HER2-positive Human Breast Cancer Research Articles

The role of autophagy in the cytotoxicity induced by trastuzumab emtansine (T-DM1) in HER2-positive breast cancer cells

Trastuzumab emtansine (T-DM1), an antibody–drug conjugate (ADC) of trastuzumab and cytotoxic agent emtansine (DM1), has been approved for the therapy of metastatic HER2-positive breast cancer after prior treatment of trastuzumab and taxane. The impressive efficacy exhibited by T-DM1 has heightened the need for more further studies on the underlying mechanisms of T-DM1 cytotoxicity. Previous research suggested that autophagy was crucial for cancer therapy, but the role of autophagy in T-DM1 treatment has not been investigated. Here, we demonstrated for the first time that T-DM1 triggered obvious autophagy in HER2-positive SK-BR-3 and BT-474 breast cancer cells. Blocking autophagy with pharmacological inhibitors chloroquine (CQ) or LY294002 partly reduced T-DM1-induced apoptosis and Caspase-3/7 activation, suggesting that autophagy played an essential role in the cytotoxicity induced by T-DM1 in HER2-positive breast cancer cells. Further investigation demonstrated that Akt/mTOR signaling pathway was involved in T-DM1-induced autophagy in a time-dependent manner. Altogether, our results highlighted the important role of autophagy as a novel mechanism for T-DM1-induced cytotoxicity and elucidated the critical relationships between T-DM1-induced autophagy and apoptosis in human HER2-positive breast cancer cells, which provides novel insight into the underlying anti-tumor mechanism of T-DM1.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

6 α-Hydroxy-4[14], 10[15]-guainadien-8β, 12-olide induced cell cycle arrest via modulation of EMT and Wnt/β-catenin pathway in HER-2 positive breast cancer cells

Cyathocline purpurea has potential biological activities and has been widely used in traditional Chinese and Ayurvedic medicine. The aim of the present study is to elucidate the anticancer effect of its 6 α-hydroxy-4[14], 10[15]-guainadien-8β, 12-olide (SRCP1) against HER-2 positive subtype of breast carcinoma. Anticancer effect of SRCP1 was assessed by cell viability, senescence, apoptosis, cell cycle, DNA synthesis, and gene expression assays. The activity was further validated by the molecular docking study. SRCP1 inhibits human HER-2 positive breast cancer growth via inhibition of DNA synthesis in a dose-dependent manner. SRCP1 induces cell cycle arrest at G2/M phase, late apoptosis, and necrosis. Further, it induces senescence causing reduction in migration via down-regulation of EMT. A remarkable increase in the number of necrotic cells and Annexin-V staining revealed that exposure to SRCP1 triggers late apoptosis. Treatment with SRCP1 increased E-cadherin, p21, p53, ER-α expression and decreased β-catenin, MMP-9, snail1, TNF-α expression. SRCP1 showed binding affinity towards an active site of the HER-2 receptor. Our results of molecular docking and biological assays demonstrated the potent anticancer activity of SRCP1 in MDA-MB-453 cells via multiple pathways including EMT, TNF-α, and Wnt/β-catenin signaling.

Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis

BackgroundHuman epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib.MethodsTBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting.ResultsTBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy.ConclusionsThe TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

Cellular uptake, intracellular distribution and degradation of Her2-targeting silk nanospheres.

BackgroundThe development of nanocarrier technology has attracted great interest in the last decade. Biodegradable spheres made of functionalized silk have considerable potential to be used as drug delivery systems for cancer treatment. A targeting ligand displayed at the surface of a carrier, with a specific affinity towards a particular receptor, can further enhance the accumulation and uptake of nanoparticles at the site of a tumor.Materials and methodsThe hybrid constructs were obtained by adding a Her2-binding peptide (H2.1) to MS1 and MS2 bioengineered silks based on the MaSp1 and MaSp2 proteins from N. clavipes, respectively. The H2.1MS1 and H2.1MS2 proteins were blended at a weight ratio of 8:2. Stable silk particles were formed by mixing a soluble protein with potassium phosphate using a micromixing technique. We used specific inhibitors of endocytosis to determine the cellular uptake pathway of the silk nanoparticles in human Her2-positive breast cancer cells. The subcellular distribution of silk particles was investigated by evaluating the signal colocalization with organelle-specific tracker. Moreover, lysosomal and exosomal inhibitors were implemented to evaluate their impact on the silk spheres behavior and degradation.ResultsThe functionalized spheres were specifically taken up by Her2-positive cancer cells. Silk particles facilitated the entry into cells through both the clathrin- and caveola-dependent pathways of endocytosis. Upon entering the cells, the particles accumulated in the lysosomes, where intracellular degradation occurred.ConclusionsThe present study demonstrated directly that the lysosomal function was essential for silk-based carrier elimination. The degradation of the carrier is of great importance to develop an optimal drug delivery system.

The d16HER2 Splice Variant: A Friend or Foe of HER2-Positive Cancers?

Human epidermal growth factor receptor 2 (ERBB2 or HER2) amplification/overexpression is associated with a particularly aggressive molecular subtype of breast cancer (BC), characterized by a poor prognosis, increased metastatic potential, and disease recurrence. As only approximately 50% of HER2-positive patients respond to HER2-targeted therapies, greater knowledge of the biology of HER2 and the mechanisms that underlie drug susceptibility is needed to improve cure rates. Evidence suggests that the coexistence of full-length, wild-type HER2 (wtHER2) and altered forms of HER2—such as carboxy-terminus-truncated fragments, activating mutations, and splice variants—significantly increases the heterogeneity of HER2-positive disease, affecting its biology, clinical course, and treatment response. In particular, expression of the d16HER2 splice variant in human HER2-positive BC has a crucial pathobiological function, wherein the absence of sixteen amino acids from the extracellular domain induces the formation of stable and constitutively active HER2 homodimers on the tumor cell surface. Notably, the d16HER2 variant significantly influences the initiation and aggressiveness of tumors, cancer stem cell properties, epithelial–mesenchymal transition (EMT), and the susceptibility of HER2-positive BC cells to trastuzumab compared with its wtHER2 counterpart, thus constituting a novel and potentially clinically useful biomarker. The aims of this review are to summarize the existing evidence regarding the pathobiological functions of the d16HER2 variant and discuss its current and future value with regard to risk assessment and treatment choices in HER2-positive disease.

GRB7 dependent proliferation of basal-like, HER-2 positive human breast cancer cell lines is mediated in part by HER-1 signaling.

GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and overexpressed with HER-2 in human breast cancer. This study addresses the role of GRB7 in HER-2 positive human breast cancers resistant to HER-2 targeted therapy. HCC1954, 21MT1, and JIMT1 are basal like HER-2 positive breast cancer cell lines based on expression profiling. These three cell lines are resistant to trastuzumab and lapatinib treatment. Knockdown of GRB7 protein expression with siRNA transfection as well as lentiviral vector mediated shRNA over-expression decreased the growth of HCC1954, 21MT1, and JIMT1 cells in vitro and the growth of tumor xenografts these cells formed in animal models. When assayed by ki-67 staining and TUNEL assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC1954 cells and increased apoptosis in JIMT1 cells. Phospho-proteome profiling found HER-1 tyrosine phosphorylation was reduced with GRB7 knock down in JIMT1 cells. Immuno-blotting and immuno-precipitation experiments found HER-1 phosphorylation was reduced with GRB7 knock down in all three cell lines. HER-1 knock down via siRNA transient transfection as well as blocking HER-1 function with panitumumab decreased proliferation of all three cell lines in vitro. Our study finds that GRB7 has an essential growth promoting function which is mediated in part by HER-1 activation. The potential of HER-1 targeting in therapy resistant HER-2 positive breast cancer merits further study.

Inhibition of ezrin causes PKCα-mediated internalization of erbb2/HER2 tyrosine kinase in breast cancer cells

Unlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKCα-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKCα-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface.

19 Autophagy induction as a new therapy for HER2+ breast tumorigenesis

IntroductionHER2 is a receptor tyrosine kinase whose gene is amplified in ~20% human breast cancer patients. Allelic loss of the autophagy gene, beclin 1/BECN1, is associated with HER2 amplification in breast cancer; low beclin 1 mRNA expression is associated with increased risk of HER2-positive breast cancer; and overexpressed HER2 and Beclin 1 interact in cultured cells. However, the functional significance of HER2/Beclin 1 interaction and of altered autophagy in HER2-driven tumorigenesis and whether autophagy induction may be beneficial in preventing HER2-positive breast tumour growth is unknown.Material and methodsWe explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects as well as genetic and pharmacological approaches to increase autophagy on HER2-driven breast cancer growth in vivo.Results and discussionsHere we show that endogenous HER2 interacts with Beclin 1 in multiple HER2 +breast cancer cells and inhibits autophagy. Mice with a knock-in mutation in Becn1 (Becn1F121A) that leads to increased basal autophagy are protected from mammary tumorigenesis when crossed with mammary-specific HER2 transgenic mice, and HER2 fails to inhibit autophagy in primary cells derived from these mice.Moreover, treatment of mice with HER2-positive human breast cancer xenografts with the Tat-Beclin 1 autophagy-inducing peptide inhibits tumour growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumour growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumours that is distinct from that observed with HER2 TKI treatment.ConclusionThese findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis. They also suggest that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.

Abstract 734: Thienoindoles: New highly promising agents for antibody-drug conjugates generation

Abstract Thienoindoles are a new proprietary class of highly potent DNA minor groove alkylating agents. This class is characterized by a fused thiophene ring whose intrinsic electron-withdrawing character provides a nearly optimal increase in stability and potency of alkylating subunits. Furthermore, the presence of a solubilizing moiety on the minor groove portion provides compounds with physicochemical properties highly compatible with deployment as antibody payloads. Extensive optimization of this class has led to the identification of the highly potent toxin NMS-P528 with sub-nanomolar IC50 in proliferation assay across a large panel of tumor cell lines. NMS-P945, a drug-linker generated from NMS-P528, is highly suited for conjugation with targeted antibodies, consistently yielding ADCs with favorable drug-antibody ratios in the absence of significant antibody aggregation and with full maintenance of antigen binding capability. Proof of concept efficacy and mechanism of action studies were performed using NMS-P945 conjugated with trastuzumab, observing for the resulting conjugate excellent in vitro target-directed cytotoxicity in HER2-positive vs. negative cancer cell lines. In animal efficacy studies against HER2-positive human breast cancer tumors, trastuzumab-NMS-P945 ADC administration yielded complete tumor regression with no effects on body weight gain, while unarmed trastuzumab and armed control antibody showed little and no effect, respectively. PK data indicated long plasma half-life of our novel ADCs in the monkey. Promising results were also obtained with a range of different antibody/antigen systems, e.g. with an antibody directed to the ALK receptor tyrosine kinase. Extensive process research has been completed for the production of NMS-P528 and NMS-P945, resulting in an efficient stereoselective method for the preparation of GMP materials on the 100g scale and both products are proposed as a licensing opportunity for conjugation with tumor-targeting antibodies. Citation Format: Michele Caruso, Fabio Gasparri, Barbara Valsasina, Clara Albanese, Italo Beria, Ilaria Candiani, Marina Ciomei, Nicoletta Colombo, Sabrina Cribioli, Ulisse Cucchi, Eduard Felder, Ivan Fraietta, Arturo Galvani, Antonella Isacchi, Aurelio Marsiglio, Paolo Orsini, Rita Perego, Simona Rizzi, Attilio Tomasi, Sonia Troiani, Carlo Visco, Daniele Donati. Thienoindoles: New highly promising agents for antibody-drug conjugates generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 734.

Abstract 2151: Development of small-molecule mTORC1 radiotracers for evaluation of targeted therapy resistance using PET

Abstract Overcoming resistance is an area of unmet clinical need for patients undergoing trastuzumab-based HER2-directed therapy. PTEN loss or PI3K mutation has been shown to induce mTOR activation and mediate trastuzumab resistance. During the BOLERO-3 phase III clinical trial, the addition of everolimus to trastuzumab plus vinorelbine significantly prolonged progression-free survival in patients with trastuzumab-resistant and taxane-pretreated, HER2-positive advanced breast cancer. It is critically important to identify HER2-positive breast cancer patients who are sensitive to mTOR inhibition. Our aim is to develop noninvasive mTORC1 binding small-molecule imaging probes to predict breast cancer patient resistance to treatment utilizing positron emission tomography (PET). Utilizing the benzofuran-based compound ChemBridge 5219657, we have developed a panel of potent inhibitors specific for mTOR complex 1. Alkyne derivatives suitable for conjugation with 2-[18F]fluoroethylazide were developed via Cu(I)-catalyzed click chemistry. Radiolabeling was performed on the automated iPhase FlexLab module providing reliable amounts of radiotracer. Synthesis of click precursor was achieved over 5 linear steps with a 24% isolated yield. Quality control was characterized using NMR, MS, UV- and radio-HPLC analysis. 18F-labeling conjugation with precursor resulted in a 40 ± 5% isolated radiochemical yield with >99% radiochemical purity and a molar activity of 12.5 ± 6.0 GBq/µmol. Initial in vitro sensitivity to everolimus was performed via SRB proliferation analysis following cold-compound treatment on HER2-positive human breast cancer cell lines (BT-474 and HCC1419) and HER2 low expressing control cell lines (MDA-MB-468 and MDA-MB-231). In vitro 18F-labeled binding experiments demonstrated an increased uptake in BT-474 and HCC-1419 cells, 1.5 and 2.5-fold compared to the negative controls, respectively. NSG mice bearing BT-474, MDA-MB-231 and MDA-MB-468 xenografts were explored for in vivo dynamic PET imaging experiments confirming this selective uptake of radiotracer in everolimus-sensitive cells. Our synthesis strategies identified a reliable radiolabeling approach capable of producing sufficient amounts of radiochemically pure radiotracer for in vivo investigation. The increased tracer uptake in mTOR treatment-sensitive cell lines in vitro as well as in vivo provides an encouraging outcome warranting further preclinical studies for the investigation of HER2-positive therapy resistance in mTOR-sensitive models. Citation Format: Adam C. Parslow, Christian W. Wichmann, Angela Rigopoulos, Uwe Ackermann, Jonathan M. White, Andrew M. Scott. Development of small-molecule mTORC1 radiotracers for evaluation of targeted therapy resistance using PET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2151.

Abstract 3092: Redirection of human HER2-positive breast cancer cells

Abstract Breast cancer is the most common form of cancer among women, affecting one eigth of the female population. While advances in clinical management and therapeutic modalities have been achieved, serious limitations in the improvement of outcomes after the diagnosis remain, largely due to the complexity of the underlying biology. Studying the mechanism that makes cancer cells tumorigenic and how they can lose the ability to cause malignant formations is the first step towards understanding complex mechanisms of this disease. The normal mammary microenvironment can redirect cells to assume a normal mammary epithelial phenotype. This phenomenon also applies to tumorigenic cells; cancer cells lose their tumor-forming capacity and differentiate, assuming a normal, non-tumorigenic phenotype. This phenomenon has been termed “cancer cell redirection.” Comparing cells that went through this transformation with original cancer cells, features that identify tumorigenic activity can be acknowledged. We have established an in vitro model of cancer cell redirection, in which HER2+ breast cancer cells are grown alone or in co-culture with normal human breast epithelial cells. The ratios of cells used in co-cultures, 1:1 and 1:50 (cancer: normal) were previously established through in vivo and in vitro studies. Redirection was confirmed by immunostaining. The metric used to determine cancer cell redirection was the absence of receptor tyrosine kinase phosphorylation without a change in surface expression of the receptor. HER2 and P-HER2 expression were both present in the HER2+ cancer cells and in the: MCF10A and 1:1 co-cultures. However, HER2 expression was observed in 1:50 co-cultures, but an absence of P-HER2 was noted. Based on attenuation of receptor phosphorylation in the 1:50 co-cultures we can confirm the breast cancer cell redirection, which matches our previous results. Furthermore, alteration of HER2 dimerization patterns in redirected cells were investigated. The capacity of cancer and redirected cells to form tumors was examined with use of mammosphere assay. Formation of mammospheres has correlated to stem cell function and tumor-initiation capacity. The HER2+ breast cancer cell lines formed significantly higher numbers of spheres compared to the non-tumorigenic breast epithelial cells. Mammosphere formation is decreased in redirected cancer cells. Breast cancer stem cells are HER2+ regardless of pathological or genetic classification. This suggests that HER2 expression is not only a target in HER2+ breast cancer but should be targeted in all breast cancers to prevent breast cancer stem cells from recapitulating tumors following treatment. Our data demonstrates that by attenuating activity of HER2 theses cancer cells lose the capacity to form tumors in vivo and lose their cancer phenotype in vitro. Citation Format: Anastasia Frank-Kamenetskii, Julia M. Mook, Brian W. Booth. Redirection of human HER2-positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3092.

Synergistic antitumor activity by combining trastuzumab with retinoic acid in HER2 positive human breast cancer cells.

Breast cancer can be classified into molecular subtypes. Tumors overexpressing HER2 protein are more aggressive and metastatic; hence, patients have a poor prognosis. Anti-HER2 strategies, such as the monoclonal antibody Trastuzumab (Tz), have therefore been developed. Despite this progress, not all patients respond to the treatment. Retinoic acid (RA) has been proposed as an adjuvant treatment of breast carcinoma because of its ability to inhibit cell growth. We evaluated the effect of Tz in combination with RA on the viability, adhesion, migration, invasion and expression of migration-related proteins in SKBR3 and BT-474 human breast cancer cells. MTT, pharmacological interaction analysis, immunofluorescence, adhesion/migration/invasion and Western blot assays were performed. The coadministration of both drugs synergistically decreased cell survival. Tz+RA significantly decreased adhesion/migration/invasion in both cell types. Tz+RA strongly reduced FAK and HER2 expression and induced nuclear FAK translocation. In addition, a granular distribution of HER2 receptor was observed after the combined treatment. In conclusion, the coadministration of both drugs in patients with this type of cancer could contribute to the improvement of their prognosis and reduce the adverse effects of therapy because the applied Tz doses would be lower due to the adjuvant effect of RA.

Increased autophagy blocks HER2-mediated breast tumorigenesis

Allelic loss of the autophagy gene, beclin 1/BECN1, increases the risk of patients developing aggressive, including human epidermal growth factor receptor 2 (HER2)-positive, breast cancers; however, it is not known whether autophagy induction may be beneficial in preventing HER2-positive breast tumor growth. We explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects of genetic and pharmacological strategies to increase autophagy on HER2-driven breast cancer growth in vivo. Our findings demonstrate that HER2 interacts with Beclin 1 in breast cancer cells and inhibits autophagy. Mice with increased basal autophagy due to a genetically engineered mutation in Becn1 are protected from HER2-driven mammary tumorigenesis, and HER2 fails to inhibit autophagy in primary cells derived from these mice. Moreover, treatment of mice with HER2-positive human breast cancer xenografts with the Tat-Beclin 1 autophagy-inducing peptide inhibits tumor growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumor growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumors distinct from that observed with HER2 TKI treatment. Taken together, these findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis and that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.

Abstract P2-08-03: An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models

Abstract Background- GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and over-expressed with HER-2 in human breast cancer. Earlier studies found a functional role of GRB7 in breast cancer. The role of GRB7 in HER-2 positive human breast cancer resistant to HER-2 targeted therapy remains unexplored however. Materials and Methods- HCC-1954, 21MT1 and JimT1 are human HER-2 positive breast cancer cell lines that are resistant to trastuzumab and lapatinib treatment. Transient knock down of GRB7 protein expression was achieved with siRNA transfection and stable knock down with lentiviral vector mediated shRNA over-expression. Cell lines transfected with non-targeting siRNA or shRNA serve as negative controls. Knock down of GRB7 protein expression is verified by Western blotting. The growth of human breast cancer cell lines after GRB7 knock down in vitro is measured with the CellTiter Glo assay as well as the Incucyte live cell imaging. Activation status of specific signaling pathways was examined with phospho-specific antibody by immune-blotting and immune-precipitation. To assess the growth promoting function of GRB7 in human breast cancer cell lines in vivo, polyclonal HCC-1954, 21MT1 and JimT1 cells, with GRB7 knock down or their corresponding negative control, were orthotopically injected into the mammary fat pads of female immune-deficient NSG mice. The growth rates of these tumors, measured serially with caliper, and final tumor weights were compared between GRB7 knock down and the negative control. The proliferation rate and apoptosis of these tumors were studied with ki-67 staining and Tunel assay.The effects of GRB7 knock down on signaling were investigated with a proteome profiler receptor tyrosine kinase kit (R&D). The role of signaling molecules differentially activated in the growth of breast cancer cells by GRB7 knock down was examined utilizing siRNA mediated knock down, and antibody and small molecule inhibitors. Results- GRB7 knock down decreased the growth of HCC-1954, 21MT1 and JimT1 cells in vitro and the growth of tumor xenograft these cells formed in animal models. When assayed by ki67 staining and Tunel assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC-1954 cells and increased apoptosis in JimT1 cells. Protein profiling found that tyrosine phosphorylation of candidate signaling molecules was reduced with GRB7 knock down in JimT1 cells. Immuno-blotting and immuno-precipitation experiments were performed to evaluate these effects in other cell lines. The effect of targeting these molecules in breast cancer cell growth by siRNA and inhibitors is being examined. Discussion- GRB7 has essential growth promoting function in therapy resistant HER-2 positive human breast cancer cells. GRB7 knock down has pleiotropic effects on signaling in various cellular contexts. The potential of targeting GRB7 signaling in treating therapy resistant HER-2 positive breast cancer merits further study. Citation Format: Luoh S-W, Wagoner W, Lai X, Hu Z, Chin K, Ramsey B. An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-03.

Doxorubicin conjugated with a trastuzumab epitope and an MMP-2 sensitive peptide linker for the treatment of HER2-positive breast cancer

HER2-positive breast cancer correlates with more aggressive tumor growth, a poorer prognosis and reduced overall survival. Currently, trastuzumab (Herceptin), which is an anti-HER2 antibody, is one of the key drugs. There is evidence indicating that conjugation of trastuzumab with chemotherapy drugs, such as doxorubicin (DOX), for multiple targets could be more effective. However, incomplete penetration into tumors has been noted for those conjugates. Compared to an antibody, peptides may represent an attractive alternative. For HER2, a similar potency has been observed for a 12-amino-acid anti-HER2 peptide mimetic YCDGFYACYMDV-NH2 (AHNP, disulfide-bridged) and full-length trastuzumab. Thus, a peptide, GPLGLAGDDYCDGFYACYMDV-NH2, which consists of AHNP and an MMP-2 cleavable linker GPLGLAGDD, was first designed, followed by conjugation with DOX via a glycine residue at the N-terminus to form a novel DOX-peptide conjugate MAHNP-DOX. Using HER2-positive human breast cancer cells BT474 and SKBR3 as in vitro model systems and nude mice with BT474 xenografts as an in vivo model, this conjugate was comprehensively characterized, and its efficacy was evaluated and compared with that of free DOX. As a result, MAHNP-DOX demonstrated a much lower in vitro IC50, and its in vivo extent of inhibition in mice was more evident. During this process, enzymatic cleavage of MAHNP-DOX is critical for its activation and cellular uptake. In addition, a synergistic response was observed after the combination of DOX and AHNP. This effect was probably due to the involvement of AHNP in the PI3K–AKT signaling pathway, which can be largely activated by DOX and leads to anti-apoptotic signals.

Defective Cyclin B1 Induction in Trastuzumab-emtansine (T-DM1) Acquired Resistance in HER2-positive Breast Cancer.

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006-19. ©2017 AACR.

Mode of action of pertuzumab in combination with trastuzumab plus docetaxel therapy in a HER2-positive breast cancer xenograft model.

In a Phase III trial for HER2-positive breast cancer (the CLEOPATRA study), the triple-drug combination arm of pertuzumab plus trastuzumab plus docetaxel showed significantly longer progression-free survival and overall survival than did the trastuzumab plus docetaxel arm. In this study, we investigated the mechanism of action of the triple-drug combination therapy in vivo. For this purpose, we established a mouse xenograft model using KPL-4, a HER2-positive human breast cancer cell line, in which the triple-drug combination treatment dramatically induced tumor regression compared with double-drug combinations (trastuzumab plus docetaxel, pertuzumab plus docetaxel, or pertuzumab plus trastuzumab). Four days after the triple-drug treatment was started, strong reduction in the phosphorylation of HER2, epidermal growth factor receptor (EGFR), HER3, extracellular signal-regulated kinase (ERK), and AKT in tumor tissues was seen, despite only weak suppression of phosphorylation seen with the single- or double-drug treatments. Histopathological analysis and flow cytometric analysis showed that the triple-drug treatment enhanced apoptosis after mitotic arrest induced by docetaxel. Furthermore, infiltration of mononuclear cells around the tumor cells was strongly induced by the triple-drug combination treatment. These results suggested that the mechanism underlying the synergistic efficacy of the triple-drug combination was attributable, at least in part, to the docetaxel-mediated apoptosis being promoted by enhanced inhibition of HER2-HER3-AKT signaling as well to the intratumor infiltration of mononuclear cells induced by anti-HER2 antibodies being enhanced by docetaxel.

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.

Abstract P2-04-10: High-affinity activated natural killer (haNK) cells augment trastuzumab efficacy in a mouse model of HER2-positive human metastatic breast cancer

Abstract Background. The ability of NK cells to kill cancer cells makes them an attractive choice for clinical immunotherapy. Early phase clinical trials in patients (pts) with advanced cancers have demonstrated the safety of activated NK (aNK [NK-92]) cells, an investigational cell line that was established from the peripheral blood mononuclear cells of a pt with non-Hodgkin lymphoma. NK cells can participate in antibody-dependent cellular cytotoxicity (ADCC) mediated by recognition of the Fc fragment of the target-bound antibody (IgG1) via the CD16 Fc receptor. Among pts with HER2-positive breast cancer treated with trastuzumab (IgG1) the high-affinity CD16 V/V genotype was significantly correlated with better clinical outcomes (Musolino. JCO. 2008;26:1789-96). To enhance the killing activity of aNK cells, we modified aNK cells to stably express high-affinity CD16 and evaluated the resulting haNK cells in combination with trastuzumab in a mouse xenograft model of HER2-positive human breast cancer. Methods. haNK cells were generated by transfection of aNK cells with a bicistronic plasmid coding for CD16 (158V) and an intracellular form of IL-2, which enables haNK cells to grow in the absence of exogenous IL-2. Female, 7 to 8-week-old NOD-scid IL2Rgammanull (NSG) mice were inoculated subcutaneously in the left and right flank area with 0.1mL of 1x108/mL MDA-MB-453 human breast cancer cells in 50% Matrigel. When tumors reached ≥100mm3, mice were randomly assigned to 10 groups of 4 mice per group and dosed (IV) with PBS, 1 or 3mg/kg IgG1 isotype control, 1 or 3mg/kg trastuzumab (determined from a dose range finding study), 1x107 non-irradiated haNK cells, or non-irradiated haNK cells in combination with IgG1 or trastuzumab. The dosing schedules were: PBS/haNK cells twice weekly for 4 weeks; IgG1/trastuzumab once weekly for 4 weeks. For the combination treatments, mice received antibodies at least 3h prior to the injection of haNK cells. Tumor growth and animal weights were measured twice weekly. Results: Results obtained after 4 weeks of treatment are shown in the table. haNK alone and both doses of trastuzumab alone significantly inhibited the growth of human MDA-MB-453 breast tumors. The combination of haNK plus 1mg/kg trastuzumab was synergistic with a T/C value of -60.1%. Conclusions: The combination of haNK cells and trastuzumab was synergistic at 1mg/kg trastuzumab resulting in tumor regressions and significantly better efficacy vs each agent alone. Trastuzumab monotherapy at 3mg/kg was very effective and likely masked any synergistic effect of haNK. This study illustrates the potential for combining haNK cells with trastuzumab in a clinical trial of pts with HER2-positive metastatic breast cancer. ResultsGroupTreatmentT/C (%)BWC (%)APBS--0.6BIgG1 (1mg/kg)40.8-4.7CTrastuzumab (1mg/kg)-34.5-2.8DhaNK (1x107)-20.3-15.9EIgG1 (1mg/kg) + haNK (1x107)-10.7-16.0FTrastuzumab (1mg/kg) + haNK (1x107)-60.1-16.6GIgG1 (3mg/kg)39.1-0.4HTrastuzumab (3mg/kg)-95.2-1.8IIgG1 (3mg/kg)+ haNK (1x107)-26.4-16.7JTrastuzumab (3mg/kg) + haNK (1x107)-93.8-18.3%T/C, percent treated/control; BWC, body weight change. Citation Format: Rabizadeh S, Zhou L, Toneguzzo F, Boissel L, Soon-Shiong P, Niazi K, Klingemann H. High-affinity activated natural killer (haNK) cells augment trastuzumab efficacy in a mouse model of HER2-positive human metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-10.