Trastuzumab-resistant Breast Cancer Cells Research Articles

Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions

We have shown that erbB2 altered breast cancer cells are less sensitive to the anti-proliferative effects of metformin than triple negative cells, and have described the differences of molecular mechanisms of metformin action by tumor subtypes. We hypothesized that metformin may be more effective against trastuzumab-resistant erbB2-overexpressing breast cancer cells because it targets the critical signaling pathways that are altered with resistance. BT474, SKBR3 and derived trastuzumab-resistant sublines BT474-HR20 (HR20) and SKBR3-pool2 (pool2) were used to test this hypothesis. Metformin treatment resulted in significantly more inhibition of proliferation and clonogenicity in resistant sublines. It decreased erbB2/insulin-like growth factor-1 receptor (IGF-1R) complexes (present only in the resistant sublines) without altering erbB2 expression, and reduced the expression and activity of erbB3 and IGF-1R in the trastuzumab-resistant but not parental cells. Trastuzumab-resistant sublines were resistant to rapamycin induced changes in mTOR activity and cell growth. In contrast, both BT474 and HR20 cells were highly sensitive to inhibitors of Src (Dasatinib) and PI-3K (LY294002). The pool2 cells showed higher sensitivity than SKBR3 cells to LY294002, but not Dasatinib. On the basis of these data, metformin appears to be significantly more effective against trastuzumab-resistant as compared to sensitive breast cancer cells. Metformin disrupts erbB2/IGF-1R complexes, erbB3 and IGF-1R expression and activity, as well as Src kinase and/or PI-3K/Akt signaling. This action appears to be independent of mTOR signaling. Our findings provide a rationale to study the effects of metformin on patients with erbB2 positive tumors treated with trastuzumab, with or without resistance.

Highlights of This Issue

As new targeted therapies enter clinical trials in the metastatic setting, using primary tumor signatures for patient selection may have profound outcome implications if biomarkers are discordant between the primary and metastatic site. A discordance between the level of potential biomarkers in primary tumor and metastasis could occur due to alterations in gene expression, different microenvironmental stimuli, stochastic events during metastasis, clonal selection during metastasis, or clonal evolution either at the primary site or in the metastasis. PIK3CA mutation and loss of PTEN expression are being pursued as potential predictors of response to novel PI3K pathway inhibitors. A high level of discordance in PTEN level, PIK3CA mutations, and receptor status between primary tumors and metastases may influence patient selection and response to PI3K-targeted therapies.The activation of insulin-like growth factor-I receptor (IGF-IR) contributes to trastuzumab resistance. To understand the roles of insulin-like growth factor binding proteins (IGFBP) in trastuzumab resistance, Dokmanovic and colleagues characterize IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant breast cancer cells. The authors show that the high levels of secreted IGFBP-3 are associated with trastuzumabsensitive breast cancer cells, whereas the secretion of IGFBP-2 is upregulated in trastuzumab-resistant breast cancer cells. Changes in secretion profiles of IGFBP-2 and IGFBP-3 warrant further clinical investigations as potential biomarkers for trastuzumab resistance.Targeted nanoparticles represent an approach to minimize dose-limiting toxicities associated with chemotherapeutics. A study by Murphy and colleagues describes the design and characterization of a versatile nanoparticle platform, described as nanogels, that combines a lipid bilayer with a cross-linked gel core. The nanogels load a wide array of chemotypes and imaging agents for use as both drug delivery systems and diagnostic tools. The nanogels improve efficacy beyond Abraxane, a clinically approved nanoparticle formulation, in murine orthotopic pancreas and breast cancer models, thereby demonstrating the benefits of targeted nanotherapies in treating malignant disease.Osteosarcoma is an aggressive, often metastatic bone cancer. Toward the goal of developing new treatments for osteosarcoma, Fu and colleagues show apoptosis of osteosarcoma cells by SCH 727965 (SCH), a new cyclindependent kinase inhibitor. Osteosarcoma cell lines resistant to doxorubicin and dasatinib, osteosarcoma cell lines prepared by the authors from patients who had received adjuvant chemotherapy, and osteosarcoma explants derived from a human xenograft in mice were responsive to SCH 727965. Apoptosis occurred at SCH concentrations of 10–20 nM and was p53-independent. SCH did not induce the apoptosis of normal osteoblasts or fibroblasts. Thus, SCH is a promising anticancer agent for osteosarcoma.

HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance

Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumab-induced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2.

Abstract 710: Gene expression profile of the Wnt signaling pathway in trastuzumab resistant breast cancer cells

Abstract Introduction: We are interested in understanding mechanisms that lead to Trastuzumab (Herceptin) resistance in HER2 overexpressing breast tumors. In this study, we examined the role of the Wnt/β-catenin signaling pathway in Trastuzumab resistance. In addition to the Wnt pathway genes, we examined gene expression profiles using 44K gene chip array and EMT associated genes in Trastuzumab resistant cells. Methods: Wnt pathway gene profiles were examined by Agilent chip (4×44K) and Wnt/β-catenin specific array. HER2 overexpressing breast cancer cells, SKBR3 and trastuzumab resistant clone, SKBR3 100-8 cells were used as cell models. The EMT (Epithelial to Mesenchymal Transition) markers Snail, Slug, Twist and E-Cadherin were examined by both real-time PCR and western blot analysis. Cell invasiveness was determined by invasive assay. HER2-HER3 dimerization was examined using immunoprecipitation and immunoblotting procedures. Results: We established a Trastuzumab resistant cell line from the HER2 overexpressing parental SKBR3 cell line. The resistant clone called SKBR3/100-8 is insensitive to 100µg/ml of Trastuzumab. It has a mesenchymal or more basal-like morphology. It overexpresses HER2 protein and mRNA more than the parental line SKBR3. This resistant clone shows increased dimerization of HER2 with HER3. The SKBR3 100-8 cells were found to overexpress EGFR1. It is more invasive compared to wt SKBR3 and shows a significant increase in EMT marker, SLUG, but a decrease in Snail and E-cadherin compared to the wtSKBR3 cells. The resistant clone shows increase in phospho-Akt, pGSK3β and a decrease in β-Catenin. Agilent chip (4×44K) array (containing 84 Wnt pathway genes) identified the following Wnt pathway genes to be upregulated by 2 or more fold in the resistant clone: AXIN1, CSNK1A1, CSNK1D, CTBP2, DAAM1, c-JUN, TLE1, WNT3 and WNT6. Conversely, the following genes were downregulated by 2 or more fold in the resistant clone: AES, CCND3, CTNNB1, FZD1, FZD4, FZD5, PITX2, PPP2CA, PPP2R1A, WIF1, WNT11, and WNT4. The resistant clone also increased Nanog and VEGF significantly. TGFβ treatment in wtSKBR3 increased SLUG, Snail, Twist, and decreased E-Cadherin. The cells showed more mesenchymal morphology than wtSKBR without TGFβ treatment. TGFβ treatment in wtSKBR3 upregulated: c-Jun, C-Myc, CCND1, FosL1, LEF1, and TCF-1. TGFβ also induced Met, MMP1, MMP7, MMP9, MMP26, Nanog, NOS2, POU5F1, PPARd, Survivin, uPAR and VEGF. In contrast, TGFβ upregulated only c-Jun, Met, Nanog, and VEGF in SKBR3 100-8 cells. Conclusion: Wnt/β-catenin signaling is highly involved in the trastuzumab resistant breast cancer cells. The resistant signature involves c-Jun, Met, Nanog and VEGF. These results suggest a very specific signature that needs further studies for target therapy for the Trastuzumab resistant tumors and implies the use of anti VEGF therapy. 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 710. doi:10.1158/1538-7445.AM2011-710

Abstract 4562: Superior targeting of the human epidermal growth factor receptor 2 (HER-2) with recombinant monoclonal antibody mixtures

Abstract Human epidermal growth factor receptor 2 (HER2) is involved in development and maintenance of malignant phenotypes of several human cancers and therefore represent an attractive therapeutic target. Trastuzumab is currently the only anti-HER2 antibody approved for treatment of human cancers and although succesful more efficacious treatments are warranted. Mixtures of recombinant monoclonal antibodies are promising candidates as the next generation of antibody therapeutics due to their multipotent activities. The aim of the present study was to identify mixtures of anti-HER2 antibodies with superior activity to existing monoclonal antibodies. Anti-HER2 antibodies were raised in mice by immunizations with HER-2 in different antigen presenting formats. A large antibody repertoire consisting of approximately 150 unique anti-HER-2 antibodies was cloned from the mice using the mSymplex™ technology. Based on a thorough sequence and binding analysis, 40 antibodies were selected for functional evaluation. The 40 antibodies were tested as individual antibodies and in mixtures of two and three for the ability to inhibit the growth of four human cancer cell lines using a standard viability assay. In total, more than 1300 mixtures were evaluated for ability to inhibit growth of the four cell lines. The 20 mixtures with the highest levels of growth inhibition were further characterized with regard to potency (IC50) and ability to engage in ADCC and CDC. The results demonstrated that HER2 mixtures were superior to trastuzumab, pertuzumab and the mixture of the two at inhibiting the growth of seven of the 10 cell lines investigated. Interestingly, the mixtures also inhibited the growth of the trastuzumab resistant breast cancer cell line HCC202, reflecting the differentiated mechanisms of anti-HER2 antibody mixtures. In vivo, anti-HER2 mixtures had superior activity compared to the monoclonal anti-HER2 antibody trastuzumab in two models of human gastric cancer. Based on these results, a candidate mixture was selected and referred to as Sym005. In conclusion, these data demonstrate that the novel antibody mixture Sym005 has a unique set of HER2 inhibitory mechanisms, which translates into superior anti-cancer activity in HER2-positive tumor xenografts. Furthermore, the results provide a clear rationale for evaluation of Sym005 in clinical trials on patients with HER2 positive tumors. 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 4562. doi:10.1158/1538-7445.AM2011-4562

Abstract 720: Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2 extracellular domain in human breast cancer cells

Abstract Human epidermal growth factor receptor 2 (HER2) is a member of a family of receptors associated with tumor cell proliferation, apoptosis, adhesion, migration, and differentiation. According to numerous preclinical and clinical studies, HER2 can be immunogenic and antibodies against this transmembrane receptor have become in use in individuals with HER2-overexpressing tumors. Trastuzumab, a monoclonal antibody that targets HER2, induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits HER2-mediated signaling. Numerous randomized trials have confirmed the utility of trastuzumab in HER2-overexpressing breast cancer in various clinical scenarios; however, HER2-overexpression has been identified only in approximately 25-30% of primary breast cancers. Moreover, it has been reported that the majority of metastatic breast cancer patients treated with trastuzumab develop acquired resistance within a year. In this study, we analyzed the mechanism of resistance to trastuzumab and examined for molecular sensitization by exogenous expression of HER2 extracellular domain in human breast cancer cells. A long-term exposure to trastuzumab induced its resistance in HER2-positive breast cancer cells. We found that trastuzumab-resistant cells exhibited down-regulation of HER2 expression in cell surface and impaired ADCC activity, suggesting that trastuzumab-resistant cells could be re-sensitized to trastuzumab by exogenous induction of extracellular domain (ECD) of HER2.We constructed the expression vector with HER2-ECD and stably transfected into low HER2-expressing human breast cancer cell line, MCF7. HER2-ECD expression had no apparent effect on the growth and signaling pathway in MCF7 cells; however, ADCC activity was strongly enhanced in HER2-ECD-expressing MCF7 cells compared to parental MCF7 cells. These results suggest that exogenous expression of HER2-ECD might be an appropriate strategy to sensitize HER2-negative or trastuzumab-resistant human breast cancer cells to trastuzumab. The modification of the tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) to contain the HER2-ECD gene for exogenous expression is currently under the investigation. We will further examine the antitumor effects of this virus in the combination with trastuzumab. 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 720. doi:10.1158/1538-7445.AM2011-720

Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo

IntroductionTrastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib.MethodsAlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry.ResultsT-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells.ConclusionsOur results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).

316 Functional and molecular MR for breast cancer characterization

We report longitudinal diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced (DCE)-MRI (7 T) studies designed to identify functional changes, prior to volume changes, in trastuzumab-sensitive and resistant HER2 + breast cancer xenografts. Athymic mice (N = 33) were subcutaneously implanted with trastuzumab-sensitive (BT474) or trastuzumab-resistant (HR6) breast cancer cells. Tumor-bearing animals were distributed into four groups: BT474 treated and control, HR6 treated and control. DW- and DCE-MRI were conducted at baseline, day 1, and day 4; trastuzumab (10 mg/kg) or saline was administered at baseline and day 3. Animals were sacrificed on day 4 and tumors resected for histology. Voxel-based DW- and DCE-MRI analyses were performed to generate parametric maps of ADC, Ktrans, and ve. On day 1, no differences in tumor size were observed between any of the groups. On day 4, significant differences in tumor size were observed between treated vs. control BT474, treated BT474 vs. treated HR6, and treated vs. control HR6 (P < .0001). On day 1, ve was significantly higher in the BT474 treated group compared to BT474 control (P = .002) and HR6 treated (P = .004). On day 4, ve and Ktrans were significantly higher in the treated BT474 tumors compared to BT474 controls (P = .0007, P = .02, respectively). A significant decrease in Ki67 staining reinforced response in the BT474 treated group compared to BT474 controls (P = .02). This work demonstrated that quantitative MRI biomarkers have the sensitivity to differentiate treatment response in HER2 + tumors prior to changes in tumor size.

Abstract 337: In vitro evaluation of dual pan-PI3-kinase/mTOR inhibitor in HER2 overexpressing breast cancer cells

Abstract The HER/EGFR is a target of significant interest in human cancer due to the high frequency and broad spectrum aberrations in the pathway observed in human tumors, including breast tumors. Following ligand induced homodimeraziation/ heterodimeraziation HER2 transmits its signal mainly via PI3Kinase-AKT-mTOR and RAS-RAF-ERK1/2 pathways that promote proliferation, survival and angiogenesis. PI3Kinase-AKT-mTOR is the most frequently activated oncopathway in human cancer (De P and Leyland -Jones B, JCO Editorial in Press; Holbro T 2003 PNAS 100: 8933). Tumor cells with hyperactive PI3Kinase-AKT-mTOR pathway are not only resistant to external stresses that can induce cell death and also to chemotherapy. Literature suggests that hyyperactivation of PI3Kinase-AKT-mTOR pathway is one the major causes for the development of de novo or acquired resistance to trastuzumab (Berns K et al 2007 Cancer Cell 12: 395-402; Junttila TT et al 2009 Cancer Cell 15: 429-440). The importance of PI3kinase pathway in Her2 amplified and/or trastuzumab resistance breast cancer implies that interruption of this pathway is important in order for HER2-directed therapies to exert an anticancer effect. The present study was carried out to investigate the preclinical efficacy of a dual pan-PI3K plus mTOR inhibitor, BEZ235 alone or in combination with trastuzumab in HER2 overexpressing trastuzumab-sensitive and trastuzumab-resistant breast cancer cell lines. Here we utilized HER2 overexpressing SKBR3, BT474 and trastuzumab -resistant BT474 cells as preclinical models. Our results show 1) BEZ235 time dependently and dose dependently suppressed the pathway downstream of PI-3Kinase including p-AKT, p-mTOR and p-P70S6Kinase in both trastuzumab-sentsitive and trastuzumab-resistance breast cancer cell lines at 50nM, 100nM and 200nM concentrations and 100% inhibition was observed at higher concentration 2) inhibition of AKT and its downstream effectors were more pronounced when trastuzumab was treated along with BEZ235 even at lower concentrations, 3) the well described AKT activation due to mTOR inhibition was prevented by higher doses of BEZ235 (at 200 nm concentration) treatment, and 4) unlike other pan-PI3Kinase inhibitor (e.g. LY294002, SF1126), BEZ235 has no effect on hypoxia-mediated stabilization of hypoxia-inducible factor −1α (HIF1α) however, heregulin-induced HIF1α synthesis which is an important angiogenic modulator in breast cancer cells, was significantly decreased (&amp;gt; 70%) following the treatment of BEZ235. Our in vitro data provide evidence that clinically pertinent dual pan-PI3K plus mTOR inhibitor can reverse trastuzumab resistance in breast cancer cells, and further support to carry out in vivo efficacy studies (tumor growth and tumor-induced angiogenesis) with BEZ 235 alone or in combination with trastuzumab. 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 337.

Targeting trastuzumab-resistant breast cancer cells with a lentivirus engineered to bind antibodies that recognize HER-2

Targeting HER-2 over-expressing breast cancer cells with trastuzumab has resulted in significant improvements in both disease-free and overall survival rates. However, despite a favorable initial response, some cancer cells become resistant and develop into fatal metastatic disease. Here we report that we can specifically target HER-2 over-expressing and trastuzumab-resistant breast cancer cells by using an engineered lentivirus which has trastuzumab bound to its envelope. In vitro, this lentiviral construct mediated both the expression of reporter genes, such as enhanced green fluorescent protein (EGFP) and firefly luciferase, as well as the therapeutic gene, herpes thymidine kinase (hTK), in HER-2 over-expressing cells. Subsequent application of the pro-drug ganciclovir selectively killed breast cancer cells in which lentivirus mediated expression of hTK. In vivo, we successfully targeted the expression of firefly luciferase to trastuzumab-resistant breast cancer tumors established in nude mice. Furthermore, we found that systemic administration of trastuzumab-bound lentivirus led to expression of EGFP in circulating trastuzumab-resistant breast cancer cells. In conclusion, HER-2 over-expressing breast cancer cells resistant to trastuzumab can be targeted for selective gene expression and destruction by viruses with envelope-proteins engineered to bind to this antibody.

Immunoconjugated gold nanoshell-mediated photothermal ablation of trastuzumab-resistant breast cancer cells

Trastuzumab is a FDA-approved drug that has shown clinical efficacy against HER2+ breast cancers and is commonly used in combination with other chemotherapeutics. However, many patients are innately resistant to trastuzumab, or will develop resistance during treatment. Alternative treatments are needed for trastuzumab-resistant patients. Here, we investigate gold nanoparticle-mediated photothermal therapies as a potential alternative treatment for chemotherapy-resistant cancers. Gold nanoshell photothermal therapy destroys the tumor cells using heat, a physical mechanism, which is able to overcome the cellular adaptations that bestow trastuzumab resistance. By adding anti-HER2 to the gold surface of the nanoshells as a targeting modality, we increase the specificity of the nanoshells for HER2+ breast cancer. Silica-gold nanoshells conjugated with anti-HER2 were incubated with both trastuzumab-sensitive and trastuzumab-resistant breast cancer cells. Nanoshell binding was confirmed using two-photon laser scanning microscopy, and the cells were then ablated using a near-infrared laser. We demonstrate the successful targeting and ablation of trastuzumab-resistant cells using anti-HER2-conjugated silica-gold nanoshells and a near-infrared laser. This study suggests potential for applying gold nanoshell-mediated therapy to trastuzumab-resistant breast cancers in vivo.

Heterotrimerization of the Growth Factor Receptors erbB2, erbB3, and Insulin-like Growth Factor-I Receptor in Breast Cancer Cells Resistant to Herceptin

Primary and acquired resistance to the breast cancer drug trastuzumab (Herceptin) is a significant clinical problem. Here, we report enhanced activation of downstream signaling pathways emanating from the growth factor receptors erbB2, erbB3, and insulin-like growth factor-I receptor (IGF-IR) in trastuzumab-resistant breast cancer cells. Interactions between IGF-IR and erbB2 or erbB3 occur exclusively in trastuzumab-resistant cells, where enhanced erbB2-erbB3 interactions are also observed. Moreover, these three receptors form a heterotrimeric complex in resistant cells. erbB3 or IGF-IR knockdown by short hairpin RNA-mediated strategies upregulates p27(kip1), inactivates downstream receptor signaling, and resensitizes resistant cells to trastuzumab. Our findings reveal a heterotrimer complex with a key role in trastuzumab resistance. On the basis of our results, we propose that trastuzumab resistance in breast cancer might be overcome by therapeutic strategies that jointly target erbB3, erbB2, and IGF-IR.

Characterization of a novel trastuzumab resistant human breast cancer cell line

HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.

Metformin Enhances Trastuzumab Efficacy and Overcomes Resistance in HER2 Type Breast Cancer Cells.

Abstract Background: HER2 alterations occur in one-fourth of breast cancers and are associated with an aggressive tumor phenotype. The anti-HER2 agent trastuzumab reduces cell proliferation, angiogenesis, DNA repair and induces antibody-dependent cellular cytotoxicity. Objective response rates and median duration of response for eligible patients given trastuzumab alone is low (12-34% and 9 months respectively), hence, it is administered in combination with other agents. Mechanisms of trastuzumab resistance include: altered receptor antibody interactions, increased signaling through other EGFR type I growth factor receptors, modulation of p27 kip1 and increased insulin-like growth factor 1 receptor (IGF-1) signaling. We have reported that metformin inhibits HER2 expression, tyrosine kinase activity (phosphorylated HER2 at both auto- and Src- phosphorylation sites), AKT/mTOR signaling, Cyclin D1 and E2F1 with G1 arrest in HER2 overexpressing breast cancer cells. We sought to determine if metformin would enhance trastuzumab associated cytotoxicity and modulate acquired resistance in vitro.Methods: We used trastuzumab sensitive (SKBR3, BT474) and resistant cell lines (BT-474/HR20, SKBR3/P2, MCF-7/713 and MCF-7/HER2Δ16) to study the effects of metformin, metformin + trastuzumab and trastuzumab alone. Assays included MTS for proliferation, clonogenicity studies, Western blots, and pull down experiments with Western blots.Results: Trastuzumab sensitive breast cancer cells were less sensitive to metformin alone, compared to trastuzumab resistant HER2 subtype breast cancer cell lines. Trastuzumab sensitive cells showed enhanced growth and clonogenicity inhibition when treated by both metformin and trastuzumab. At the molecular level, these bi-treated cells showed decreases in HER2/pHER2, erbB3/perbB3, and inactivation of AKT and MAPk signaling. Metformin as low as 20mM increased the efficacy of trastuzumab. All HER2 resistant cell lines showed higher levels of IGF1R and HER2/IGF1R complexes, as compared to sensitive parental lines. In these resistant lines Metformin decreased cell proliferation and clonogenicity, HER2/IGF1R complexes (identified through pull down experiments) and protein expression of HER2/pHER2, erbB3/perbB3, IGF1R/pIGF1R as well as downstream signaling via Akt and IGF1 pathways. Metformin overcame trastuzumab resistance as demonstrated by growth and clonogenicity assays.Conclusions: HER2 amplified trastuzumab resistant breast cancer cells showed greater sensitivity to metformin than sensitive parental lines. Metformin reversed trastuzumab resistance and decreased HER2:IGF1R complexes, HER2/pHER2 and IGF1R expression, whereas sensitive parental lines showed no complex formation. These preclinical data suggest a combination of metformin and trastuzumab may have clinical benefit, improving the efficacy and reducing the emergence of or reversing trastuzumab resistance in HER2 positive breast cancer patients.Herceptin graciously provided by Genentech Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1133.

MIC A/B Protein Expression Associates with Trastuzumab-Resistant Breast Cancer Cells Leading to Effective Immunotherapy by Cytokine Induced Killer Cells.

Abstract 4092Poster Board III-1027Citokine Induced Killer (CIK) cells are a heterogenous subset of T lymphocytes sharing phenotype and functional properties with natural killer (NK) cells. CIK cells can be massively ex-vivo expanded and are capable of antitumor activity in both hematological and solid malignancies. The antitumor activity of CIK cells is classically MHC-unrestricted and mostly mediated by the interaction of CIKs' membrane receptor NKG2D with its ligands on tumor cells. The main ligand for NKG2D is MHC class I-related chain (MIC) A/B. The expression of this protein is potentially induced by pathological stimuli and is reported to be associated to some malignant phenotypes. The presence of MIC A/B on tumor targets is crucial for an effective killing by CIK cells and its accurate evaluation might help identifying the tumors, and potentially the patients, more sensitive to a CIK based immunotherapy approach.Trastuzumab, is a humanized monoclonal antibody directed against the extracellular portion of the tyrosine kinase HER2. Although Trastuzumab has been successfully used in HER2-overexpressing breast cancer (BC) patients, resistance is a frequent problem that culminates in treatment failure. The scarsity of treatment options for resistant patients renders crucial the exploitation of new potential targets for tumor treatment.In this context we hypothesized that MIC A/B expression might be associated with the acquisition of aggressive characteristics by BC cells, like the emersion of resistance to Trastuzumab treatment.Flow-cytometry analysis showed that the expression of MIC A/B was virtually absent on HER2-overexpressing BC cells (SKBR3). These cells showed a high sensitivity to treatment with Trastuzumab in vitro, detected through cell proliferation test in soft agar and bioluminescent essays for ATP release. Similarly to what happens in the clinic, a prolonged treatment (> 6 months), determined the selection of cell clones resistant to Trastuzumab exposure. This event was found to be associated with the acquisition of a high expression of MIC A/B on cancer cells' membrane (> 95%). A high expression of MIC A/B (>95%) was confirmed also on a different BC cell line (JIMT-1) that is characterized by a primitive resistance to Trastuzumab. As hypothesized, CIK cells, generated from healthy donors, efficiently killed Trastuzumab-resistant (TR), MIC A/B positive, BC cells (86%, 85%, 81% and 68% of specific killing at a 40:1, 20:1, 10:1 and 5:1 effector/target ratio respectively) while were minimally effective against MIC A/B negative, Trastuzumab-sensitive (TS), BC cells (30%, 23%, 15% and 11% of specific killing at a 40:1, 20:1, 10:1 and 5:1 effector/target ratio respectively, p < 0.0001). The test was performed by staining target cells with Carboxyfluorescein Succinimidy ester (CFSE) and co-culturing them with CIKs at different ratios; data were analyzed after 4 and 12 hours by flow cytometry (Fig.1).Our data describe for the first time the association of MIC A/B upregulation on HER2 overexpressing BC cells and Trastuzumab resistance. This biological event induces the susceptibility of cancer cells to tumor killing operated by CIK cells. If confirmed on breast cancer tissues, our data might provide a rationale for CIK based immunotherapy in HER2 overexpressing breast cancer patients developing clinical resistance to Trastuzumab. Additional studies will investigate the possible molecular links between Trastuzumab resistance and MIC A/B expression.CIK vs Trastuzumab sensitive BCCIK vs Trastuzumab resistant BCP< 0.0001 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Novel Toll-Like Receptor 9 Agonist Cooperates with Trastuzumab in Trastuzumab-Resistant Breast Tumors through Multiple Mechanisms of Action

Resistance to anti-HER2 monoclonal antibody trastuzumab is a relevant issue in breast cancer patients. Among the mechanisms implicated in trastuzumab resistance, increasing evidence supports a role of tumor microenvironment. We previously found that a novel toll-like receptor 9 agonist, referred to as immune modulatory oligonucleotide (IMO) and currently under clinical investigation, acts through epidermal growth factor receptor (EGFR) and shows direct antiangiogenic effects by cooperating with anti-EGFR or anti-VEGF drugs, thus interfering with cancer cells and microenvironment. In this study, we used KPL-4 and JIMT-1 trastuzumab-resistant breast cancer cells to evaluate the combination IMO plus trastuzumab as a therapeutic option for trastuzumab-resistant breast cancers. IMO inhibits KPL-4 and JIMT-1 xenografts growth and potentiates trastuzumab antitumor effect, with complete suppression of tumor growth, potent enhancement of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity, and strong inhibition of EGFR/HER2-related signaling. In KPL-4 xenografts, IMO alone interferes with HER signal transduction, whereas trastuzumab is ineffective. IMO induces an HER-dependent signal inhibition also in vitro by modulating a functional interaction between toll-like receptor 9 and HER receptors occurring at membrane level. Finally, IMO plus trastuzumab produces a cooperative antiangiogenic effect related to suppression of endothelial HER-related signaling. We showed a cooperative effect of IMO plus trastuzumab in trastuzumab-resistant breast cancers due to IMO direct antitumor and antiangiogenic activity and antibody-dependent cell-mediated cytotoxicity enhancement. Moreover, we provided first evidence of a toll-like receptor 9/HER interaction at membrane level as novel mechanism of action. Altogether, we propose IMO plus trastuzumab as an effective strategy in trastuzumab-resistant breast cancers.

Selective death of human breast cancer cells by lytic immunoliposomes: Correlation with their HER2 expression level

Trastuzumab (Herceptin™) targets the human epidermal growth factor receptor 2 (HER2), which is overexpressed in 20–30% of breast and ovarian cancers carrying a bad prognosis. Our purpose was to target HER2-overexpressing human breast cancer cells with pegylated immunoliposomes bearing trastuzumab and containing melittin, which has recently shown anticancer properties. Using a panel of human breast cancer cells with different HER2 expression levels, these immunoliposomes decreased cancer cells viability in a dose–response manner and in correlation to their level of HER2 expression. Specific binding of the immunoliposomes to SKBr3 breast cancer cells was shown by ImageStream-based analysis. The morphological changes observed in the treated cells suggested a cytolytic process. This preclinical approach may suppose an effective strategy for the treatment of HER2-overexpressing tumors, and can support the development of an early phases I–II clinical trial. Trastuzumab resistant breast cancer cells (JIMT-1), can also be targeted using this approach.

Deterministic Effects Propagation Networks for reconstructing protein signaling networks from multiple interventions

BackgroundModern gene perturbation techniques, like RNA interference (RNAi), enable us to study effects of targeted interventions in cells efficiently. In combination with mRNA or protein expression data this allows to gain insights into the behavior of complex biological systems.ResultsIn this paper, we propose Deterministic Effects Propagation Networks (DEPNs) as a special Bayesian Network approach to reverse engineer signaling networks from a combination of protein expression and perturbation data. DEPNs allow to reconstruct protein networks based on combinatorial intervention effects, which are monitored via changes of the protein expression or activation over one or a few time points. Our implementation of DEPNs allows for latent network nodes (i.e. proteins without measurements) and has a built in mechanism to impute missing data. The robustness of our approach was tested on simulated data. We applied DEPNs to reconstruct the ERBB signaling network in de novo trastuzumab resistant human breast cancer cells, where protein expression was monitored on Reverse Phase Protein Arrays (RPPAs) after knockdown of network proteins using RNAi.ConclusionDEPNs offer a robust, efficient and simple approach to infer protein signaling networks from multiple interventions. The method as well as the data have been made part of the latest version of the R package "nem" available as a supplement to this paper and via the Bioconductor repository.

HSP90 inhibition in HER2-positive breast cancer cells

e14573 Background: HSP90 is required for the stability and activity of HER2 and downstream proteins, such as Akt, which play a key role in survival. We aimed to assess the anti-tumor effect of the HSP90 inhibitor NVP-AUY922 in HER-2 positive breast cancer cell lines. Methods: HER2 positive breast cancer cell lines with varied sensitivity to trastuzumab (Sensitive: BT474, SKBR3; acquired resistance: BT474Res, SKBR3Res; innate resistance: HCC1419, HCC1954, MDA-MB-453) were treated with the HSP90 inhibitor NVP-AUY922 (Novartis) and trastuzumab. IC50s were determined using the acid phosphatase assay. HER2, Akt and HSP90 levels were determined by immunoblotting after treatment with NVP-AUY922. Combinations of NVP-AUY922 with docetaxel, cisplatin and 5'-deoxy-5-fluorouridine (5-DFUR) were tested in BT474 and SKBR3 cells. Results: All of the HER2 positive cells were sensitive to NVP-AUY922, with IC50s ranging from 5.5 to 16.4 nM. Combined treatment with NVP-AUY922 (10 nM) and trastuzumab (10 nM) showed significantly greater inhibition of growth than either trastuzumab or NVP-AUY922 alone in BT474 and BT474Res cell lines (p&lt;0.005). In SKBR3 and SKBR3Res cells, dual treatment with NVP-AUY922 and trastuzumab did not significantly increase response compared to NVP-AUY922 alone ( Table 1 ). Treatment with NVP-AUY922 resulted in a dose-dependent decrease in HER2 and Akt levels in trastuzumab-sensitive and -resistant cells. Combinations of docetaxel, cisplatin or 5-DFUR with NVP- AUY922 were antagonistic in both BT474 and SKBR3 cells (CI values &gt;1). Conclusions: This study demonstrates that NVP-AUY922 has anti-tumor activity in trastuzumab-sensitive, and in both innate and acquired trastuzumab-resistant HER2 positive breast cancer cells. The antagonistic interactions observed for combinations of NVP-AUY922 with chemotherapy do not favour clinical evaluation of such combinations. However, combinations with other targeted therapies, such as trastuzumab, warrant further investigation. [Table: see text] [Table: see text]

Trastuzumab-Resistant Breast Cancer Cells Remain Sensitive to the Auger Electron–Emitting Radiotherapeutic Agent 111In-NLS-Trastuzumab and Are Radiosensitized by Methotrexate

Our goals in this study were to determine whether (111)In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with (111)In. HER2 expression was determined by Western blot and by radioligand binding assay using (111)In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of (111)In-NLS-trastuzumab and (111)In-trastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with (111)In-NLS-trastuzumab, (111)In-trastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. The expression of HER2 was highest in SK-BR-3 cells (12.6 x 10(5) receptors/cell), compared with 231-H2N and TrR1 cells (6.1 x 10(5) and 5.1 x 10(5) receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 x 10(5) and 0.6 x 10(5) receptors/cell, respectively). NLS peptides increased the nuclear uptake of (111)In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1%+/-0.01%, 2.5%+/-0.2%, 2.8%+/-0.7%, and 0.5%+/-0.1% to 0.5%+/-0.1%, 4.6%+/-0.1%, 5.2%+/-0.6%, and 1.5%+/-0.2%, respectively. The cytotoxicity of (111)In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for (111)In-NLS-trastuzumab was 9- to 12-fold lower than for (111)In-trastuzumab and 16- to 77-fold lower than for trastuzumab. MDA-MB-231 and TrR2 cells were less sensitive to (111)In-NLS-trastuzumab or (111)In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to (111)In-NLS-trastuzumab was 1.42 and 1.68, respectively. Targeted Auger electron radioimmunotherapy with (111)In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.