Mouse Model Of HER2 Research Articles

Mouse Models of HER2+ Human Breast Cancer: A Literature Review

Introduction: HER2+ breast cancer (BC) makes up 15-20% of BC cases, where HER2 overexpression drives BC progression via proliferative signals. Targeted therapies inhibit HER2's activity but face challenges like resistance and metastasis. Thus, HER2+ BC is a developing research area where many preclinical studies are being conducted, and mice are often used due to their genetic and physiological similarities to humans. This study aims to review and compare existing mouse models to determine the best model of HER2+ BC. Methods: A literature search on PubMed in February 2024 using search terms including HER2, neu, neuNT, MMTV, and mammary tumors identified 16 primary research articles that used Neu-overexpressing mice. The papers were categorized under five models: MMTV-Neu, MMTV-NeuNT, FloxNeo-NeuNT, MMTV-NIC, and MMTV-HER2. Results: Among examined studies, Andrechek et al.'s FloxNeo-NeuNT model had the longest average tumor onset at 447 days, while Kuang et al.'s MMTV-NIC had the shortest at 126 days. Ursini-Segal et al.'s MMTV-NIC mice showed 100% mammary tumor incidence. Most models resulted in mammary adenocarcinomas, with lung metastases commonly observed, especially in papers that used MMTV-NIC. Discussion: Human HER2+ BC is commonly adenocarcinoma and often metastasizes to lungs, bones, liver, and brain. The mouse models were also found to be adenocarcinomas, but they primarily showed lung metastasis, possibly due to insufficient tumor detection methods for brain and bone metastasis. Neu copy numbers in Andrechek et al.’s FloxNeo-NeuNT model also align with findings on HER2 copy number amplification in human HER2+ BC. Conclusion: We have found that MMTV-NIC is the optimal mouse model for HER2+ BC research due to its short latency, 100% tumor incidence, and high rate of lung metastasis.

Abstract 3380: HER2 overexpression induces breast tumorigenesis in a non cell autonomous manner by inducing oxidative stress in the tissue microenvironment

Abstract Breast cancer is the most common cancer type in the world. Approximately 70% of pre-malignant breast tumours, known as ductal carcinoma in situ (DCIS), and 20% of invasive breast tumours overexpress HER2. Although molecular mechanisms of HER2 function in breast tumour cells have been extensively studied, our knowledge on how HER2 drives breast tumourigenesis at its pre-malignant stage is quite limited. Here, we investigate the HER2-overexpression driven cellular events at the earliest stages of breast tumourigenesis using in vitro, in vivo and ex vivo models. Using mouse models of HER2+ breast cancer and clinical samples of patients with DCIS, we showed that breast cancer stem cells (BCSCs) reside in HER2-negative population of tumour cells and arise from a HER2-negative cell-of-origin. Proteomics and metabolomics analyses of mammary ducts isolated from the MMTV-Neu mice at the pre-cancerous stage showed that HER2/Neu overexpression alters the energy metabolism in mammary epithelial cells in vivo. Furthermore, we have observed an accumulation of reactive oxygen species levels in pre-cancerous mammary ducts, which consequently causes oxidative stress induction and DNA damage in both HER2-positive and HER2-negative epithelial cells. Collectively, our findings indicate that HER2 overexpression in normal mammary epithelia may lead to a genotoxic tissue microenvironment that may consequently result in the accumulation of further somatic mutations in HER2-negative cell populations, which contain BCSCs and their cell-of-origin. Our findings therefore illustrate a novel mechanism explaining the HER2-driven breast tumour initiation and thus provide new avenues of research for developing novel preventive approaches against breast cancer. Citation Format: Sevim Beyza Gurler, Oliver Wagstaff, Lili Dimitrova, Fuhui Chen, Robert Pedley, William Weston, Ian Donaldson, Brian A. Telfer, David Novo, Kyriaki Pavlou, George Taylor, Yaqing Ou, Kaye Williams, Andrew Gilmore, Keith Brennan, Ahmet Ucar. HER2 overexpression induces breast tumorigenesis in a non cell autonomous manner by inducing oxidative stress in the tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3380.

Abstract 6939: Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program

Abstract Background: In HER2+ breast cancer (BC), residual disease (RD) after neoadjuvant HER2 inhibition is associated with increased risk of recurrence. However, the genetic and non-genetic resistance mechanisms underlying RD remain incompletely characterized. Here we used single nuclei (sn) RNA and DNA sequencing to profile genetic and transcriptional changes in growing, residual, and recurrent tumors from an inducible mouse tumor model of HER2+ breast cancer. Methods: We used mammary tumors/tumor bed (n=2-3/time point) from female MMTV-rtTA; TetO-ERBB2 mice at 9 weeks DoxOn (HER2on), 7 days DoxOff (HER2off) and 12-21 weeks Doxoff (recurrent). Samples were snap frozen and single nuclei isolation was performed. snRNAseq was performed using 10 × 5’ v2 kits for library preparation and sequencing. snDNAseq was performed using a custom amplicon panel targeting the mouse genome and human ERBB2 locus via the MissionBio Tapestri platform. Results: For snRNAseq analysis, tumor cell clusters were identified by Epcam and ERBB2 gene expression. Tumor cells were further subclustered into 18 unique clusters that separated by ERBB2 expression. Seven subclusters of HER2on tumor cells were characterized by low ERRB2 expression: 1.38 vs 2.94 (median normalized log counts) in HER2on-ERBB2high cells. Top 5 differentially expressed genes (DEG) per condition (log2 fold change >1.5, adjusted p value < 0.05) were: HER2on: Erbb2, Csn3, Lcn2, Csn2, Wfdc18; HER2on-ERBB2high: Csn3, Igfbp5, Csn2, Lcn2, Trf; HER2on-ERBB2low: Lars2, Gm47283, Cmss1, Gm42418, Neat1; HER2off: Lars2, Hexb, Cmss1, Gphn, Gm42418; Recurrent: Capza2, Vim, Lgals1, Crabp1, S100a6. HER2on-ERBB2low cells were more closely related in transcriptional space to HER2off residual tumor cells than HER2on-ERBB2high tumor cells. Trajectory analysis suggested that HER2off RD evolved from HER2on-ERRB2low cells and formed the basis of recurrent disease. To determine clonal evolution over time, we used common top 10 DEGs between HER2on vs HER2off vs HER2low snRNAseq data and intersected that set with significantly enriched single nucleotide variants (SNVs) from our snDNAseq data. We identified 16 clones containing at least 1 SNV at >1% frequency across all samples. We observed clonal persistence, relative enrichment and extinguishment between HER2on, HER2off and recurrent samples but without clonal dominance. Conclusion: Time-series analysis of genetic and transcriptional evolution in a mouse model of HER2+ BC RD suggests that a tumor cell subpopulation characterized by an ERBB2low-associated transcriptional program drives RD after HER2 inhibition. Citation Format: Sheheryar K. Kabraji, Weihua Wang, Yali Zhang, Johann Bergholz, Tao Jiang, Vittal Kurisetty, Maia Homsi, Jianmin Wang, Jean Zhao. Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6939.

Abstract P6-12-05: The role of endothelial-specific AXL and its associated signaling pathways in the tumor microenvironment

Abstract Most breast cancer deaths result from the development of metastases. The complex tumor microenvironment provides signals that can instruct both breast cancer cells to invade and tumor blood vessels to be leaky, hence supporting metastasis. We identified the receptor tyrosine kinase (RTK) AXL to be essential for metastasis. Genetic ablation of Axl in mouse models of HER2+ breast cancer, either globally or specifically in mammary epithelial cells, blunts metastasis but not primary tumor growth. We found that Axl expressed in tumor cells contributes to the remodeling of the tumor microenvironment, including immune cell recruitment and abnormal blood vessels. Hence, AXL in epithelial cells promotes cell invasion and shapes a pro-metastatic microenvironment that would be poorly responding to treatments such as immunotherapy. While AXL is known to be expressed on endothelial cells, its endothelial function has yet to be clearly defined. This project aims to address the hypothesis that AXL expressed on endothelial cells promotes processes that lead to abnormal blood vessel formation to promote metastasis. To address this hypothesis, we first studied the specific intracellular interactions between two RTKs, AXL and the known pro-angiogenic receptor VEGFR. Our preliminary analyses indicates that GAS6 can not only induce the phosphorylation of the permeability marker eNOS on its own but can also potentiate the VEGF’s response. The index of linearity of the endothelial tight junctions upon GAS6 stimulation show increased permeability of the endothelial cells. To further characterize the signaling pathways controlled by these receptors, we plan to perform a phosphoproteomics screen. We will focus on studying the AXL+VEGFR phosphoproteome and define the signaling pathways that impact vascular permeability. The most interesting candidate(s) will be studied in the context of mouse tumor models. In parallel, we are generating a conditional deletion of Axl in endothelial cells by crossing Axlfx/fx mice with Pdgfb:iCreER animals. With this, we will study the specific endothelial effects of Axl in vivo, specifically with vessel permeability and retinal angiogenesis assays. Multiple anti-angiogenic therapies for cancer failed in the clinic highlighting the need for new strategies to target endothelial cells in cancer. This project will provide essential information on the role of endothelial specific AXL, in the context of the HER2 breast cancer, and of its downstream effectors. With more specific targets, anti-angiogenic therapies could decrease their off-target effects and increase their efficacy in the clinic. This project has the potential to uncover therapeutic targets that could reduce the metastatic burden in breast cancer patients, and therefore better the prognostic for the HER2 cancer subtype. Citation Format: Andréane Lalonde, Jean-François Côté. The role of endothelial-specific AXL and its associated signaling pathways in the tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-05.

HER2-driven breast cancer suppression by the JNK signaling pathway

The HER2+ subtype of human breast cancer is associated with the malignant transformation of luminal ductal cells of the mammary epithelium. The sequence analysis of tumor DNA identifies loss of function mutations and deletions of the MAP2K4 and MAP2K7 genes that encode direct activators of the JUN NH2-terminal kinase (JNK). We report that invitro studies of human mammary epithelial cells with CRISPR-induced mutations in the MAPK and MAP2K components of the JNK pathway caused no change in growth in 2D culture, but these mutations promoted epithelial cell proliferation in 3D culture. Analysis of gene expression signatures in 3D culture demonstrated similar changes caused by HER2 activation and JNK pathway loss. The mechanism of signal transduction cross-talk may be mediated, in part, by JNK-suppressed expression of integrin α6β4 that binds HER2 and amplifies HER2 signaling. These data suggest that HER2 activation and JNK pathway loss may synergize to promote breast cancer. To test this hypothesis, we performed invivo studies using a mouse model of HER2+ breast cancer with Cre/loxP-mediated ablation of genes encoding JNK (Mapk8 and Mapk9) and the MAP2K (Map2k4 and Map2k7) that activate JNK in mammary epithelial cells. Kaplan-Meier analysis of tumor development demonstrated that JNK pathway deficiency promotes HER2+-driven breast cancer. Collectively, these data identify JNK pathway genes as potential suppressors for HER2+ breast cancer.

BSCI-17 TOPIRAMATE DECREASES RADIATION-INDUCED CYTOTOXIC EDEMA IN HER2+ BRAIN METASTASES VIA AQUAPORIN 4 INHIBITION

Abstract Brain metastasis (BM) occurs in 30-40 % of breast cancer patients with Her2+ tumors, and radiation is part of the standard treatment for BM. About 10% of BM patients treated with radiation develop brain edema. We have shown that combination of Ado-trastuzumab Emtansine (T-DM1)-the main targeted therapy for metastatic Her2+ Breast Cancer- and radiation increases the risk of developing radionecrosis by 13.5-fold (Stump et al., 2019). We also showed that T-DM1 enhances radiation-induced astrocytic toxicity and cytotoxic edema through upregulation of aquaporin-4 water-transporter (AQP4). Here, we determined whether blockage of AQP4 would prevent astrocytic swelling –cytotoxic edema- in vitro and in vivo models of Her2+ BM. Results: Electron microscopy of brain cortex from mice treated with 35 Gy (single dose), showed acute astrocytic end-feet swelling and a significant increase in AQP4 expression compared with non-irradiated mice. Consistent with prior findings in murine astrocytes, primary human astrocytes (huAST) also upregulated AQP4 levels 24 h post-radiation (8 Gy), and T-DM1 treatment exacerbated this effect. AQP4 upregulation was concomitant with 4.8 fold increase in the astrocytic area (indicative of cytotoxic edema). The FDA-approved anti-epileptic and migraine prevention drug, Topiramate (TPM), which works as an AQP4 inhibitor, blocked radiation-induced astrocytic swelling in huAST in vitro. Thus, we tested whether pre-treatment with TPM could prevent radiation-induced edema in a mouse model of HER2+BMs. Mice were injected intracardially JmT1BR3 brain metastatic cells and ten days later randomized based on the total head flux to (1) Radiation + vehicle, (2) Radiation + TPM (2 days prior to irradiation), (3) Non-Radiation + vehicle, and (4) Non-radiation + TPM. TPM decreased brain-water content (a marker of brain edema) in irradiated mice as compared with vehicle-treated mice, without alteration of metastatic burden 21 days post-injection. These results suggest TPM could be repurposed as a preventive agent of radiation-induced brain edema.

Abstract 3564: Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses

Abstract Background: Immune checkpoint blockade (ICB) antibodies have demonstrated that effective induction of T cell responses can elicit tumor regression of different metastatic cancers, although these responses are restricted to certain individuals with specific types of cancer. To enhance these responses, there has been renewed emphasis in developing different types of cancer-specific vaccines to stimulate and direct T cell and B cell immunity to important oncologic targets, such as the oncogene human epidermal growth factor receptor 2 (HER2), which is expressed in ~20% of breast cancers (BC). In our previous work we used a DNA plasmid vaccine targeting HER2, which utilized a lysosome-associated membrane protein 1 (LAMP) domain to traffic HER2 antigen to endolysosomal compartments. We demonstrated that this approach was more effective at eliciting antigen specific CD4+ and CD8+ T cell responses, and provided a significant but limited survival benefit in an endogenous mouse model of HER2+ BC compared to a HER2-wild type vaccine. Methods: In our current study, we explored the use of this modified antigen construct using different vaccine modalities, including DNA plasmid, adenoviral (Ad), modified vaccinia ankara (MVA), and self-replicating RNA vaccines to identify effective vaccine platforms to elicit HER2-specific immunity and anti-tumor immunity. Additionally, we have tested the combination of these modalities in homologous and heterologous vaccine boosting regimens to determine an optimal strategy to elicit anti-tumor immunity in vivo. Results: In our studies, plasmid and MVA vectors elicited suboptimal immune priming responses, in comparison to Ad and self-replicating RNA vectors that elicit significant HER2-specific T and B cell responses upon vaccination. While timing of boosting is critical, our results reflect that certain platforms (e.g., plasmid DNA vectors) are capable of homologous boosting capacity, while others (e.g., Ad vectors) elicit more robust T and B cell responses to capsid epitopes, limiting homologous boosting capacity for HER2-specific immunity. Significantly, using an endogenous model of metastatic HER2+ BC, we have found that heterologous vaccination with different HER2-LAMP targeted vectors can elicit significantly augmented HER2-specific T and B cell responses that translated into more effective anti-tumor immunity. This immunity proved capable of eliciting tumor regression in ~80% of vaccinated mice, compared to 0% using homologous HER2-WT plasmid vaccination, 30% using homologous HER2-LAMP plasmid vaccination, or 50% with HER2-WT heterologous vaccination. Conclusions: These data demonstrate the potential of utilizing heterologous vaccine platforms encoding LAMP-based endolysosomal trafficking vaccines to elicit HER2-specific immunity and effect anti-tumor responses. Citation Format: Robert D. Marek, Alan Chen, Renhuan Xu, Junping Wei, Tao Wang, Xiao Yang, Gangjun Lei, Teri Heiland, Zachary C. Hartman. Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3564.

A triple enhanced permeable gold nanoraspberry designed for positive feedback interventional therapy.

Due to the unique microenvironment, nanoparticles cannot easily penetrate deeply into tumours, which decreases their therapeutic efficacy. Thus, new strategies should be developed to solve this problem and increase the efficacy of nanomedicine. In this study, gold nanoraspberries (GNRs) were constructed using ultrasmall gold nanospheres (UGNPs) with a matrix metalloproteinase (MMP)-2/9-sensitive peptide as a cross-linking agent. These UGNPs were then modified with trastuzumab (TRA) and mertansine derivatives (DM1) via the AuS bond. TRA targets the human epidermal growth factor receptor-2 (Her-2) which is overexpressed on Her-2+ breast cancer cells. The AuS bond in GNRs-DM1 can be replaced by the free sulfhydryl group of GSH, which could achieve GSH dependent redox responsive release of the drug. In the mouse model of Her-2+ breast cancer, a "positive feedback" triple enhanced penetration platform was construct to treat tumours. Firstly, near-infrared light-triggered photothermal conversion increased vascular permeability, resulting in nanoparticle penetration. Secondly, GNRs disintegrated into UGNPs in response to stimulation with MMPs. GNRs with larger particle sizes reached the tumour site through EPR effect and active targeting. Meanwhile, UGNPs with smaller particle sizes penetrated deeply into the tumour through diffusion. Thirdly, the UGNPs transformed activated cancer-associated fibroblasts to a quiescent state, which reduced intercellular pressure and promoted the penetration of the UGNPs into the interior of the tumour. In turn, an increase in the number of nanoparticles penetrating into the tumour led to a "positive feedback" loop of triple enhanced photothermal effects and further self-amplify the permeability in vivo. Interventional photothermal therapy (IPTT) was used to improve the therapeutic efficacy by reducing the laser power attenuation caused by percutaneous irradiation. The GNRs also showed excellent multimode imaging (computed tomography, photoacoustic imaging and photothermal imaging) capabilities and high anti-tumour efficacy due to efficient tumour targeting and triple enhanced deep penetration into the tumour site. Thus, these MMP-2/redox dual-responsive GNRs are promising carriers of drugs targeting human epidermal growth factor receptor 2+ breast cancer.

Abstract PS3-03: [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models

Abstract Introduction: Paclitaxel (PTX) is one of the most commonly used first-line chemotherapies in the treatment of breast cancer. However, it is reported that overall PTX response rate is between 30-60% for the treatment of metastatic breast cancer. Thus, novel discoveries of molecular mechanisms that account for ineffective response is critical in breast cancer treatment. [89Zr]-Pertuzumab (antibody that targets HER2 receptors) and 2-deoxy-2- [fluorine-18] fluoro-D-glucose ([18F]-FDG) positron emission tomography/computed tomography (PET/CT) imaging allows us to noninvasively measure the amount and heterogeneity of HER2 expression and tumor glucose metabolism. These methods are clinical translatable and can provide insight into tumor biology changes during the course of therapy. This study evaluates whether HER2 expression level is correlated with PTX treatment efficacy in controlled pre-clinical mouse models of HER2+ breast cancer. Experimental Design: BT474 (1×107), MDA-MB-361 (6×106), or MDA-MB-231 (2×106) cells were subcutaneously injected into athymic nude mice (N = 7 per cell line). When the tumor volume reached approximately 200 mm3, mice were enrolled in the study. Tumor size was measured with calipers weekly until enrolled and every 3 days after experiment started. In vivo HER2 expression level was determined by [89Zr]-Pertuzumab PET/CT imaging at one week prior to initiation of treatment and confirmed with immunohistochemistry staining. PTX (15 mg/kg) was administered via i.v. on days 0 and 3. In vivo tumor metabolism was quantified by [18F]-FDG PET/CT imaging on day 0, 3 and 6. Mean, standard deviation, sum, and frequency histogram of standard uptake value (SUV) were quantified. Tumors were harvested at day 6 for histological analysis. Hematoxylin and eosin (H&E) and cleaved caspase 3 immunohistochemistry (IHC) staining were used to determine tumor apoptosis. Pearson’s correlation and ANOVA were used as statistical analysis. Results: [89Zr]-Pertuzumab SUVmean of BT474 (HER2+) tumors were 4.9±1.5, MDA-MB-361 (HER2+) tumors were 1.4±0.2 (p<0.0001, compared with BT474), and MDA-MB-231 (HER2-) tumors were 1.1±0.4 (p<0.0001, compared with BT474). [18F]-FDG SUV sum was positively correlated with tumor volume (R2=0.1669, p=0.0250). Tumor volumes showed no significant changes during the treatment. However, normalized [18F]-FDG SUV concentration changes from day 0 to day 3 was negatively correlated with baseline [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show paclitaxel treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. [89Zr]-Pertuzumab PET/CT imaging quantitively measured HER2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. This discovery will help identify chemotherapy responders and potentially enhance clinical decision making. Acknowledgments: We thank funding sources American Cancer Society RSG-18-006-01-CCE and NIH NCI R01CA240589. Citation Format: Yun Lu, Adriana Massicano, Patrick Song, Meng Li, Suzanne Lapi, Anna Sorace. [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-03.

Abstract PR04: Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer

Abstract Integrin-mediated adhesion and signaling via focal adhesion kinase is required for mammary tumorigenesis in the PyMT mouse model. Moreover, PTK2 encoding FAK is amplified in a substantial fraction of human breast cancers. We have investigated the mechanism through which integrin signaling controls mammary tumorigenesis and found that integrin-mediated mechano-transduction controls invasive growth in stiff 3D matrices through activation of YAP/TAZ. Mechanistic studies revealed that integrin signaling induces—via activation of FAK and Src family kinases (SFKs) —tyrosine phosphorylation and inactivation of LATS. The ensuing activation of YAP/TAZ is necessary for invasive growth of HER2+ breast cancer cells. Engagement of HER2/HER3 with neuregulin impinges on SFKs to amplify activation of YAP/TAZ in HER2+ breast cancer cells, suggesting that integrin-mediated mechano-transduction functions as a rheostat to regulate HER2 oncogenic signaling. Finally, administration of dasatinib significantly increases the efficacy of HER2 inhibition in the MMTV-Neu mouse model of HER2+ breast cancer. These findings reveal a major mechanism through which the Hippo pathway is disabled in breast cancer and suggest that targeting HER2 and SFKs simultaneously may be a rational strategy in selected cases of breast cancer. This abstract is also being presented as Poster A22. Citation Format: Shimin Wang, Young-Mi Kim, Ralph Garippa, Larry Norton, Filippo G. Giancotti. Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR04.

Abstract IA28: Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer

Abstract Integrin-mediated adhesion and signaling via focal adhesion kinase is required for mammary tumorigenesis in the PyMT mouse model. Moreover, PTK2 encoding FAK is amplified in a substantial fraction of human breast cancers. We have investigated the mechanism through which integrin signaling controls mammary tumorigenesis and found that integrin-mediated mechano-transduction controls invasive growth in stiff 3D matrices through activation of YAP/TAZ. Mechanistic studies revealed that integrin signaling induces—via activation of FAK and Src family kinases (SFKs) —tyrosine phosphorylation and inactivation of LATS. The ensuing activation of YAP/TAZ is necessary for invasive growth of HER2+ breast cancer cells. Engagement of HER2/HER3 with neuregulin impinges on SFKs to amplify activation of YAP/TAZ in HER2+ breast cancer cells, suggesting that integrin-mediated mechano-transduction functions as a rheostat to regulate HER2 oncogenic signaling. Finally, administration of dasatinib significantly increases the efficacy of HER2 inhibition in the MMTV-Neu mouse model of HER2+ breast cancer. These findings reveal a major mechanism through which the Hippo pathway is disabled in breast cancer and suggest that targeting HER2 and SFKs simultaneously may be a rational strategy in selected cases of breast cancer. Citation Format: Shimin Wang, Young-Mi Kim, Ralph Garippa, Larry Norton, Filippo G. Giancotti. Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA28.

Genetic disruption of calpain-1 and calpain-2 attenuates tumorigenesis in mouse models of HER2+ breast cancer and sensitizes cancer cells to doxorubicin and lapatinib

Calpains are a family of calcium activated cysteine proteases which participate in a wide range of cellular functions including migration, invasion, autophagy, programmed cell death, and gene expression. Calpain-1 and calpain-2 isoforms are ubiquitously expressed heterodimers composed of isoform specific catalytic subunits coupled with an obligate common regulatory subunit encoded by capns1. Here, we report that conditional deletion of capns1 disrupted calpain-1 and calpain-2 expression and activity, and this was associated with delayed tumorigenesis and altered signaling in a transgenic mouse model of spontaneous HER2+ breast cancer and effectively blocked tumorigenesis in an orthotopic engraftment model. Furthermore, capns1 knockout in a tumor derived cell line correlated with enhanced sensitivity to the chemotherapeutic doxorubicin and the HER2/EGFR tyrosine kinase inhibitor lapatinib. Collectively, these results indicate pro-tumorigenic roles for calpains-1/2 in HER2+ breast cancer and provide evidence that calpain-1/2 inhibitors could have anti-tumor effects if used either alone or in combination with chemotherapeutics and targeted agents.

Macrophages orchestrate breast cancer early dissemination and metastasis

Cancer cell dissemination during very early stages of breast cancer proceeds through poorly understood mechanisms. Here we show, in a mouse model of HER2+ breast cancer, that a previously described sub-population of early-evolved cancer cells requires macrophages for early dissemination. Depletion of macrophages specifically during pre-malignant stages reduces early dissemination and also results in reduced metastatic burden at end stages of cancer progression. Mechanistically, we show that, in pre-malignant lesions, CCL2 produced by cancer cells and myeloid cells attracts CD206+/Tie2+ macrophages and induces Wnt-1 upregulation that in turn downregulates E-cadherin junctions in the HER2+ early cancer cells. We also observe macrophage-containing tumor microenvironments of metastasis structures in the pre-malignant lesions that can operate as portals for intravasation. These data support a causal role for macrophages in early dissemination that affects long-term metastasis development much later in cancer progression. A pilot analysis on human specimens revealed intra-epithelial macrophages and loss of E-cadherin junctions in ductal carcinoma in situ, supporting a potential clinical relevance.

Abstract 2993: STING signaling in breast tumor microenvironment modulates immune checkpoint blockade efficacy in the neu-N mouse model of breast cancer

Abstract Background: The presence of tumor infiltrating T cells (TILs) is associated with improved clinical outcomes in multiple tumor types and is also necessary for response to immune checkpoint blockade. While T cell responses to some tumors occur spontaneously, the majority of cancers are not naturally recognized by the immune system. The lack of response is attributed primarily to insufficient T cell infiltration into the tumor microenvironment (TME). Activating the STING pathway represents one strategy for increasing infiltration of T cells into the TME. This induces interferon-β (IFN-β) production, leading to dendritic cell (DC) activation and priming of tumor antigen specific CD8+ T cells that mediate tumor regression. The intratumoral injection (IT) of ML-RR-S2-CDA (ADU-S100), a synthetic cyclic dinucleotide STING agonist, has antitumor efficacy in several aggressive mouse tumor models, including B16 melanoma, CT26 colon carcinoma, Panc02 pancreatic carcinoma, and 4T1 triple negative breast cancer. However, the impact of antigen-specific tolerance on tumor regression associated with STING-activation remains poorly understood. Therefore, we evaluated the efficacy of IT ADU-S100 in both non-tolerant parental FVB/N and the immune tolerant neu/N transgenic mice bearing established HER-2+ breast tumors. Methods: First we first evaluated the impact of IT ADU-S100 on tumor regression, survival, innate sensing, and priming of HER-2 specific CD8+ T cells in both tumor-bearing non-tolerant FVB/N control and tolerant neu/N mice. Then we determined whether modulating the most highly expressed immune checkpoints on tumor infiltrating CD8+ T cells enhanced intratumoral STING activation with ADU-S100 in the tolerant neu/N mouse model of HER-2+ breast cancer. Results: ADU S-100 induced HER-2-specific CD8+ T cell priming and durable tumor clearance in 100% of non-tolerant, FVB/N mice. In contrast, ADU S-100 failed to sufficiently prime HER-2-specific CD8+ T cells in tolerant neu/N mice, delaying tumor growth and clearing tumors in only 10% of mice. No differences in IFN-β production, DC priming, or HER-2-specific CD8+ T cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2-specific CD8+ T cells was defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high levels of PD1 and OX40 on CD8+ T cells, and high levels of PD-L1 on both myeloid and tumor cells. Modulating PD-L1 and OX40 signaling combined with IT ADU S-100 enhanced HER-2-specific CD8+ T cell activity, clearing tumors from 40% of neu/N mice. Conclusions: Intratumoral STING activation synergizes with PD1 pathway-blockade and OX40 receptor stimulation to overcome immune tolerance and prime tumor antigen-specific CD8+ T cell responses that mediate effective tumor regression. Citation Format: Jeremy B. Foote, Marlene Kok, James M. Leatherman, Todd D. Armstrong, Bridget Marcinkowski, David B. Kanne, Elizabeth M. Jaffee, Thomas W. Dubensky, Leisha A. Emens. STING signaling in breast tumor microenvironment modulates immune checkpoint blockade efficacy in the neu-N mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2017-2993

Abstract 4337: PDGFR-α induced stiffness abrogates mammary ductal development and enhances tumorigenesis in vivo

Abstract Breast cancer is a leading cause of mortality in women worldwide, in part due to the tumor microenvironment which increases tumor heterogeneity and abets tumor growth. Fibroblasts are cells of mesenchymal origin that are an important component of normal and tumor stroma. Genetic alterations in these cells were shown by several groups including our own to cause fibroblast activation and fuel tumor progression giving rise to more aggressive disease. Platelet-Derived Growth Factor Receptor (PDGFR) alpha is a receptor tyrosine kinase that is chiefly expressed in mesenchymal cells such as fibroblasts. Ligand binding (PDGFAA) activates this receptor. PDGFRα signaling plays critical roles in development and aberrant signaling is seen in several types of cancer, such as lung, pancreas, GI and brain. The central goal of this study was to elucidate the role of stromal PDGFRα in breast cancer development and metastasis, where its role remains largely unknown. To address this goal, we developed a genetic mouse model of stromal activation of PDGFRα in the mammary gland by crossing an auto-activating Pdgfra mutant allele with a mesenchymal specific Cre recombinase. We found that stromal PDGFRα activation completely abrogated postnatal mammary gland ductal formation, with significantly reduced terminal end bud formation. PDGFRα activation also led to progressive fibrosis in the mouse mammary fat pad. As early as four weeks of age, mammary collagen (trichrome staining; second harmonic generation) and hyaluronan deposition (Alcian Blue) was greatly increased in vivo. In fact, this increase in collagen and hyaluronan deposition in mutant animals is believed to be responsible for the observed increased in stiffness of mutant mammary tissue (atomic force microscopy {AFM}). pFAK, which can be activated due to mechanical stress, was increased in mammary epithelia of the mutant mice in vivo corroborating the AFM results. Further, when tumor cells were injected into the mammary glands of the PDGFRα mutants, tumors grew faster as compared to controls. Importantly, we found that mRNA expression of PDGFRA correlates with worsened patient outcomes in HER2+ disease, while expression of both the ligand (PDGFA) and the receptor were found to correlate with increased incidence of lung metastases. We further discovered that in HER2+ patients, PDGFRA levels correlate with breast density. Breast density is the third strongest risk factor for breast cancer, and is directly related to collagen deposition and breast stiffness, thus suggesting a novel predictive role of PDGFRA as a molecular readout of stiffness and density. Studies are underway to utilize mouse models of HER2+ breast cancer to study both primary tumor growth and metastases. Taken together, our mouse studies and paralleling human data analyses suggest that the stromal PDGFRα signaling provides a novel theranostic window in breast cancer treatment and prognosis. Citation Format: Anisha Mathur Hammer, Gina M. Sizemore, Vasudha Shukla, Steven T. Sizemore, Maria Cuitino, Cynthia J. Timmers, Quinn Verfurth, Arnab Chakravarti, Gustavo W. Leone, Samir N. Ghadiali, Michael C. Ostrowski. PDGFR-α induced stiffness abrogates mammary ductal development and enhances tumorigenesis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2017-4337

A mouse-human phase I co-clinical trial of taselisib in combination with TDM1 in advanced HER2-positive breast cancer (MBC).

1030 Background: PI3K blockade has the potential to revert resistance to anti-HER2 therapies. Methods: Pre-clinical experiments were conducted in parallel to a phase Ib study with the PI3-kinase inhibitor Taselisib + T-DM1 in HER2+ MBC. Results: Pre-clinically, HER2+ cell lines (HCC1954, MDA-MB-361, MDA-MB453) treated with T-DM1 until resistance had decreased HER2 expression. Tx with Taselisib restored HER2 levels in T-DM1 resistant cells. In vivo, the combination of T-DM1 + Taselisib was superior to either agent alone in transgenic mammary carcinomas driven by HER2 with or without a PIK3CA H1047R transgene. The phase Ib study enrolled 26 pts with HER2+ MBC. Median age was 57 (38-95), ER+ (58%), ER- (42%). PIK3CA wild type (35%) mutant (23%) pending results (42%). Pts had a median of 2 lines of prior tx for MBC. Prior T-DM1 was given to 38.5% of pts. Median duration of tx among 18 pts was 4 cycles (2-19). 8 pts remain on tx. No DLTs were observed in the 2 dose levels explored: T-DM1 (3.6mg/Kg) q3 wk with either Taselisib 2mg QD or Taselisib 4mg QD. 20 patients were included in the expansion phase. Most common G3 G4 AEs were thrombocytopenia (19.2%), diarrhea (15.4%), vomiting (7.7%), lipase increase (7.7%) and hyperglycemia (7.7%). Pneumonitis was reported in 4 cases (3 G1, 1 G2); further evaluation revealed elevated beta-glucan in 3 of the 4 cases raising suspicion for pneumocystis pneumonia (PJP). 24/26 pts were evaluable for confirmed response: (4% CR, 29% PR, 50% SD). Confirmed PR/CR according to prior TMD1 (yes 30% no 36%) and PIK3CA mut status (40% mut, 22% wild type; 9/23 PIK3CA results pending). Median PFS was 7.6 mos (95% CI 2.9 to NR). Conclusions: The combination of Taselisib + T-DM1 is superior to either agent alone in mouse models of HER2+ BC. In the phase Ib study, T-DM1 + Taselisib had an acceptable safety profile although PJP prophylaxis should be considered for this regimen. Thrombocytopenia was reversible with no bleeding events. Preliminary evidence of efficacy was observed regardless of PIK3CA status or prior tx with T-DM1. This co-clinical trial allowed for the rapid translation of lab observations to the clinical setting and provided data to facilitate interpretation of the clinical results. Clinical trial information: NCT02390427.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract P2-11-12: Novel protocol combining metronomic nant-paclitaxel with HER2-targeted natural killer cells (innate immunotherapy) for HER2-positive metastatic breast cancer

Abstract Background. Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Phase 1 clinical trials in patients with advanced cancers demonstrated the safety of unmodified, activated NK-92 cells (aNK), with no evidence of cytokine storm from 18 infusions delivered over 6 months; clinical responses were observed in a subset of patients. Like T cells, NK cells can be engineered to express chimeric antigen receptors (CARs) to enhance their antitumor activity. A stable clonal HER2-specific NK-92 cell line (HER2.taNK) mediated selective and sequential killing of HER2-expressing MDA-MB-453 cells in vitro (Schönfeld. MolTher. 2015;23:330-338). In addition, HER2.taNK cells were enriched in MDA-MB-453/EGFP xenografts and reduced the number of pulmonary metastasis in a renal cell carcinoma model, suggesting that HER2.taNK cells are a promising clinical candidate for use in adoptive cancer immunotherapy. Metronomic (low-dose, continuous) chemotherapy can be more effective than high-dose therapy in patients with advanced breast cancer (Montagna. Canc. Treat. Rev. 2014;40:922-950). Here we evaluate HER2.taNK cells in combination with metronomic nant-paclitaxel (lyophilized polymeric micellar formulation of paclitaxel) in a mouse model of HER2-positive breast cancer to determine the feasibility of a human clinical trial of HER2.taNK in combination with metronomic nant-paclitaxel. Methods. HER2.taNK cells were generated as described previously (Schönfeld. MolTher. 2015;23:330-338). MDA-MB-453 cells were implanted into the mammary fat pads of female nude mice. When tumors reached 100 mm3, mice were divided into 6 groups of 5 mice and dosed (IV) with saline (10 mL/kg, qd x 15), nant-paclitaxel (2.5-4 mg/kg q2d x 15), γ-irradiated (5 Gy) HER2.taNK cells (1 x 107 cells, days 1, 3, 5, and 8), or nant-paclitaxel + γ-irradiated (5 Gy) HER2.taNK cells–γ-irradiation is a potential safety measure for clinical application and prevents HER2.taNK cell replication while preserving antitumor activity. Tumor size and animal weights were measured every other day post-implantation. Results: Results obtained 20 days post-treatment are shown in the table. Nant-paclitaxel alone and HER2.taNK alone significantly inhibited tumor growth. The combination of nant-paclitaxel + HER2.taNK led to significant tumor regressions (p<0.05). Treatment Dose T/C (%) P-ValueSalinenant-paclitaxel5 mg/kg-26.7 P < 0.05 (vs saline) HER2.taNK1 x 107 cells-22.2 P < 0.05 (vs saline)nant-paclitaxel +5 mg/kg +-60.0P < 0.05 (vs nant-paclitaxel)HER2.taNK1 x 107 cellsP < 0.05 (vs HER2.taNK) Conclusions: Single agent nant-paclitaxel and HER2.taNK were similarly effective at inhibiting tumor growth in this mouse model of HER2+ breast cancer. The combination of nant-paclitaxel + HER2.taNK appeared to be synergistic resulting in tumor regressions and significantly better efficacy vs each agent alone. This study illustrates the potential for combining metronomic low-dose chemotherapy with NK-based immunotherapy in a clinical trial of patients with metastatic breast cancer. Citation Format: Rabizadeh S, Simon B, Klingemann H, Sims D, Weiss R, Soon-Shiong P. Novel protocol combining metronomic nant-paclitaxel with HER2-targeted natural killer cells (innate immunotherapy) for HER2-positive metastatic 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 P2-11-12.

Abstract A19: Stathmin plays a role in both normal and transformed mammary gland

Abstract Normal mammary gland is a highly dynamic organ and displays complex tissue architecture, precise breast epithelial cell hierarchy and lineage plasticity regulation. These features are thought to underlie the high degree of both inter- and intra-tumor heterogeneity of breast cancers (BC). We have recently made the observation that stathmin knock-out (STMKO) female mice do not breast feed their first litter. Stathmin (also called Oncoprotein 18, OP18) is a microtubule-destabilizing protein. It is overexpressed in many human malignancies, including breast cancer, especially in advanced/metastatic disease. The phenotype that we observed suggested that complete development of lactating mammary gland was not properly and/or timely achieved in the absence of stathmin. To evaluate if this was the case, we analyzed mammary glands from WT and STMKO mice at prepuberal stage, at puberty, at day 13.5 of pregnancy and one day post-partum. Whole mount observation of mammary glands indicated that stathmin loss greatly impacted on the development of the mature gland, which displayed lower number of branches and defective alveolar buds. Most evident defects were displayed in the architecture of one day post-partum glands. Our molecular analyses showed that especially the polarity and the proliferation processes were affected by the absence of stathmin in the breast tissue. Loss of polarity and deregulated proliferation are profoundly linked to epithelial tumorigenesis. We thus tested whether loss of stathmin had an impact on BC onset and/or progression, using a mouse model of HER2+ BC. Among the different BC subtypes, HER2+ tumors represent ~20% of invasive BC and display particularly aggressive phenotypes and poor prognosis. A splice variant of the human HER2 gene, called Delta16 HER2 (d16HER2), displaying enhanced transforming activity and associated with worse patients' survival, has been identified. We intercrossed STMKO with d16HER2 transgenic mice and analyzed tumor onset, growth and progression in the different backgrounds. Our analysis showed that STMKO profoundly impacted on both tumor initiation and proliferation. The effect of stathmin on proliferation seemed to be mainly cell-autonomous, as observable by injecting BC cells extracted from d16HER2-STMKO, in either WT or STMKO recipient mice. Thus, our results suggest that stathmin may play multiple roles in mammary epithelial cells. By regulating mammary epithelial cell polarity, it may be determinant in the definition of differentiation hierarchy, in cellular transformation and, eventually, tumor initiation. By regulating proliferation, it can affect tumor growth and burden. The unbalanced regulation of these critical processes certainly represents fertile ground for tumor initiation and may conceivably contribute to the great degree of tumor heterogeneity observed in breast cancer. Citation Format: Ilenia Segatto, Stefania Berton, Sara D'Andrea, Gustavo Baldassarre, Barbara Belletti. Stathmin plays a role in both normal and transformed mammary gland. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A19.

Abstract B29: Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer

Abstract Background: Obesity is associated with increased breast cancer mortality in premenopausal women, who predominately develop estrogen receptor (ER)-α negative breast tumors. New mechanism-based targets and intervention strategies for offsetting the procancer effects of obesity are urgently needed given the rising rates of obesity in women throughout the world and the lack of effective targeted therapies for ER-negative breast cancer. Unfortunately, the mechanisms by which obesity impacts ER-negative breast cancer prognosis remain unclear. Methods: In this study, we utilized the MMTV-neu transgenic mouse model of Her2+ breast cancer to test the hypothesis that dietary energy balance modulation alters mammary tumor development and progression through epigenetic regulation of ER in the mammary epithelium of these mice. MMTV-neu transgenic mice form spontaneous mammary tumors that progress from an ERα-positive hyperplasia to aggressive ERα-negative ductal adenocarcinomas. Female MMTV-neu transgenic mice were randomized to 3 diet regimens (30/regimen), resulting in either an obese, overweight or lean phenotype. A subset of mice were killed at baseline, 1, 3, and 5 months following diet initiation, and tissues were collected for analysis; remaining animals were followed for up to a 22 month survival study. Results: Gene and protein expression analysis revealed that mammary ERα expression, known to be lost by 8 weeks of age in normoweight MMTV-neu transgenic mice, was maintained in lean mice but lost in the overweight and obese mice (which did not statistically differ in any parameter studied). Additionally, we found lean mice had significantly increased mammary ERβ gene expression and delayed onset of hyperplasia relative to the overweight/obese mice. ERα and ERβ gene intron methylation increased from 5 months to 12 months on diet in lean mice and conversely decreased in overweight/obese mice, which parallels the ER gene expression data. After 22 months of feeding, lean mice had significantly increased tumor-free survival in relative to the overweight/obese mice. Interestingly, human and mouse HER2+ (but not but not HER2-) breast cancer cell lines, when treated with serum from obese (BMI>30), women resulted in a significant down-regulation of ERβ gene expression. Conclusions: In MMTV-neu transgenic mice, dietary energy balance modulation impacts spontaneous mammary tumor development and ERα and ERβ levels in normal and tumor tissue, possibly through epigenetic mechanisms. Moreover, increased mammary ERβ expression may represent a novel target for interventions to offset the enhancing effects of obesity on breast cancer. Citation Format: Emily L. Rossi, Sarah M. Dunlap, Nikki A. Ford, Laura W. Bowers, Stephen D. Hursting. Maintenance of a lean phenotype is associated with increased ERβ expression and ERβ gene intron methylation in murine MMTVneu luminal mammary cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B29.