HER2-positive Breast Cancer Cell Lines Research Articles

Abstract 5428: Lactate production as a potential marker of HER2-addiction and Trastuzumab susceptibility

Abstract Background: Although the introduction of the HER2-specific humanized monoclonal antibody Trastuzumab (T) in clinical practice revolutionized the treatment of HER2-positive breast cancer (BC), about half of the T-treated patients do not benefit or become refractory after its administration. Despite different molecular tumor profiles significantly associated with T benefit were reported, the identification of a non invasive clinical approach to select patients responsive or not to T is still a clinical unmet task. We recently reported that mammary cancer cell lines derived from spontaneous nodules arisen in mice transgenic for the human d16HER2 splice variant (HER2-addicted) were significantly enriched in genes involved in glycolysis pathway versus those developed in mice transgenic for the human full-length HER2-positive (WTHER2) (HER2-non addicted) (1,2). Accordingly, the aim of our study was to define whether tumor lactate levels could reflect HER2 addiction and, in turn, T susceptibility. Methods: The metabolite content in murine and human HER2-positive cell lines and specimens was determined by quantitative Nuclear Magnetic Resonance (NMR) spectroscopy. The optical metabolic imaging that quantifies the levels of the anaerobic glycolysis-related metabolite nicotinamide adenine dinucleotide (NADH) and the aerobic metabolite flavin adenine dinucleotide (FAD) was represented by Optical redox ratio (NADH/FAD). The extracellular levels of lactate were calculated by blood gas analyzer. Results: The metabolic profile of HER2-addicted (MI6, MI7) versus -non-addicted (WTHER2_1, WTHER2_2) murine tumor cells revealed higher intracellular levels of metabolites associated to glycolysis in MI6 and MI7 cells versus WTHER2-positive cells. In keeping, also the evaluation of the metabolic profile of human HER2-positive BC cell lines classified as addicted (SKBR3 and BT474) or not (MDAMB453 and MDAMB361) to HER2-signaling (3) confirmed significant higher intracellular and extracellular levels of lactate in HER2-addicted versus -non-addicted cells. The extracellular levels of NADH and NADH/FAD ratio further sustained a significantly higher activity of an anaerobic metabolism in HER2-addicted cells. Noteworthy, the metabolic profile of 27 HER2-positive BC cases treated with T, classified as HER2-addicted or not on the basis of the activation status of d16HER2 (1), provided further evidence of significantly higher levels of lactate in tumors HER2-addicted. Conclusions: Overall, even if a dysregulated glucose metabolism is a tumor hallmark, our results provide relevant in vitro and in vivo evidences of the potential future non-invasive lactate detection as a possible biomarker of HER2-addiction and T-responsiveness. Supported by the Italian Ministry of Health and AIRC.

Biological function of long noncoding RNA snaR in HER2-positive breast cancer cells.

Long noncoding RNA, snaR (small NF90-associated RNA), has been reported to be upregulated in various cancer cell lines. We evaluated the additional role of snaR in HER2-positive breast cancer cell lines. We explored changes of expression of snaR among the selected long noncoding RNAs which have a potential in cancer proliferation or progression. The proliferation, migration, and invasion of HER2-positive breast cancer cells (SK-BR3) were evaluated by snaR with RNA interruption in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, wound-healing assay, and Transwell assay. The expression of snaR was remarkably upregulated in SK-BR3 cell lines together with ANRIL, while the SFMBT2 was downregulated in SK-BR3 cell lines. Although Nespas, 7SK, PSF inhibiting RNA, mascRNA, Hoxa11as, NRON, AK023948, MER11C, p53 mRNA, CAR Intergenic 10, HUC 1 and 2, ZFAS1, SCA8, and SNHG5 were also upregulated and UCA1 was downregulated, the differences were not dominent. Based on the expression result, we explored the functional role of snaR in HER2-positive breast cancer. Downregulation of snaR with small interfering RNA was identified to significanlty inhibit migration as well as proliferation of SK-BR3 cells. In this study, snaR was identified as upregulated and to play a role in cancer progression of HER2-positive breast cancer cells. These results suggest snaR as a potential biomarker for HER2-positive breast cancer.

Dynamic Phenotypic Transition of Breast Cancer Cells In Vitro Revealed by Self-floating Cell Culture

Breast tumor heterogeneity leads to phenotypic diversity, such as tumor-initiating and metastatic properties and drug sensitivity. We found that a self-floating cell (SFC) culture enriches a drug-resistant subpopulation in a HER2-positive breast cancer cell line. SFCs were analyzed for cancer stem cell markers, gene expression profiles, and sensitivity for anticancer drugs. SFCs expressed cancer stem cell markers, such as aldehyde dehydrogenase (ALDH) activity and elevated HER2 autophosphorylation. Gene expression profiles of SFCs showed a dramatic difference compared to those of parental or forced floating cells. SFCs also expressed CD133, a marker of drug resistance, and resisted cytotoxic drugs by drug efflux transporters. In contrast, HER2 kinase inhibitors efficiently reduced SFC viability. SFCs enrich drug-resistant subpopulations even in vitro and might reflect the highly plastic nature of breast cancer cells even in vitro.

Drug-screening and genomic analyses of HER2-positive breast cancer cell lines reveal predictors for treatment response.

BackgroundApproximately 15%–20% of all diagnosed breast cancers are characterized by amplified and overexpressed HER2 (= ErbB2). These breast cancers are aggressive and have a poor prognosis. Although improvements in treatment have been achieved after the introduction of trastuzumab and lapatinib, many patients do not benefit from these drugs. Therefore, in-depth understanding of the mechanisms behind the treatment responses is essential to find alternative therapeutic strategies.Materials and methodsThirteen HER2 positive breast cancer cell lines were screened with 22 commercially available compounds, mainly targeting proteins in the ErbB2-signaling pathway, and molecular mechanisms related to treatment sensitivity were sought. Cell viability was measured, and treatment responses between the cell lines were compared. To search for response predictors and genomic and transcriptomic profiling, PIK3CA mutations and PTEN status were explored and molecular features associated with drug sensitivity sought.ResultsThe cell lines were divided into three groups according to the growth-retarding effect induced by trastuzumab and lapatinib. Interestingly, two cell lines insensitive to trastuzumab (KPL4 and SUM190PT) showed sensitivity to an Akt1/2 kinase inhibitor. These cell lines had mutation in PIK3CA and loss of PTEN, suggesting an activated and druggable Akt-signaling pathway. Expression levels of five genes (CDC42, MAPK8, PLCG1, PTK6, and PAK6) were suggested as predictors for the Akt1/2 kinase-inhibitor response.ConclusionTargeting the Akt-signaling pathway shows promise in cell lines that do not respond to trastuzumab. In addition, our results indicate that several molecular features determine the growth-retarding effects induced by the drugs, suggesting that parameters other than HER2 amplification/expression should be included as markers for therapy decisions.

Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas

Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d=6.33±0.86nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490nm wavelength) in the dynamic range of 100-700nM ECD_Apt1 with a detection limit of 12.5±2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. ©2016 AACR.

Herceptin-functionalized pure paclitaxel nanocrystals for enhanced delivery to HER2-postive breast cancer cells

The objective of this study was to prepare Herceptin (HCT)-functionalized paclitaxel nanocrystals and evaluated their cell-specific interactions, cellular accumulation, and growth inhibition in HER2-positve breast cancer cells as a tumor-targeted delivery module. Paclitaxel (PTX) was fabricated in the form of nanocrystals (PNCs) by a sono-precipitation method, and HCT were coated using a facile non-covalent method (PNCs-HCT). Our results showed that the PNCs-HCT were stable for at least 1month at 4°C with no noticeable desorption of HCT. The release test showed that PNCs-HCT exhibited sustained drug release similar to only PNCs but with a higher release rate than only PTX powder. Cellular uptake, cytotoxicity, and cell cycle arrest studies revealed that PNCs-HCT exhibit greater binding affinity and higher cell-specific internalization to HER2-positive breast cancer cell lines as compared to PNCs, followed by enhanced cell growth inhibition. HCT-functionalized PNCs presented in this study offer a promising strategy for targeted pure drug nanocrystal delivery and enhancing the efficiency of anticancer therapy.

Abstract 352: HER2 protein isoform heterogeneity investigated by single-cell western blotting

Abstract Accounting for ∼20% of breast cancers, half of metastatic HER2-positive breast cancer patients treated with trastuzumab plus chemotherapy will experience progression of disease within one year. Trastuzumab treatment, though associated with improvement in median overall survival, still sees drug resistance as an unmet challenge. Differential response to therapy may arise from tumor heterogeneity. Further complicating the situation is the fact that HER2/erbB2 biology is complex. HER2 is not simply one full-length protein (p185) overexpressed on tumor cell surfaces, but also includes truncated isoforms, t-erbB2 (e.g., 95 and 110 kDa isoforms) lacking the extracellular domain targeted by trastuzumab (and pertuzumab and T-DM1). The t-erbB2 isoforms are implicated in tumorigenesis, metastasis, and therapeutic response. Given existing pathology and life sciences tools, the high heterogeneity and need for single cell resolution presents a tremendous challenge. We introduce here a novel microfluidic device to quantitatively assess HER2 protein isoforms in heterogeneous tumor biopsies with single-cell resolution. The single-cell western blot (scWB) provides both the sensitivity and specificity needed to detect t-erbB2 isoforms owing to use of a single-cell electrophoretic protein separation followed by immunoprobing. The assay further provides enough sensitivity to obviate the need for cell pooling, which obscures cell-to-cell heterogeneity. We assay ∼1000's of single cells on one microscope slide in a 4-6 hr workflow. First, we validated that the scWB can separate full length (p185) from truncated (p95) HER2 in single p185- and p95-genetically engineered CHO cells. The p95 was resolved from p185 protein in a short ∼1 mm separation lane (resolution = 0.97±0.03, n>500 cells). Next, we assessed a HER2-positive breast cancer cell line (BT474) and found <3% t-erbB2 expressing cells. To further investigate the effect of t-erbB2 in HER2-related signaling, we examined phospho-proteins including p-HER2, p-HER3, p-ERK Thr187/Tyr185, p-ERK Thr202/Tyr204, p-rs6 from each t-erbB2 expressing BT474 cell. Protein activation in t-erbB2 expressing cells was similar to cells expressing full length HER2. This first-in-kind study demonstrates a powerful single-cell resolution multiplexing capability including for phospho-proteins. We are now applying the novel scWB to measure t-erbB2 in HER2-positive and triple positive (ER/PR/HER2) breast tumor biopsies and will report our results at AACR. Taken together, the microfluidic single-cell resolution western provides a sensitive, quantitative, and multiplexed tool to assay clinically-relevant oncoprotein isoforms that are currently difficult or impossible to assay. Correlating isoform expression ratios with clinical outcomes may confer a more comprehensive understanding of drug resistance responses derived from oncoprotein isoform profiles within heterogeneous cancer cell populations. Citation Format: Ginny (Chi-Chih) Kang, Toby M. Ward, Jessica Bockhorn, Mark D. Pegram, Amy E. Herr. HER2 protein isoform heterogeneity investigated by single-cell western blotting. [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 352.

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.

Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: therapeutic implications.

BackgroundMolecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here we investigated the pathophysiologic role of Mcl-1 in Her2-positive BC cells under hypoxic conditions.MethodsRNA interference and a novel small molecule inhibitor, EU-5346, were used to examine the role of Mcl-1 in Her2-positive BC cell lines and primary BC cells (sensitive or intrinsically resistant to Her2 inhibitors) under hypoxic conditions (using a hypoxic incubation chamber). Mechanisms-of-action were investigated by RT-PCR, mitochondrial isolation, as well as immunoprecipitation/blotting analysis, and microscopy. The specificity against Mcl-1 of the novel small molecule inhibitor EU5346 was verified in Mcl-1Δ/nullversus Mcl-1wt/wt Murine Embryonic Fibroblasts (MEFs). Proliferation, survival, and spheroid formation were assessed in response to Mcl-1 and Her2 inhibition.ResultsWe demonstrate for a strong correlation between high Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line.ConclusionOur data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0686-4) contains supplementary material, which is available to authorized users.

TIRF high-content assay development for the evaluation of drug efficacy of chemotherapeutic agents against EGFR-/HER2-positive breast cancer cell lines.

Elevated expression of epidermal growth factor receptor (EGFR) is reported to be associated with poor prognosis in breast cancer. EGFR subtype identification plays a crucial role in deciding the drug combination to treat the cancer patients. Conventional application of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) produces more discordance results in EGFR subtype identification of cancer specimens. The present study is designed to develop an analytical method for simultaneous identification of cell surface biomarkers and quantitative estimation of drug efficacy in cancer specimens. For this study, we have utilized a total internal reflection fluorescence microscope (TIRFM), Qdot molecular probes and chemotherapeutic agent camptothecin (CPT)-treated breast cancer cell lines namely MCF-7, SK-BR-3 and JIMT-1. Highly sensitive detection signals with low background noise generated from the evanescent field excitation of TIRFM make it a highly suitable tool to detect the cell surface biomarkers in living cells. Moreover, single wavelength excitation of Qdot probes offers multicolour imaging with strong emission brightness. In the present study, TIRF high-content imaging system simultaneously showed the expression pattern of EGFRs and EC50 value for CPT-induced apoptosis and necrosis in MCF-7, SK-BR-3 and JIMT-1 cancer cell lines.

Abstract P3-06-05: Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs

Abstract Aim: The aim of this study was to elucidate the role of transforming growth factor β (TGFβ) in the resistance of HER2-positive breast cancer cells to anti-HER2 drugs including trastuzumab and lapatinib. Methods: A HER2-positive breast cancer cell line, SKBR3, was cultured in the presence or absence of TGFβ for 14 days. Subsequently, TGFβ-treated cells were cultured for seven days with or without the anti-HER2 drugs. Sensitivity to trastuzumab and lapatinib was estimated by the WST-8 cell viability assay or absolute cell counts using In Cell Analyzer (GE Healthcare). Proportion of CD44+ CD24– breast cancer stem cells was estimated by flow cytometry of cells immunostained with anti-CD44 and anti-CD24 antibodies. For clinical study, 33 patients with HER2-positive breast cancer receiving neoadjuvant paclitaxel plus trastuzumab in our institution were analyzed. Among the cases, 27 biopsy samples obtained before any treatment from 27 patients who completed 12 cycles of weekly paclitaxel and trastuzumab were subjected to CD24 immunohistochemistry. Results: SKBR3 cells cultured with TGFβ for 14 days exhibited decreased sensitivity to both trastuzumab and lapatinib. Time course study revealed that continuous stimulation for 14 days with TGFβ was required for the resistance to anti-HER2 drugs. Activation of SMAD3, a downstream target molecule of TGFβ, was enhanced over time, judged by the increase in phosphorylation and in nuclear translocation. During 14 day culture with TGFβ, proportion of CD44+ CD24– cells were dramatically increased, and mammosphere formation, another marker of breast cancer stem cells, was significantly enhanced compared to cells treated without TGFβ. Among four HER2-positive breast cancer cell lines, only SKBR3 cells showed increased proportion of CD44+ CD24– cells and resistance to the anti-HER2 drugs, while other two cell lines exhibited epithelial-mesenchymal transition (EMT) in response to TGFβ. To explore the possibility of targeting TGFβ-SMAD3 axis to overcome resistance to anti-HER2 therapy, we used SIS3, a specific inhibitor of SMAD3. Importantly, SIS3 completely restored the sensitivity to both trastuzumab and lapatinib of TGFβ-treated SKBR3 cells, with the decrease in the proportion of CD44+ CD24– cells. These in vitro results suggest that CD24 downregulation can be a surrogate marker of resistance to anti-HER2 therapy. To establish this, we evaluated the CD24 expression in tumor samples of breast cancer patients who received paclitaxel plus trastuzumab in the neoadjuvant setting. Weak CD24 expression in tumor cells in biopsy samples obtained before any treatment was significantly correlated with poorer response to the drugs. Conclusion: These data clearly indicate the importance of TGFβ-SMAD3 axis in the acquired resistance to anti-HER2 drugs. Moreover, resistance to anti-HER2 therapy is associated with the property of breast cancer stem cells rather than EMT. Targeting TGFβ-SMAD3 axis warrants further investigation for overcoming resistance to anti-HER2 therapeutics. Citation Format: Shimoda M, Chihara Y, Kagara N, Naoi Y, Shimomura A, Shimazu K, Kim SJ, Noguchi S. Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-05.

Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors.

Abstract B17: Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model

Abstract Sphingosine kinase 1 (SphK1) phosphorylates pro-apoptotic sphingosine to form pro-survival sphingosine-1-phosphate (S1P), leading to cancer progression. In breast cancer, high levels of SphK1 mRNA are found in 80% of the patients, with average increase of 2-fold in breast tumors compared with normal tissue from the same patient. Recent studies suggest that SphK1 expression is associated with disease-free survival in ER-negative and HER2-positive breast tumors. In addition, inhibition of SphK1 in HER2-positive breast cancer cell line reduced S1P-induced HER2 and ERK1/2 activation. While these evidences suggest that SphK1 may play an important role in HER2-positive breast cancer, the exact role of SphK1 in breast tumorigenesis remains unclear. The aim of this project is to define the role of SphK1 in HER2-positive breast tumorigenesis and to investigate whether the pathway components are potential target for breast cancer prevention and therapies. Here, we report that SphK1 is required for HER2/neu-induced breast tumorigenesis and SphK1 regulates claudin-2 (CLDN2) to promote tumor development perhaps by mediating HER2. In MMTV-neu transgenic mouse model, genetic deletion of SphK1, which was accompanied by reduced S1P and increased C16-ceramide levels, significantly inhibited breast tumor development, with reduced incidence (SphK1-/-, 19%; SphK1+/-, 26%; and SphK1+/+, 57%; p < 0.05) and multiplicity (SphK1-/-, 0.27 ± 0.13; SphK1+/-, 0.32 ± 0.07; and SphK1+/+, 0.67 ± 0.14 tumors per animal; p < 0.05). Unexpectedly, SphK1 deletion did not affect tumor growth as results showed no differences in tumor volume. However, this is not surprising when considering the fact that the tumors were allowed to grow until tumor reached 20 mm in longer diameter, at which they were sacrificed regardless of age. Interestingly, whole genome expression analysis by Illumina MouseWG-6 v2.0 Expression BeadChip Array revealed significant reduction of CLDN2 expression in tumors from SphK1 deficient mice (p < 0.0001). In support to these findings, overexpression of SphK1 in MDA-MB-453 human breast cancer cells, which HER2 is amplified but lacks hormone receptors, lead to increase in CLDN2 expression (p < 0.05). Furthermore, overexpression of SphK1 in MDA-MB-453 cells also enhanced cell proliferation, colony formation, and migration/invasion, and drug resistance to cisplatin. The relevance of this finings in humans was confirmed by immunostaining of SphK1 and CLDN2 in HER2-positive breast cancer tumors and normal tissues (n = 93 and 35 subjects, respectively). The results showed that the expression of SphK1 was higher in tumors compared to adjacent normal tissue (p < 0.0001). Furthermore, higher SphK1 expression was observed in high grade tumors (vs low grade; p < 0.001) and ER-PR- tumors (vs ER+PR+; p < 0.0001). Interestingly, we observed similar staining pattern of CLDN2 proteins, where CLDN2 expression was high in tumors (p < 0.0001), particularly in high grade tumors (p < 0.01) and ER-PR- tumors (p < 0.0001). Moreover, we found a strong correlation between SphK1 and CLDN2 expressions in tumors but not in normal tissues, confirming the results obtained in animal study. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis, and targeting SphK1 and/or CLDN2 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment. Citation Format: Yoshiko Shimizu, Hideki Furuya, Paulette M. Tamashiro, Steve Goodison, Owen Chan, Ian Pagano, Kayoko Iino, Rafael Peres, Kanani Hokutan, Lenora W.M. Loo, Brenda Hernandez, Aung Naing, Charles J. Rosser, Toshihiko Kawamori. Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B17.

Enriched DNA aptamer pool for HER2-positive breast cancer cell line

Enriched DNA aptamer pool for HER2-positive breast cancer cell line

Safety and efficacy of gemcitabine and trastuzumab in HER2-directed therapy pretreated patients with HER2-positive metastatic breast cancer: SBP-01 study.

142 Background: Prognosis of HER2-positive metastatic breast cancer (MBC) has been dramatically improved by trastuzumab (Tmab). More recently, newer anti-HER2 agents such as lapatinib, pertuzumab and T-DM1 have prolonged survival. Despite the efficacy of these drugs, most patients develop progressive disease during or after treatment, and alternative anti-HER2 agents plus chemotherapies are required in subsequent lines of treatment. However, there are few evidence on efficacy of Tmab-containing regimens after disease progression. Gemcitabine (GEM) is non-cross resistant to anthracycline and taxane. Preclinical studies have shown that the combination of Tmab and GEM has synergistic effect against HER2-positive breast cancer cell lines. SBP-01 study assessed the efficacy and safety of the combination of Tamb and GEM in patients with HER2-positive MBC previously treated with anti-HER2 therapy. Methods: SBP-01 study included patients treated with one or more anti-HER2 directed regimens for MBC. Patients were administered with GEM 1250 mg/m2 on days 1 and 8 of each 21-day cycle and Tmab 4mg/kg loading dose and then 2mg/kg weekly. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression free survival (PFS), overall survival, and safety. Results: Between June 2011 and June 2014, 35 patients were enrolled. Patients had ER positive tumor (37.1%), a median of 2 metastatic organ sites, visceral metastasis (80.0%), prior (neo) adjuvant Tmab (22.9%) and a median of 2 prior chemotherapy regimens for MBC. Previous HER2-directed drugs included Tmab (94.3%), lapatinib (37.1%), T-DM1 (8.6%) and pertuzumab (2.9%). ORR was 22.9% (95% CI, 8.6%-36.8%). Median PFS was 146 days. Patients with stable disease response received a median of 7 cycles (6-28 cycles) of treatment. Grade3/4 leukopenia (20.0%) and neutropenia (48.6%) were observed. All non-hematological toxicities were less than grade3. Conclusions: The Combination Tmab and GEM is effective and well-tolerated regimen for patients previously treated with HER2-directed therapy, and appears to make disease stable for long time period. Clinical trial information: UMIN000005881.

Abstract 3120: miR-489 function as tumor suppressor miRNA by targeting HER2 in HER2-positive breast cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in 20-30% of all breast cancer and associated with poor prognosis and outcome. Previous studies have shown that dysregulation of several micro RNAs (miRs), play a key role in breast cancer progression and metastasis. In this study, we evaluated ∼330 miRs, to find specific miRs dysregualted by HER2 signaling. After further screening we focused our study on miR-489, whose expression was ∼6-7 fold downregulated specifically by HER2 signaling. Restoration of miR-489 expression in several HER2-positive breast cancer cell lines resulted in increased cell death and cell cycle arrest. To investigate the molecular mechanism of action of miR-489, we studied effect of miR-489 on HER2 signaling pathway and found that higher expression of miR-489 led to the decreased levels of HER2 (both mRNA and protein) and decreased Akt activation. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2 and miR-489 signaling pathways form a mutual regulatory loop. To further demonstrate clinical relevance of miR-489, we analyzed both normal and tumor human breast tissue samples using Fluorescent in situ hybridization technique (FISH). Our imaging data indicated that miR-489 is expressed at much lower level in tumor tissues compared to that of the normal breast tissue. Overall, our data indicates that miR-489 acts as a tumor suppressor miR at least partially by directly targeting HER2 in HER2 positive breast cancer cells. Citation Format: Yogin Patel, Nirav Shah, Racheal Botbyl, Jishin Lee, Hexin Chen. miR-489 function as tumor suppressor miRNA by targeting HER2 in HER2-positive breast cancer. [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 3120. doi:10.1158/1538-7445.AM2015-3120

Abstract 211: A novel FRET assay for the intracellular activation of ADC linkers

Abstract Evaluating the intracellular activation of ADC by a novel FRET assay. Despite the recent success of ADCs as cancer therapeutics, their mechanisms of action are not fully understood. In this study, we developed ADCs using a novel fluorescence resonance energy transfer (FRET) linker in order to facilitate monitoring the details of intracellular uptake, vesicular trafficking and payload release. In the FRET linker, the cathepsin-cleavable dipeptide of val-cit was inserted between a fluorescence donor Alexa Fluor 488 (or later, fluorescein) and an acceptor tetramethylrhodamine (TAMRA). Upon cleavage of the val-cit linker, a fluorescence signal from Alexa Fluor 488 or fluorescein is expected to appear as a probe for monitoring intracellular activation of ADC. We used two in-vitro human cancer cell lines, SKBR3 and PC3. SKBR3 is a Her2-positive breast cancer cell line and PC3 is a prostate cancer cell line that has been engineered to express a prostate cancer-specific growth factor receptor, tomoregulin-2 (TenB2). We assessed the intracellular processing of the FRET conjugates using flow cytometry. We found that 74% and 41% of the linker was cleaved in PC3 and SKBR3 cells after 20 hours of treatment, respectively. In the live-celling imaging of the FRET probe in SKBR3 and PC3 cells, Anti-Her2 and TenB2 FRET ADCs showed a remarkable difference of internalization pathways and cleavage following receptor-mediated endocytosis. In PC3 cells, FRET ADCs were internalized and congregated into a certain localized area during the early stage of uptake in 2 to 3 hours. The ADC-containing vesicles were spread over the cytosol and near the plasma membrane in a subset of cells. A similar directed localization at the early stage of uptake was not observed for the SKBR3 cells. The endosomal vesicles in SKBR3s were spread over the cytosol during the course of ADC internalization. The amount of the release payload in the cytosol of SKBR3s was 3-fold greater than that of PC3 cells, suggesting that the endosomal/lysosomal membrane of SKBR3 may be more permeable than that of PC3. In summary, our ADC FRET probes allowed for the facile, robust and non-invasive evaluation of intracellular processing for ADC linkers. Quantitative measurements for the rate of antigen-mediated intracellular cleavage of the FRET linker as well as cytosolic release could be evaluated by our FRET probes. Citation Format: Byoung-Chul Lee, Cecile Chalouni, Sam Nalle, Sophia Doll, Martine Darwish, Ira Mellman, Richard Vandlen. A novel FRET assay for the intracellular activation of ADC linkers. [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 211. doi:10.1158/1538-7445.AM2015-211

Abstract 2357: Knockdown of neuropilin-1 in monocytes impaired lymphocyte migration and anti-tumor activity in a humanized mouse model

Abstract Background Antibody therapy such as trastuzumab is imperative for HER2-positive breast cancer patients. Although its antitumor action is not completely understood, the role of immune cells in antibody therapy is indispensable. Neuropilin-1 (NRP1) in monocytes induces high in vitro anti-tumor activity. Here, we showed the role of NRP1 in monocytes in HER2-positive breast cancer, using a tumor-bearing NOD/Shi-SCID, IL-2Rγnull(NOG) mouse model. Experimental procedures A tumor-bearing NOG mouse was made using BT474-sGL containing transfected HER2-positive human breast cancer cell lines that secreted Gaussia luciferase (Gluc). Tumor volumes were measured, using Gluc relative light units (RLUs), in 5 treatment arms (Table). Each arm was allocated 5 NOG mice, randomized by body weight and RLUs. Cytokines released into culture supernatants were determined using human Bio- Plex Cytokine Assay Kits (Bio-Rad, Hercules, CA), according to the manufacturer's instructions. mRNA was measured using TAQMAN array (LifeTechnologies, Carlsbad, CA) gene expression studies, according to the manufacturer's instructions. Results Knockdown of NRP-1 in monocytes impaired anti-tumor activity: After 2 cycles of treatment, RLUs increased significantly from baseline in Arm E, compared with those in Arm D. There was no significant difference between Arms B and E. Knockdown of NRP-1 in monocytes impaired migration activity: Staining for human-specific CD45 revealed that the tumor infiltrating lymphocytes of Arm E decreased compared with those of Arm D; CD45 mRNA was significantly lower in Arm E than in Arm D. Chemokine secretion is regulated by NRP-1: Tumor chemokine (e.g., IP-10, MIP-1α, MIP-1β and RANTES) secretion was significantly decreased due to knockdown of NRP-1. Conclusion Our data suggest that NRP-1-expressing immune cells initiate anti-tumor activity via migration and chemokine secretion. Summary of resultsArmHumanizedTrastuzumabNRP1 KD% change RLU (mean ± SEM)CD45 mRNA relative quantities (mean ± SEM)IP-10 pg/mL (mean ± SEM)MIP-1α pg/mL (mean ± SEM)MIP-1β pg/mL (mean ± SEM)RANTES pg/mL (mean ± SEM)ANoNoNo495 ± 76.00.75 ± 0.3822.2 ± 6.351.16 ± 0.013.51 ± 3.515.36 ± 2.68BNoYesNo49.6 ± 43.20.50 ± 0.2617.1 ± 13.91.20 ± 0.275.75 ± 5.757.87 ± 4.40CYesNoNo566 ± 145504 ± 2421361 ± 4538.31 ± 3.1582.9 ± 43.659.4 ± 42.6DYesYesNo-45.7 ± 12.91650 ± 4055880 ± 26006.43 ± 1.3767.0 ± 13.9171 ± 35.4EYesYesYes46.2 ± 15.4307 ± 121285 ± 73.21.60 ± 0.3010.7 ± 2.3229.7 ± 9.69p value of D vs. Ep < 0.01p = 0.04p = 0.03p < 0.01p < 0.01p < 0.01 Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Isao Kii, Tatsuki R. Kataoka, Masahiro Hirata, Hironori Haga, Masatoshi Hagiwara, Masakazu Toi. Knockdown of neuropilin-1 in monocytes impaired lymphocyte migration and anti-tumor activity in a humanized mouse model. [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 2357. doi:10.1158/1538-7445.AM2015-2357

MRNA profiling reveals determinants of trastuzumab efficiency in HER2-positive breast cancer.

Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed SNPs. These results contribute to a better understanding of trastuzumab action and resistance mechanisms.