI3K inhibition to allow maximal dosage and efficacy.

Abstract P6-05-08: Nuclear HER3 localisation plays a role in trastuzumab resistance in HER2-positive breast cancer

Abstract Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

Effect of afatinib alone and in combination with trastuzumab in HER2-positive breast cancer cell lines.

632 Background: Trastuzumab and lapatinib have been shown to significantly improve the prognosis for HER2 positive breast cancer patients. However, resistance is a significant clinical problem. The aim of this study is to assess the activity of afatinib, an irreversible pan-HER tyrosine kinase inhibitor, in HER2 overexpressing breast cancer cell lines, including trastuzumab and/or lapatinib resistant cells. Methods: Using proliferation assays, the effect of afatinib was assessed alone and in combination with trastuzumab in HER2 positive cell lines. The effect of afatinib on HER2, Erk and Akt was determined by immunoblotting. Results: The eight HER2 positive breast cancer cell lines tested, including trastuzumab and/or lapatinib resistant cells, responded to afatinib with IC50values ranging from 5 to 80 nM. The combination of afatinib and trastuzumab was additive in four trastuzumab sensitive cell lines and one model of acquired trastuzumab resistant HER2 positive breast cancer. In the remaining three trastuzumab and/or lapatinib resistant cell lines, combined treatment with trastuzumab and afatinib showed no enhancement compared to afatinib alone. Finally, afatinib decreased the phosphorylation of HER2 and Erk in all cell lines tested. Conclusions: Our results suggest that afatinib has activity in HER2 positive breast cancer, including trastuzumab and/or lapatinib resistant breast cancer. We also demonstrate that afatinib in combination with trastuzumab may be more effective than either agent alone in trastuzumab sensitive breast cancer. [Table: see text]

Radiosensitization of HER2-positive breast cancer cell lines with trastuzumab.

e11501 Background: HER2 is a member of the human epidermal growth factor (EGF) receptor family that is blocked by a monoclonal antibody, Trastuzumab (Herceptin). In daily clinical practice, Trastuzumab is being used concurrently with radiotherapy (RT) in breast cancer patients. Administration of Trastuzumab with RT appear to be safe in regard to cardiac morbidity and mortality in patients with relatively modest follow-up duration (less than 5 years). In this study we aimed to determine the possible interactions between RT and Trastuzumab in HER2 positive breast cancer cell line MDA-MB-453. Methods: MDA-MB-453 cells were treated with increased dose of Trastuzumab (10 – 200 µg/mL, 72 hours) and irradiation (0-8 Gy, 4 Gy/min, 48 hours). XTT (Roche) viability assay was used to measure the cytotoxicity of Trastuzumab, and trypan blue method was used to measure the cytotoxicity of irradiation. The sensitization of MDA-MB-453 cell lines was done by IC50 of Trastuzumab with 24 hours treatment followed by 6 and 8 Gy irradiation. Results: Cytotoxicity was increased in a dose and time dependent manner by Trastuzumab and irradiation treatment in MDA-MB-453 cell lines. IC50 values of Trastuzumab and irradiation were found to be 167 µg/mL and 8 Gy, at 48 hours, respectively.Cells were pretreated with IC25 and IC50 doses of Trastuzumab for 24 hours and then irradiated with 6 and 8 Gy doses. The cell viability at 24 and 48 hours were significantly decreased (p=0.0012) compared to single exposures (Trastuzumab or irradiation), indicating that Trastuzumab sensitizes HER2 positive breast cancer cells to irradiation. Conclusions: This preliminary study showed the sensitization effect of Trastuzumab to irradiation in HER2 positive breast cancer cells. Further studies are warranted for the optimal dosing and normal tissue toxicity of this combination.

Abstract 1702: Metabolic heterogeneity of HER2-positive breast cancers.

Abstract Breast cancers are at least five molecularly distinct diseases with reported metabolic differences. Basal-like breast cancers are typically more glycolytic as compared to luminal-like breast cancers. However, HER2-positive breast tumors have variable FDG-PET maximum uptake values. We hypothesized that HER2-positive breast cancer cell lines would reflect the metabolic heterogeneity reported in breast cancer tumors. In the current study, a panel of HER2-positive breast cancer cell lines (N = 8) was profiled by their metabolic properties. To evaluate glycolytic dependency for ATP production, ATP-synthase dependent oxygen consumption rates were normalized to extracellular acidification rates using the Seahorse Bioscience XF analyzer. The glycolytic dependency was highly variable (12-fold range); ER-negative lines including AU565 and HCC1954 were the most glycolytic. Consistent with these results, HER2-positive/ER-negative cell lines produced more lactate compared to HER2-positive/ER-positive cell lines (1.5 versus 1.2 lactate produced/glucose consumed, p = 0.02). The glycolytic phenotypes of HER2-positive/ER-negative breast cancer cell lines were similar to that of basal-like breast cancer cell lines. For the HER2-positive lines, the highest rates of glutamine consumption were in ER-positive lines BT474 and HCC1419. Differential expression of mitochondrial glutaminase 2 (GLS2, p = 0.02), but not cytosolic glutaminase (GLS) or glutamine synthetase (GLUL), may contribute to higher rates of glutamine consumption by HER2-positive/ER-positive breast cancers. Consistent with higher rates of mitochondrial respiration, ER-positive/HER2-positive cells were also associated with higher expression of carbonic anhydrase 12 (CA12) and lower expression of the lactate transporter MCT1 (SLC16A1). Metabolic heterogeneity of HER2-positive cells was only evident in normoxic conditions. Per the “Pasteur effect,” all cell lines cultured under hypoxia (1% O2) were equally glycolytic (average = 1.8 lactate production/glucose consumption ratio) with correspondingly reduced rates of glutamine uptake. We conclude that the heterogeneity observed in FDG-PET may be influenced by fundamental metabolic differences between HER2-positive breast cancers, in particular, for well-oxygenated regions of the tumor. Citation Format: Jennifer B. Dennison, Gordon B. Mills. Metabolic heterogeneity of HER2-positive breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1702. doi:10.1158/1538-7445.AM2013-1702

Abstract 4329: Novel HER2 targeting antibody-drug conjugates based on DNA-interacting duocarmycin and an unique linker technology with great potential in breast cancer and NSCLC.

Abstract We have generated cleavable linker-duocarmycin payloads that previously have been shown to be highly stable in human plasma and to cause excellent in vitro and in vivo efficacy when conjugated to antibodies directed against multiple targets in different models. In our HER2 program, we have used these linker-duocarmycin payloads to generate HER2-targeting antibody-drug conjugates (ADCS) and prepare these for FIM studies. HER2 is a validated target in breast (BC) and gastric cancer (GC) that is currently used to target trastuzumab towards HER2-positive BC and GC tumors. We have generated a set of cysteine-coupled ADCs using newly developed duocarmycins conjugated to a therapeutic HER2/neu antibody. In vitro studies with the anti-Her2 ADCs showed that compared to the naked Ab, the ADCs tested have gained a more than 100-fold potency and more than two-fold efficacy in cell killing capacity. Studies in HER2 positive and negative cell lines indicated that cytotoxicity was HER2- mediated. HER2 binding and ADCC activity was not affected by conjugation. Single dose and multiple dose xenograft studies in mice using a human HER2-positive breast cancer cell line showed dose-dependent anti-tumor activity of the ADCs in vivo. Patient-derived xenografts (PDx) using primary tumors from HER2-positive breast cancer and NSCLC patients demonstrated the ability of the ADC to induce complete remission of tumors that remained unresponsive to the naked Ab trastuzumab. Also taken into account toxicology studies in rodents and cynomolgous monkeys, we conclude that we have generated a new class of HER2-based ADCs that have great potential to become effective treatment for HER2-positive tumors, including breast cancer and NSCLC. From a series of compounds, a preclinical candidate was selected that is currently being prepared for Phase I clinical trials that will start in 2014. Citation Format: Wim Dokter, Patrick Beusker, Gijs Verheijden, Ruud Ubink, Miranda van der Lee, Diels van den Dobbelsteen, Peter Goedings, Jacques Lemmens, Vincent de Groot, Marco Timmers. Novel HER2 targeting antibody-drug conjugates based on DNA-interacting duocarmycin and an unique linker technology with great potential in breast cancer and NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2013-4329

Abstract 1536: Neurropilin-1 high monocytes play an important role in trastuzumab mediated antibody-dependent cellular cytotoxicity in HER2 positive breast cancer cell lines.

Abstract Purpose Neuropilin-1 (NRP-1) was initially characterized as a neuronal receptor for specific secreted members of the semaphorin family involved in axon repulsion. However, it was recently reported to play an important role in the immune system. This study aimed to investigate the role of NRP-1 expression on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cell line SKBR3. Methods Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were prepared using Lymphoprep. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD14 and allophycocyanin (APC)-conjugated anti-NRP-1 antibodies, and an NRP-1high and NRP-1low monocyte subset was sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay using SKBR3 as target cells and trastuzumab as an antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. A CD107a degranulation assay was also used to determine the role of NRP-1 expression on monocytes in ADCC. PBMCs were co-cultured with target SKBR3 cells and trastuzumab for 2 h, stained with APC-conjugated anti-NRP-1 and PE-Cy5-conjugated anti-CD107a antibodies, and analyzed by flow cytometry. Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted NRP-1 high monocytes (12%) than in NRP-1 low monocytes (4%). Interestingly, microscopic findings showed that most NRP-1 high monocytes covered trastuzumab-treated target cancer cells (showing higher cellular cytotoxicity) while NRP-1 low monocytes did not cover the target cells but changed their morphology from a round to a dendritic shape (showing less cellular cytotoxicity) during ADCC. Moreover, the degranulation assay showed a higher expression of CD107a in NRP-1 high monocytes (17.4%) than in NRP-1 low monocytes (1.16%). Mean fluorescence intensity of CD107a in NRP-1 high and NRP-1 low monocytes was 92.6 and 26.3, respectively. Conclusions These data suggest that NRP-1 high monocytes exhibit higher trastuzumab-mediated ADCC than NRP-1 low monocytes in HER2-positive breast cancer cell lines. It may therefore be possible to differentiate HER2-positive breast cancer patients for whom trastuzumab will be less effective by testing for monocyte NRP-1 expression levels. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Tomoharu Sugie, Fumiaki Sato, Masakazu Toi. Neurropilin-1 high monocytes play an important role in trastuzumab mediated antibody-dependent cellular cytotoxicity in HER2 positive breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2013-1536

Abstract 1980: Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells.

Abstract Aneuploidy is a hallmark of solid tumors, contributing to tumorigenesis and resistance to chemotherapy. Aneuploidy is prevented by two centrosomes directing a bipolar mitotic spindle. Centrosome amplification (CA), the presence of supranumerary centrosomes within a cell, has been detected in both premalignant lesions and breast tumors, supporting the role of this phenomenon in breast cancer initiation and progression. Moreover, CA has been associated with HER2 overexpression. CA is initiated by abnormal signals from oncogenes or altered tumor suppressors that affect regulatory molecules shared by both cell and centrosome cycles. Elucidating the molecular circuitry and functional consequences of impaired centrosome cycle is therefore important for a complete understanding of its role in breast cancer. Nucleophosmin (NPM) is a widely expressed nucleolar phosphoprotein that has several functions, including the early regulation of centrosome duplication. Recent reports from breast cancer patients indicate the presence of NPM antigens in HER2-negative cases and low NPM expression as a prognostic marker of poor survival in the luminal A subtype. We therefore hypothesized that NPM expression and activity might directly impact the CA in HER2-positive breast cancer cells. To test this hypothesis, we have used genetic alteration of total and phospho-NPM to investigate its function in human mammary epithelial cell lines that includes non-tumorigenic and HER2-positive tumorigenic lines. Our initial findings revealed significantly elevated CA percentages and higher expression level of p-NPM (T199) in the HCC1954, JIMT1, and SKBR3 cells (HER2-positive) compared to other subtypes and non-tumorigenic MCF10A cells. This residue is known to be responsible for regulating the function of NPM in centrosome duplication. Conversely, HER2-positive cells displayed lower protein levels of total NPM compared to the other cell lines investigated. Expression of a deletion mutant phospho-NPM (T199A) reduced the percentage of CA in HCC1954 cells. In addition, an HCC1954 derivative with primary resistance to Herceptin showed augmented frequency of CA versus sensitive parental cells. Ongoing experiments involve the inhibition of p-NPM at T199 in JIMT1 and SKBR3 cells as well as the overexpression of a wild type form in all of the three HER2-positive breast cancer cell lines followed by the assessment of CA. In parallel, the non-tumorigenic MCF10A cells have been transfected with a phospho-mimetic NPM (T199D) and will be subjected to similar investigations. Finally, the correlation between p-NPM-driven CA and Herceptin resistance is being studied in all of our HER2-positive cell models of breast cancer. These results suggest that the high NPM expression could have a protective effect against CA and a more aggressive phenotype displayed by HER2-positive breast cancer cells. Citation Format: Mihaela Marina, Harold Ivan Saavedra. Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2013-1980

Abstract 1894: The lipogenic phenotype of HER2/neu-positive breast cancer cells.

Abstract 15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. Kourtidis et al. (2010) have recently shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The nuclear receptors NR1D1 and PPARγ enable these cells to upregulate de novo fatty acid synthesis while avoiding palmitate-induced lipotoxicity by storing neutral fats (TAGs). The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis of BT474 cells. Metabolomic analysis of NR1D1 overexpressing cells compared to normal cells reveals profound differences in their metabolite profiles. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis suggests that palmitate induces an ER-stress response in the HER2-positive cells, ultimately resulting in cell death. Preliminary results suggest that this response is CHOP-dependent. Combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. The uptake of exogenous fatty acids as well as the de novo fatty acid synthesis are dependent on the essential co-factor coenzyme A (CoA), which is generated from the precursor pantothenate (vitamin B5). Based on our genomic analysis we predicted that HER2-positive breast cancer cells are more sensitive to changes in intracellular CoA levels. Our results show that HER2-positive breast cancer cells are sensitive to shRNA-mediated knockdown of coenzyme A pathway enzymes whereas MCF-7 cells are not. We observe the same pattern of sensitivity using the competitive CoA pathway inhibitor homopantothenate. Interestingly, limiting the supply of CoA in HER2-positive cells reduces the toxicity of exogenous palmitate. Our data suggest that HER2-positive breast cancer cells are metabolically different from other breast cancer cell lines and that these differences offer new possibilities for therapeutic applications. Citation Format: Jan M. Baumann, Antonis Kourtidis, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2013-1894