Trastuzumab-resistant Cell Lines Research Articles

571 - Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer

The HER2 receptor is overexpressed in 7-34% of gastric cancer (GC) tumors, being a potential therapeutic target. Trastuzumab, an anti-HER2 monoclonal antibody, in combination with platinum therapy was approved in 2010 as first-line treatment for advanced GC tumors overexpressing HER2. Despite the clinical benefit of trastuzumab, development of acquired resistance to treatment limits the efficacy of trastuzumab. To study the mechanisms of trastuzumab acquired resistance we generated, upon chronic exposure to trastuzumab, a trastuzumab-resistant cell line derived from the human HER2-positive GC cell NCI-N87, hereafter named NTR2.4. The resistant phenotype of NTR2.4 cell line was associated with an increased basal activation of SRC, MAPK/ERK and PI3K/mTOR HER2 downstream pathways that persisted upon exposure to trastuzumab. Trastuzumab treatment induced a compensatory upregulation of HER3 and an increased expression of the HER family ligands EGF, AREG, HB-EGF, and NRG1 in NTR2.4 compared with the trastuzumab-sensitive NCI-N87 cell line. Ligand stimulation induced resistance to trastuzumab in NCI-N87 cells. These results were replicated in another model of trastuzumab-resistance, OTR6 cell line, suggesting an important role of HER receptor plasticity and HER family ligand in trastuzumab efficacy and resistance. Analysis of clinical samples from HER2-positive GC patients treated with trastuzumab showed higher EGF concentration in serum samples and increased EGF mRNA expression in tumor biopsies at trastuzumab progression compared with samples previous to trastuzumab treatment. Treatment with Pan-HER, a novel antibody mixture against EGFR, HER2, and HER3, was effective in both trastuzumab-sensitive and trastuzumab-resistant cell lines and also in the presence of ligands. Altogether, our results suggested that Pan-HER would be a potential candidate to overcome trastuzumab resistance in advanced GC patients.

The voltage gated Ca(2+)-channel Cav3.2 and therapeutic responses in breast cancer.

BackgroundUnderstanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca2+)-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca2+-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca2+-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome.MethodsExpression levels of a panel of Ca2+-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort).ResultsIncreased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers.ConclusionOur study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca2+-signalling in breast cancer progression and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0299-0) contains supplementary material, which is available to authorized users.

Abstract P5-08-22: DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer

Abstract Background. The majority of patients develop resistance against suppression of HER2-mediated signaling by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Methods. Using Affymetrix HGU133plus2 gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further constricted to those markers significantly associated to worse survival. Functional significance of the two strongest predictive biomarkers was evaluated in vitro experiments after gene silencing in the HER2 overexpressing, trastuzumab resistant breast cancer cell lines SKBR-3-TR and JIMT-1. Results. Out of 10 investigated MKPs, the strongest predictive genes were DUSP4/MKP2 (AUC=0.75, p=0.0096) and DUSP6/MKP3 (AUC=0.77, p=5.29E-05). Furthermore, higher expression for these correlated to worse survival in 221 HER2 positive BC patients (DUSP4: HR=1.6, p=0.04 and DUSP6: HR=1.8, p=0.0053). Silencing of DUSP4 had significant sensitization effects - viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected, trastuzumab treated controls (SKBR-3-TR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Conclusions. Our findings suggest that DUSP4 is involved in the development of trastuzumab resistance in HER2 positive BC. Citation Format: Györffy B, Munkacsy G, Esteva FJ, Miquel TP, Menyhart O. DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer. [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 P5-08-22.

Trastuzumab-Resistant Luminal B Breast Cancer Cells Show Basal-Like Cell Growth Features Through NF-κB-Activation.

A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(( 1 )) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed "progression through grade" or "evolution of resistance" hypothesis. Furthermore, we identified IKK-α/IKK-β and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation.

Abstract 724: Anti-tumor effect of Dasatinib in HER2 positive breast cancer with Trastuzumab resistance

Abstract [Background] Overexpression of human epidermal growth factor receptor 2 (HER2) was observed in approximately 20%-25% of breast cancers and they are classified in HER2-positive breast cancer. Trastuzumab is a therapeutic drug for the first choice for HER2-positive breast cancer, showing good response. However, acquired resistance to trastuzumab is one of the critical clinical issues in breast cancer treatment, especially in the patients with recurrent breast cancer. Therefore it is necessary to develop the effective therapy to overcome resistance. In this study, we established a trastuzumab-resistant breast cancer cell line from a trastumab-sensitive cell line with HER2 amplification (BT-474), and demonstrated anti-tumor effect of dasatinib in the breast cancer cell with trastuzumab resistance. [Methods] Trastuzumab-resistant breast cancer cell line (BT-474-R) was established by treating BT-474 cells for long-term exposing with trastuzumab. We performed cell viability assay to analyze drug sensitivity. Then, we performed quantitative PCR and western blotting to investigate expression and activation of HER2 and related molecules. Cell viability assay and signal analysis were performed with or without trastuzumab and/or dasatinib treatment. [Results] Proto-oncogene tyrosine-protein kinase Src was up-regulated and activated in BT-474-R. Signaling molecules, such as Akt and MAPK, were also activated. Dasatinib, which is known as a Src inhibitor, suppressed the activation of signal molecules and cell survival of BT-474-R. Moreover, dasatinib treatment recovered sensitivity to trastumab in the BT-474-R with down-regulation of Src phosphorylation. [Conclusion] Src plays an important role in acquired resistance to trastumab in breast cancer. Our data also suggest that inhibition of Src may become the new strategy to overcome trastuzumab resistance. Citation Format: Tatsuaki Takeda, Hirotaka Kanzaki, Shinichi Toyooka, Mototsugu Watanabe, Tomoaki Ohtsuka, Ken Suzawa, Shinsuke Hashida, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Yoshihisa Kitamura, Toshiaki Sendo. Anti-tumor effect of Dasatinib in HER2 positive breast cancer with Trastuzumab resistance. [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 724. doi:10.1158/1538-7445.AM2015-724

Abstract 726: Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer

Abstract [Purpose] Luminal B breast cancer patients are significantly at the risk of relapse with in 5 years. Acquired resistance to the receptor tyrosine kinase HER2 targeting antibody trastuzumab is a major clinical problem in the treatment of recurrent HER2-positive breast cancer. The molecular mechanisms leading to trastuzumab resistance (TZR) remain unclear in luminal B breast cancer. Here, we investigated the mechanism of TZR in HER2-positive luminal B breast cancer cells with acquired resistance. [Methods and Results] We generated trastuzumab resistant breast cancer cell line (BT-474-R) by treating BT-474 cells with trastuzumab for over long-term. Initial validation showed that HER2 overexpression in the BT-474-R cells remained unaltered suggesting that probably HER2 independent signaling pathway is involved in the development of TZR. We demonstrated that NF-κB is constitutively activated in the BT-474-R cells by quantitative realtime RT-PCR, western blotting and immunofluorescent microscopy. Expression of pro-inflammatory cytokines were also demonstrated. Pharmacologic inhibition of NF-κB improved sensitivity to trastuzumab in BT-474-R cells. We identified both IKK-α and IKK-β as the major determinants of the activation of the NF-κB signaling in the resistant cells. [Conclusion] Our study suggests by activating NF-κB pathway, luminal B cells may facilitate acquiring basal-like, Her2+ phenotype a notion consistent with “progression through grade” hypothesis. Finally, our study suggests that blocking NF-κB will provide a new treatment option to limit relapse in HER2 positive breast cancer patients treated with trastuzumab. Citation Format: Hirotaka Kanzaki, Nishit Makhopadhya, Xiaojiang Cui, Krishnan V. Ramanujan, Tatsuaki Takeda, Yoshihisa Kitamura, Toshiaki Sendo, Ramachandran Murali. Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B 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 726. doi:10.1158/1538-7445.AM2015-726

Abstract 3919: Inhibition of HER2 and IGF2 in trastuzumab-resistant HER2 positive JIMT1 cells decreases cell growth and reduces activation of MAPK and AKT

Abstract Breast cancer is the second leading cause of death in women. Overexpression of human epidermal growth factor receptor 2 (HER2) is detected in 20-30% of breast cancers. Increased levels of HER2 are linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four-member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity. Dimerization of HER2 activates the MAPK and AKT pathways. The HER2 receptor also hetero-dimerizes with the IGF1 receptor (IGF1R) in trastuzumab-resistant cells. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the IGF1R, insulin receptor (InsR), and a cross-talk with the estrogen receptors alpha and beta (ERα, ERß). Trastuzumab resistance occurs in 70% of patients with HER2 positive breast cancer. Therefore it is important to understand the mechanism of resistance so we can develop new therapies to help patients with trastuzumab-resistant rumors. Previous findings in our lab demonstrate an increased expression of IGF2 in the trastuzumab-resistant cell line JIMT1 when compared to trastuzumab-sensitive cell lines BT474, SKBR3, and ZR75-30. We hypothesize that to overcome trastuzumab-resistance in JIMT1 cells, dual inhibition of the HER2 and IGF2 is necessary to induce cell death. When JIMT1 cells were treated with trastuzumab and an IGF2 blocking antibody (IBA) a decrease in cell viability was observed, but changes in PARP cleavage were not present. We also analyzed the protein and phosphorylation levels of HER2, IGF1R, MAPK, and AKT. Our studies suggest that blocking both HER2 and IGF2 decreases cell viability and reduces phosphorylation of MAPK and AKT. Citation Format: Xousaen M. Helu, Daisy DeLeon. Inhibition of HER2 and IGF2 in trastuzumab-resistant HER2 positive JIMT1 cells decreases cell growth and reduces activation of MAPK and AKT. [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 3919. doi:10.1158/1538-7445.AM2015-3919

Abstract LB-261: Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer

Abstract Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [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 LB-261. doi:10.1158/1538-7445.AM2015-LB-261

T-Darpp Promotes Enhanced EGFR Activation and New Drug Synergies in Her2-Positive Breast Cancer Cells.

Trastuzumab has led to improved survival rates of HER2+ breast cancer patients. However, acquired resistance remains a problem in the majority of cases. t-Darpp is over-expressed in trastuzumab-resistant cell lines and its over-expression is sufficient for conferring the resistance phenotype. Although its mechanism of action is unknown, t-Darpp has been shown to increase cellular proliferation and inhibit apoptosis. We have reported that trastuzumab-resistant BT.HerR cells that over-express endogenous t-Darpp are sensitized to EGFR inhibition in the presence (but not the absence) of trastuzumab. The purpose of the current study was to determine if t-Darpp might modulate sensitivity to EGFR inhibitors in trastuzumab-resistant cells. Using EGFR tyrosine kinase inhibitors AG1478, gefitinib and erlotinib, we found that trastuzumab-resistant SK.HerR cells were sensitized to EGFR inhibition, compared to SK-Br-3 controls, even in the absence of trastuzumab. t-Darpp knock-down in SK.HerR cells reversed their sensitivity to EGFR inhibition. Increased EGFR sensitivity was also noted in SK.tDp cells that stably over-express t-Darpp. High levels of synergy between trastuzumab and the EGFR inhibitors were observed in all cell lines with high t-Darpp expression. These cells also demonstrated more robust activation of EGFR signaling and showed greater EGFR stability than parental cells. The T75A phosphorylation mutant of t-Darpp did not confer sensitivity to EGFR inhibition nor activation of EGFR signaling. The over-expression of t-Darpp might facilitate enhanced EGFR signaling as part of the trastuzumab resistance phenotype. This study suggests that the presence of t-Darpp in HER2+ cancers might predict the enhanced response to dual HER2/EGFR targeting.

Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.

Trastuzumab has been successfully employed for the treatment of Her-2-positive gastric cancer. However, there are problems with both primary and secondary resistance to trastuzumab. In this study, we employed the human gastric carcinoma cell line NCI-N87 with high Her-2 expression to create trastuzumab-resistant NCI-N87/TR cells by stepwise exposure to increasing doses of trastuzumab. Western blotting and Real-time PCR were conducted to detect protein and gene levels. Compared with NCI-N87 cells, the expression of P-IGF-1R and P-AKT proteins was significantly increased in NCI-N87/TR cells (both P = 0.000), while PTEN gene and protein expression showed a significant decrease (both P = 0.000). In addition, mutations of the PTEN gene were detected at exons 5, 7, and 8. The sensitivity of NCI-N87/TR cells to trastuzumab was increased by transfection with the PTEN gene, or by incubation with a PI3K inhibitor (LY294002) or an IGF-IR inhibitor (AG1024), as well as siRNA targeting PI3K p110 or IGF-1R. Taken together, our findings showed that activation of the PI3K-AKT signaling pathway was one of the major mechanisms leading to resistance of NCI-N87/TR gastric cancer cells to trastuzumab, which was probably associated with PTEN gene down-regulation and mutation, as well as with over-activity of the IGF-1R signaling pathway.

CD44 expression contributes to trastuzumab resistance in HER2-positive breast cancer cells.

Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. CD44, a putative breast cancer stem cell (CSC) marker, is overexpressed in trastuzumab-resistant breast cancer cells. While CSC-related genes may play a role in the development of trastuzumab resistance, conflicting results have been published about CSC response to trastuzumab. We hypothesized that CD44 contributes to trastuzumab resistance independently of its role as a CSC marker. We used trastuzumab-sensitive breast cancer cell lines and their trastuzumab-resistant isogenic counterparts to evaluate the role of CD44 in response to trastuzumab. miRNA and mRNA expression were analyzed using microarray chips. A gene set enrichment analysis was created and matched with response to trastuzumab in cells and patient samples. The proportions of CSC in trastuzumab-resistant cells were similar to or lower than in the trastuzumab-sensitive cells. However, CD44 expression levels were significantly higher in both trastuzumab-resistant cell lines and its knockdown led to an increased response to trastuzumab. The invasiveness and anchorage-independent growth of trastuzumab-resistant cells were higher and blocked by downregulation of CD44. Results also showed that CD44-related resistance to trastuzumab is regulated by miRNAs. We identified a CD44-related gene expression profile that correlated with response to trastuzumab in cell lines and breast cancer patients. CD44 mediates trastuzumab resistance in HER2-positive breast cancer cells independently of its role as a CSC marker and that this role of CD44 is partly regulated by miRNA.

Abstract P4-07-06: miR-9 expression, retinoids and their potential role in trastuzumab resistance

Abstract HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, the majority of metastatic HER2 positive breast cancers progress on trastuzumab treatment due to either innate or acquired resistance. The aim of this study is to investigate the role of microRNAs (miRNAs) in a cell line model of acquired trastuzumab resistance. The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. miRNA extracted from SKBR3 and SKBR3-T was profiled using Taqman low density arrays (TLDA). Differentially regulated miRNAs were selected using >2-fold change and a P-value of <0.05. Individual quantitative RT-PCR (qRT-PCR) was performed to confirm miRNA alterations. Proliferation assays were performed using the acid phosphatase method. Taqman gene expression assays for retinoic acid receptor alpha (RARA) were performed using GAPDH as an endogenous control. Immunodetection of RARA was performed using α-tubulin as a control. Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in SKBR3-T compared to SKBR3 cells including miR-9 which was 2.2 fold up-regulated (p=0.04). Utilising miRWalk, we identified RARA as a target for miR-9. We confirmed that RARA mRNA and protein expression are reduced in SKBR3-T cells compared to SKBR3 cells. Treatment with all-trans retinoic acid (ATRA) (0.2 µM–0.025 µM) alone inhibited growth of the SKBR3 cell line in a dose dependent manner but not in the SKBR3-T cell line. Combined treatment with trastuzumab (10 µg/uL) and ATRA (0.2 µM) had a significantly greater growth inhibitory effect on the SKBR3 cell line (90.1±5.2 %) (p=0.0002) than either trastuzumab (39.1±4.0 %) (p= 0.0004) or ATRA alone (57.9±4.2 %) (p=0.001). Interestingly, despite relative insensitivity to ATRA in the SKBR3-T cells (23.7±5.0%) (p=0.86), combined treatment with trastuzumab produced significant growth inhibition in the SKBR3-T cells (74.2±5.1%) compared to either trastuzumab (24.1±4.4%) (p=0.0001) or ATRA (23.7±5.0%) (p=0.86) alone. miR-9 is up-regulated in the acquired trastuzumab resistant SKBR3-T cell line, RARA expression is downregulated, and SKBR3-T cells are resistant to ATRA treatment compared to SKBR3 cells. However, SKBR3-T cells are sensitive to combined treatment with trastuzumab and ATRA. Thus combined treatment with ATRA and trastuzumab may overcome acquired resistance to trastuzumab in HER2 positive breast cancer. Citation Format: Karen Howe, Brigid C Browne, Sinead Aherne, John Crown, Norma O'Donovan. miR-9 expression, retinoids and their potential role in trastuzumab resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-06.

Abstract B38: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

Abstract Trastuzumab is a humanized recombinant monoclonal antibody directed against extracellular region of ErbB2 and used for the treatment of different ErbB2-positive cancers. Based on literature reports and our clinical data from trastuzumab neoadjuvant studies, increased ErbB2-Y1248 phosphorylation is associated with a better response to trastuzumab in patient samples. Also, in trastuzumab-sensitive cell lines (SKBR3 and BT474), increased basal level and trastuzumab-mediated increase in ErbB2-Y1248 phosphorylation was readily detectable compared to trastuzumab-resistant cell line model (JIMT1). The mechanisms of trastuzumab-induced ErbB2-Y1248 phosphorylation and the role of ErbB2-Y1248 phosphorylation in trastuzumab sensitivity/resistance are still incompletely understood. We demonstrate that trastuzumab activates ErbB2 kinase activity in trastuzumab-sensitive cells, which may further contribute to trastuzumab-mediated ErbB2-Y1248 phosphorylation. However, blocking ErbB1/ErbB2 kinase activity with Lapatinib, a small molecule tyrosine kinase inhibitor only partially reduced trastuzumab-mediated ErbB2-Y1248 phosphorylation. This led to the discovery of a previously unappreciated relationship between trastuzumab and Csk-homologous kinase (CHK), a non-receptor tyrosine kinase also known as MATK. We establish that trastuzumab treatment promotes interaction between ErbB2 and CHK in trastuzumab-sensitive but not in trastuzumab-resistant cells despite similar CHK expression levels in both types of cells. Mimicking trastuzumab, CHK overexpression also induces ErbB2-Y1248 phosphorylation, Akt downregulation and cell growth inhibition and further stimulated trastuzumab-mediated reduction in total ErbB2 levels in trastuzumab sensitive cells. Also, CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition in trastuzumab-sensitive but not in trastuzumab-resistant cells. Understanding of the molecular mechanisms of trastuzumab mediated ErbB2 phosphorylation changes may suggest novel therapeutic approaches in the treatment of trastuzumab resistant cancers. Disclaimer: The information presented in this article reflects the views of the authors and does not represent the policy of the U.S. Food and Drug Administration. Citation Format: Milos Dokmanovic, Yun Wu, Yi Shen, Jieqing Chen, Dianne S. Hirsch, Wen Jin Wu. Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B38.

Anti-IGF-1R monoclonal antibody inhibits the carcinogenicity activity of acquired trastuzumab-resistant SKOV3.

BackgroundAntibody resistance, not only de novo but also acquired cases, usually exists and is related with lower survival rate and high risk of recurrence. Reversing the resistance often results in better clinical therapeutic effect. Previously, we established a trastuzumab-resistant ovarian cancer cell line, named as SKOV3-T, with lower HER2 and induced higher IGF-1R expression level to keep cell survival.MethodsIGF-1R was identified important for SKOV3-T growth. Then, a novel anti-IGF-1R monoclonal antibody, named as LMAb1, was used to inhibit SKOV3-T in cell growth/proliferation, migration, clone formation and in vivo carcinogenicity.ResultsIn both in vitro and in vivo assays, LMAb1 showed effective anti-tumor function, especially when being used in combination with trastuzumab, which was beneficial to longer survival time of mice as well as smaller tumor. It was also confirmed preliminarily that the mechanism of antibody might be to inhibit the activation of IGF-1R and downstream MAPK, AKT pathway transduction.ConclusionWe achieved satisfactory anti-tumor activity using trastuzumab plus LMAb1 in trastuzumab-resistant ovarian cancer model. In similar cases, not only acquired but also de novo, good curative effect might be achieved using combined antibody therapy strategies.

ADAM10 and ADAM17: New Players in Trastuzumab Resistance

The availability of the anti-HER2 monoclonal antibody, trastuzumab (Herceptin) has transformed the outcome of a subgroup of patients with breast cancer that previously had a poor prognosis, i.e., those with HER2-positive disease [1]. Although HER2 gene amplification/overexpression is necessary for breast cancer patients to receive trastuzumab, more than 50% of patients positive for this biomarker are intrinsically resistant to the treatment. Furthermore, almost all patients who initially respond, develop resistance which frequently occurs within the first year of treatment. Using mostly preclinical models, several different mechanisms have been proposed for conferring resistance to trastuzumab. These include hyperactivation of the PI3K pathway (due to PI3K mutations or PTEN loss), activation of alternative pathways (SRC, IGFR1), increased levels of EGFR/HER ligands and presence of HER2 isoforms [1]. In this issue of Oncotarget, Feldinger et al [2] describe a potential new mechanism of acquired resistance to trastuzumab, i.e., increased expression of ADAM10. ADAM10, together with the related ADAM17, are responsible for the release of all the ligands that bind to, and activate the EGFR/HER family of proteins [3]. Thus, ADAM17 is the primary sheddase for TGF-alpha, amphiregulin, HB-EGF, and epiregulin, while ADAM10 is believed to be primarily responsible for the release of EGF and betacellulin [3]. Previous evidence implicated high levels of these ligands in conferring resistance to anti-HER2 therapies, including trastuzumab [4,5]. Feldinger et al [2] now reports that trastuzumab increases ADAM10 levels in cell culture, in an animal xenograft model and importantly, also in patients. Furthermore, knockdown of ADAM10 or treatment with a selective low molecular weight ADAM10 inhibitor (INCB8765; Incyte) increased trastuzumab response in both naive and trastuzumab-resistant HER2-positive cell lines. This enhanced response appeared to result from the inhibition of betacellulin release and subsequent reduced activation of EGFR, although this was not investigated in detail. Consistent with these preclinical findings, the authors showed, using a small number of patients, that pretreatment ADAM10 levels were associated with a poor response and shorter relapse-free interval following treatment with trastuzumab. Previous research by the same group implicated increased levels of ADAM17 in conferring resistance to trastuzumab [6]. Taken together, these 2 reports [2,6] suggest that inhibition of ADAM10, ADAM17 or preferably both ADAMs is a potential new approach for minimizing resistance to trastuzumab. Currently, several ADAM10/17 inhibitors are available and indeed some of these have shown anti-cancer activity in preclinical systems [3,7]. To our knowledge, only one of these has undergone investigations in a clinical trial, for potential anti-cancer activity, i.e., the dual ADAM10/17 inhibitor, INCB7839 (Incyte) [8]. Preliminary results suggest that this drug is generally well tolerated with no major musculoskeletal side effects or anti-EGFR-related side effects such as skin rash. Furthermore, there were no reports of drug-induced increases in liver enzymes, bone marrow toxicity or increase in cardiomyopathy [8]. Evidence of target inhibition was the finding that administration of INCB7839 decreased shedding of different HER ligands as well as the extracellular domain of HER2. The time has now come to further investigate ADAM10/17 inhibitors in animal models for minimizing resistance to trastuzumab. Hopefully, the preliminary results of Kong and colleagues [2,6] can be confirmed and that we can then move on to clinical trials using ADAM10/17 inhibitors in combination with trastuzumab.

Moving a Carbohydrate Mimetic Peptide into the clinic

Tumor-Associated Carbohydrate Antigens (TACAs) are broad-spectrum targets for immunotherapy. Immunization with Carbohydrate Mimetic Peptides (CMPs) is a strategy to induce broad-spectrum TACA-reactive antibodies hypothesized to interfere with cellular pathways involved in tumor cell survival. A Phase I study was conducted with a first-in-man CMP referred to as P10s, conjugated to the Pan T cell carrier PADRE, along with MONTANIDE™ ISA 51 VG as adjuvant over a course of 5 immunizations. While designed as a safety and tolerability study, the potential for therapeutic impact was observed in a subject with metastatic lesions as evaluated before and after vaccine treatment. The subject received Vinorelbine and Trastuzumab (VT) for two months prior to study eligibility. PET scans showed partial response in the lungs and complete resolution of a previously enlarged subpectoral lymph node. Immunization with P10s vaccine resulted in responses to P10s, with serum and plasma antibodies reactive with and cytotoxic to human breast cancer cells in vitro, including the Trastuzumab-resistant HCC1954 cell line. However, the patient developed cystic masses in the brain parenchyma with no apparent evidence of metastases. The subject was switched to Docetaxel, Pertuzumab and Trastuzumab a year later, and her last PET scan showed a complete response in the lungs and lymph nodes. Incubation of cancer cells with a combination of vaccine-induced serum and docetaxel suggests that the induced antibodies sensitize tumor cells for more efficient killing upon administration of docetaxel. The data suggest that P10s-PADRE induces anti-tumor antibody response that in combination with chemotherapy can affect metastatic lesions in breast cancer patients.

Abstract 1826: Altered expression of miRNAs in acquired trastuzumab resistance

Abstract HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, patients with metastatic HER2 positive breast cancer frequently develop resistance to trastuzumab. The aim of this study is to investigate microRNAs (miRNAs) in cell line models of acquired trastuzumab resistance to identifty altered miRNAs which may be biomarkers of resistance. The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. MiRNA extracted from SKBR3, and SKBR3-T was profiled using Taqman low density arrays (TLDA). Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in the SKBR3-T compared to SKBR3 cells: miR-224 1.6 fold down-regulated (p=0.01), miR-221 5.8 fold up-regulated (p=0.04), miR-222 was 3.8 fold up-regulated (p=0.01) and miR-9 was 2.2 fold up-regulated (p=0.04). In another model of acquired trastuzumab resistance, BT474-Tr, miR-221 and -222 were also up-regulated, miR224 expression was switched off. No significant change in miR-9 was observed in BT474-Tr. We analysed expression of the four miRNAs in a panel of HER2 positive cell lines which included 6 innate trastuzumab resistant cell lines and 3 innately sensitive cell lines. miR-224 was significantly down-regulated in the 6 resistant cell lines and miR-221 was significantly up-regulated in 3 of the 6 resistant cell lines, compared to the sensitive cell lines. miR-222 and miR-9 were not consistently altered in the resistant cell lines compared to the sensitive cell lines. We analysed expression of these miRNAs in a small cohort of patients who showed durable complete response (pCR) (9 %) following treatment with trastuzumab and chemotherapy [1], and a control group of patients whose disease progressed following treatment. miRNA was extracted from FFPE tumor samples (n=12) and analysed by qRT-PCR. The mean levels of expression of miR-9, -221, and -222 were higher in non-responders (n=6) than responders (n=6) and the mean levels of miR-224 were lower in the non-responders compared to responders. These results suggest that miR-9, -221, -222 and -224 may be potential biomarkers of trastuzumab resistance. 1. Gullo, G., et al., Durable complete response following chemotherapy and trastuzumab for metastatic HER2-positive breast cancer. Annals of Oncology, 2012. Citation Format: Karen Howe, Alex J. Eustace, Giuseppe Gullo, Brigid Browne, Sinead Aherne, John Crown, Norma O'Donovan. Altered expression of miRNAs in acquired trastuzumab resistance. [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 1826. doi:10.1158/1538-7445.AM2014-1826

Abstract 1827: Inhibition of Src, HER2, and EGFR by the multi-kinase inhibitor KD019 overcomes trastuzumab resistance in breast cancer

Abstract KD019 (previously EXEL-7647) is an orally bioavailable spectrum selective small molecule kinase inhibitor of Src family nonreceptor tyrosine kinases (Src) as well as HER2, EGFR, and VEGFR2/KDR. Because of its complement of targets KD019 is able to disrupt multiple components of tumor pathology and, in earlier studies, KD019 demonstrated robust activity in multiple in vivo tumor models. In recent years Src has emerged as a critical convergent node downstream of multiple signaling cascades initiated by transmembrane receptor tyrosine kinases (RTKs), most notably HER2, EGFR and IGF-1R. Crosstalk between Src and RTKs facilitates RTK-dependent tumor growth and, in patients with HER2-amplified (HER2+) breast cancer, where Src is upregulated, may confer resistance to anti-HER2 therapies. Based on the target specificity of KD019, including Src, we hypothesized that KD019 may prove to be an effective treatment in overcoming trastuzumab resistance in HER2+ breast cancer. Here we present data showing that KD019 effectively inhibited growth of several trastuzumab resistant cell lines with IC50 of 0.3 uM in vitro. Not only did KD019 potently inhibit HER2 and EGFR phosphorylation in these cells, but it also efficiently inhibited the phosphorylation of c-Met through receptor hetero-oligomerization mechanism, thus downregulating another pathway by which tumors may escape HER2 targeted therapies. Further, we demonstrate that once daily dosing of KD019 strongly inhibited tumor growth in xenograft models of trastuzumab resistance and that Src activity was inhibited in KD019 treated tumors even 24 hrs after drug administration. The significance of inhibiting the specific combination of targets is underscored by the fact that KD019 was 10 fold more potent when compared to saracatinib (a selective Src inhibitor) in inhibiting growth of trastuzumab resistant cells, which are also insensitive to blockade of individual RTKs. Together, these results provide a rational for clinical investigation of KD019 in patients whose tumors are resistant to anti-HER2 therapies. Citation Format: Rigen Mo, Nishta Rao, James Tonra, Samuel Waksal, Masha Poyurovsky. Inhibition of Src, HER2, and EGFR by the multi-kinase inhibitor KD019 overcomes trastuzumab resistance in 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 1827. doi:10.1158/1538-7445.AM2014-1827

Abstract 4244: Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 cells

Abstract Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Increased levels of HER2 are linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity and dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor(IGF1R) in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the IGF1R, insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). Previous findings in our lab demonstrate an increased expression of IGF2 in trastuzumab-resistance cell line JIMT1 when compared to trastuzumab-sensitive cell lines BT474, SKBR3, and ZR75-30. We hypothesis that to overcome trastuzumab-resistance in JIMT1 cell lines, dual inhibition of HER2 and IGF2 is necessary to induce cell death. When JIMT1 cells were treated with resveratrol, both HER2 and IGF2 mRNA and protein levels were reduced and a decrease in cell viability was observed. We also analyzed the phosphorylation levels of HER2 and IGF1R. Our studies suggest that blocking both HER2 and IGF2 is necessary to decrease cell viability in trastuzumab-resistant JIMT1 cells. Citation Format: Xousaen M. Helu, Daisy De Leon. Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 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 4244. doi:10.1158/1538-7445.AM2014-4244

Abstract 4661: The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer

Abstract Fibronectin (FN) is a glycoprotein of the extracellular matrix and plays a major role in cell adhesion, migration and oncogenic transformation. Aberrant FN expression is associated with poor prognosis in a variety of cancers including breast cancer. However, the regulatory mechanism between FN and HER2 is not fully elucidated in breast cancer. Our clinical data showed that both disease free survival (DFS) and overall survival (OS) was lower in EGFR (+) and HER2 (+) breast cancer patients than in EGFR (+) breast cancer patients. To verify the causing of poor prognosis, we analyzed secreted proteins in EGFR and/or HER2 overexpressed MDA-MB231 cells. Our results showed that the level of FN expression was significantly increased in HER2 overexpressed MDA-MB231 cells. On the other hand, the induction of FN by HER2 overexpression was decreased by trastuzumab (HER2 monoclonal antibody) and AG825 (HER2 inhibitor), respectively. In addition, we observed that the expression of FN was dose-dependently increased by EGFR ligands in SKBR3 cells which are expressed endogenous EGFR and HER2. EGF ligands-induced FN expression also decreased through the suppression of HER2 in SKBR3 cell. However, these effects did not detect in trastuzumab-resistant breast cancer cell line, JIMT-1. Taken together, we demonstrated that the induction of FN by HER2 correlates poor clinical outcome in breast cancer patients. Citation Format: Myeongjin Jeon. The regulatory mechanism of fibronectin in EGFR and/or HER2 positive 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 4661. doi:10.1158/1538-7445.AM2014-4661