Abstract
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in about 20% of invasive breast cancers. Anti-HER-2 monoclonal antibody therapy is effective, but its utility is ...limited by high costs, side effects and development of resistance, thus underlining the need of new therapeutic approaches. A novel anti-HER-2 vaccine made of virus-like particles (VLPs) displaying the extracellular domain (ECD) of the human oncogene/antigen HER-2 induced protective immune responses against transgenic mouse mammary carcinomas expressing human HER-2. We have developed a versatile antigen display platform that, unlike existing technologies, effectively facilitates directional covalent attachment of large antigens at high density on the surface of VLPs (J. Nanobiotechnology 14: 30, 2016). The system uses a tag/catcher conjugation system that was developed by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes into a highly reactive peptide (SpyTag) and a protein (SpyCatcher) binding partner. Interaction between SpyTag and SpyCatcher results in the spontaneous formation of an isopeptide bond, occurring at high efficiency in a wide variety of protein contexts and buffer conditions. Here, we genetically fused with SpyCatcher the full extracellular domain (subdomains I-IV) of human HER-2, and bound the fusion antigen (SpyCatcher-HER2) to the surface of VLPs (derived from the AP205 phage), each presenting 360 SpyTag peptides. The vaccine, referred to as HER2-VLP, effectively overcame immune tolerance and induced Th1 cytokines and high-titer, high affinity, therapeutically potent anti-HER-2 antibodies which inhibited tumor growth in wild-type FVB mice implanted with transgenic mammary carcinomas expressing human HER-2. Furthermore, vaccination with HER2-VLP prevented spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice. Vaccination with a control preparation of untagged VLP and HER-2 ECD did not induce protective immune responses. Polyclonal IgG antibodies elicited by HER2-VLP vaccination had an affinity for human HER-2 comparable to trastuzumab and inhibited the 3D growth in vitro of both trastuzumab-sensitive and trastuzumab-resistant BT-474 human breast cancer cells. In conclusion, the HER2-VLP vaccine has the potential to become a tool in the fight against HER-2-positive human cancer. The results also provide strong proof-of-concept for the use of the versatile VLP platform to develop a variety of vaccines against other tumor antigens. Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, the Danish Research Council, the Eurostars program and the European Research Council (ERC).
Citation Format: Arianna Palladini, Susan Thrane, Christoph M. Janitzek, Jessica Pihl, Stine B. Clemmensen, Wilhelm A. de Jongh, Thomas M. Clausen, Giordano Nicoletti, Lorena Landuzzi, Manuel L. Penichet, Tania Balboni, Marianna L. Ianzano, Veronica Giusti, Thor H. Theander, Morten A. Nielsen, Ali Salanti, Pier-Luigi Lollini, Patrizia Nanni, Adam F. Sander. A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma 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 716.
Abstract
ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2, developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibits mammary carcinoma growth ...and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system: Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administered alone, thanks to the intrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16 (FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fat pad with 1 million QD cells; vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas 70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free one year after cell challenge. Mice challenged intravenously (i.v.) developed more than 300 lung metastases, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 and develop aggressive mammary carcinomas with a median latency of 5 months. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lung nodules, whereas 73% of mice therapeutically vaccinated without adjuvant, and all mice vaccinated with E2SB-C001+ISA 51, were free from metastases. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL in Delta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++ breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated by ELISPot (21±3 spots/2x105 cells). The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2.
Citation Format: Francesca Ruzzi, Arianna Palladini, Stine Clemmensen, Annette Strobaek, Nicolaas Buijs, Tanja Domeyer, Jerzy Dorosz, Vladislav Soroka, Dagmara Grzadziela, Christina Jo Rasmussen, Ida Busch Nielsen, Max Soegaard, Maria Sofia Semprini, Laura Scalambra, Stefania Angelicola, Lorena Landuzzi, Pier-Luigi Lollini, Mette Thorn. Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 687.
Abstract
Human breast cancer cells express full-length HER-2 along with proteins resulting from mutation, alternative splicing, alternative initiation of translation and post-translational ...modification. The Delta16 splice variant, lacking exon 16, has the properties of an activated oncogene, but it could also play beneficial roles in the response to targeted therapeutic agents. To study mammary carcinogenesis in a mouse model that mimics human coexpression of full-length HER-2 and Delta16 isoforms, we produced hybrid mice bearing heterozygous copies of both human transgenes (F1 mice), and we compared them to parental mice (Delta16 and HER-2 transgenic mice, respectively). Tumor onset in F1 and Delta16 mice was much faster than in HER-2 mice (30 vs >80 weeks), but the growth of established tumors and metastatic spread were not enhanced. Each mammary carcinoma of F1 mice expressed both isoforms at variable ratios. Most (∼80%) expressed high levels of Delta16 and low levels of full length HER-2, a few (∼5%) expressed full-length HER-2 and little, if any, Delta 16, and the remainder (∼15%) coexpressed at high level both isoforms. The study of tumor vascularization showed that full-length HER-2 tumors mainly contained few large vessels or vascular lacunae, whereas Delta16 tumors were perfused by numerous endothelium-lined small vessels. F1 tumors with high full-length HER-2 expression made few large vessels, whereas tumors with low full-length HER-2 and high Delta16 contained numerous small vessels and expressed high levels of both VEGF and VEGFR2. Administration of trastuzumab to young F1 mice effectively prevented mammary carcinoma onset in ∼85% of mice at 1 year of age. The preventive effect of trastuzumab was stronger in F1 mice than in both parental strain. To analyze the intrinsic sensitivity of F1 mammary carcinoma cells to targeted drugs in 3D, we established cell lines expressing different HER-2 isoform ratios. High Delta16 expression caused high sensitivity to HER-2 inhibitors (trastuzumab, neratinib, lapatinib), whereas high full-length HER-2 was associated with a lower sensitivity. Interestingly, high levels of both isoforms was associated with resistance to trastuzumab, but sensitivity to small molecule inhibitors. In conclusion, the coexpression of full-length HER-2 and Delta16 controls several determinants of mammary carcinoma development, progression and sensitivity to targeted agents, as revealed by the study of F1 mice.
Citation Format: Arianna Palladini, Massimiliano Dall’Ora, Tania Balboni, Giordano Nicoletti, Marianna Ianzano, Roberta Laranga, Lorena Landuzzi, Veronica Giusti, Alessia Lamolinara, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni, Pier-Luigi Lollini. HER-2 isoform interaction in mammary carcinoma onset and progression. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1200.
The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active ...immunopreventive strategies targeting the human HER2 molecule in a tolerant host.
FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy.
Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells.
Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity.
We previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the ...release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention.
HER2/neu transgenic mice with homozygous knockout of IL-15 (here referred to as IL15KO/NeuT mice) were compared to IL-15 wild-type HER2/neu transgenic mice (NeuT) regarding mammary carcinogenesis, profile of peripheral blood lymphocytes and splenocytes and humoral and cellular responses induced by the vaccine.
IL15KO/NeuT mice showed a significantly earlier mammary cancer onset than NeuT mice, with median latency times of 16 and 20 weeks respectively, suggesting a role for IL-15 in cancer immunosurveillance. Natural killer (NK) and CD8+ lymphocytes were significantly lower in IL15KO/NeuT mice compared to mice with wild-type IL-15. The IL-12-adjuvanted allogeneic HER2/neu-expressing cell vaccine was still able to delay mammary cancer onset but efficacy in IL-15-lacking mice vanished earlier: all vaccinated IL15KO/NeuT mice developed tumors within 80 weeks of age (median latency of 53 weeks), whereas more than 70 % of vaccinated NeuT mice remained tumor-free up to 80 weeks of age. Vaccinated IL15KO/NeuT mice showed less necrotic tumors with fewer CD3+ lymphocyes and lacked perforin-positive infiltrating cells compared to NeuT mice. Concerning the anti-vaccine antibody response, antibody titer was unaffected by the lack of IL-15, but less antibodies of IgM and IgG1 isotypes were found in IL15KO/NeuT mice. A lower induction by vaccine of systemic interferon-gamma (IFN-γ) and interleukin-5 (IL-5) was also observed in IL15KO/NeuT mice when compared to NeuT mice. Finally, we found a lower level of CD8+ memory cells in the peripheral blood of vaccinated IL15KO/NeuT mice compared to NeuT mice.
We demonstrated that IL-15 has a role in mammary cancer immunosurveillance and that IL-15-regulated NK and CD8+ memory cells play a role in long-lasting immunoprevention, further supporting the potential use of IL-15 as adjuvant in immunological strategies against tumors.
Abstract
Almost 90% of human primary breast cancers (BCs) express 4-9% of total wild-type (WT) HER2 as a splice variant lacking exon 16 (d16HER2). Consequent in-frame activating deletion of 2 ...cysteine residues causes an imbalanced conformation resulting in receptor constitutive homodimerization, enhanced signaling activity, transformation and altered trastuzumab (T) binding. We recently produced a human d16HER2 transgenic (tg) mouse characterized by a rapid multifocal mammary tumors onset and the expression of active d16HER2 dimers on tumor cells, whose downstream signaling was found coupled to multiple activated nodes that include Src kinase. In order to dissect d16HER2 role in the aggressiveness and in the susceptibility to anti-HER2 therapy, we focused on the generation of stable d16HER2-expressing mammary tumor cell lines to be used in different bioassays. An immunomagnetic purification procedure was applied to generate primary homogeneously d16HER2-expressing cell cultures. These purified tumor cell lines were analyzed by flow cytometry and immunofluorescence and their migration/invasion ability was assessed through Boyden chamber test. d16HER2 downstream signaling was evaluated by western blot and T, Lapatinib (L) and Dasatinib (D) drugs sensitivity was measured with WST-1 and soft-agar bioassays. As controls, we compared in vitro d16HER2-models oncogenic features to those of the human BC BT474, which also co-expresses a low amount of d16HER2 transcript (4%), and to a murine mammary carcinoma cell line (wtHER2), derived from a primary mammary tumor developed in human WT HER2 tg mice. We found that d16HER2 in vitro models expressed high levels of stable homodimers combined with the recruitment of activated Src, STAT3, MAPK and Akt, as in vivo primary mammary tumors. Both in bidimensional (2D) and matrigel-cultured tumor cells, we confirmed the T lower binding capability for d16HER2 than other anti-HER2 MAbs directed to different extracellular domain epitopes. d16HER2 tumor cells had an enhanced migratory and invasive capacity compared to wtHER2 and BT474 cells and, notably, were completely resistant to T treatment and less responsive to L. In virtue of a highly activated Src kinase expression in d16HER2-positive models, we tested in 2D the therapeutic effects of D and observed that d16HER2-cells were significantly more sensitive than wtHER2 and BT474 cells. Preliminary 3D-drug susceptibility assays showed that the sensitivity of d16HER2 cells increased for all the tested drugs, if assessed in a suitable environment such as soft-agar. Our findings further indicate that the constitutive expression of d16HER2 variant identifies an aggressive tumor phenotype and confirm, at least in vitro in 2D conditions, that this isoform is resistant to T and L, whereas is sensitive to D. Further analyses are ongoing to analyze in vivo drug sensitivity of d16 in comparison to WT HER2 model.
Supported by AIRC and Ministry of Health
Citation Format: Gaia C. Ghedini, Arianna Palladini, Valentina Ciravolo, Lorenzo Castagnoli, Giulia Marzano, Roberta Zappasodi, Guido Santilli, Augusto Amici, Alessia Lamolinara, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Role of d16HER2 splice variant in HER2-positive breast cancer. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2014-2637
Abstract
Progression of HER-2+ breast cancer can result in the emergence of HER-2-negative tumor variants that activate alternative mitogenic pathways, either spontaneously or after therapy. We found ...that HER-2 loss occurs even in transgenic mouse models in which the oncogene is driven by viral promoters, thus mammary carcinoma of human HER-2 transgenic mice (huHER-2 mice) can be used to study not only the early phases of HER-2-driven mammary carcinogenesis, but also tumor progression beyond HER-2 addiction. Primary mammary carcinomas of huHER-2 mice express high levels of the oncogene, with a marked intratumoral heterogeneity. Cell lines grown from HER-2+ mammary carcinomas frequently undergo a progressive loss of expression. We have established a model system consisting of cell lines, clones and variants that exhibit one of three phenotypes: a) high and stable HER-2 expression in vitro and in vivo, b) high but labile HER-2 expression which is lost either during in vitro culture, after tumor growth in mice or after in vitro treatment with trastuzumab, and c) complete loss of HER-2 expression After HER-2 loss, most variants displayed a transition to an elongated, motile phenotype (epithelial to mesenchymal transition), an increased ability to generate mammospheres, a reduced expression of CD24 and an increased expression of CD44 (denoting mammary cancer stem cells). Tumorigenic and metastatic ability of HER-2-negative cells was increased in comparison to HER-2+ cells. As expected, HER-2 loss was accompanied by resistance to HER-2 targeted monoclonal antibodies and small molecule inhibitors. The study of therapeutic agents directed against downstream targets showed that HER-2-loss was accompanied by a loss of sensitvity to a Src inhibitor, whereas a PI3K inhibitor was highly effective regardless of HER-2 expression. Our results indicate that human HER-2 transgenic mice are a useful model to study the dynamics of HER-2 loss in advanced HER-2+ mammary carcinoma, and to analyze alternative therapeutic strategies.
Supported by grants from the Italian Association for Cancer Research (AIRC).
Citation Format: Patrizia Nanni, Arianna Palladini, Lorena Landuzzi, Massimiliano Dall'Ora, Marianna Ianzano, Valentina Grosso, Dario Ranieri, Giordano Nicoletti, Roberta Laranga, Carla De Giovanni, Manuela Iezzi, Pier-Luigi Lollini. Dynamics of HER-2 loss in mammary carcinoma of human HER-2 trangenic mice. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2014-1820
Abstract
The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of ...breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-driven activation of BCICs. In this context, our preliminary analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from spontaneous transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, including CD44, Wnt, Notch and Bmi1, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing breast cancer cell lines BT474 and MDA-MB-361, which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions, as indicated by qPCR analyses. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a consistently higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a greater role than WTHER2 in regulating BCICs of HER2-driven mammary tumors.
Supported by Minister of Health and AIRC
Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314
Abstract
Homozygous knockout of p53 in mice leads to early mortality from lymphoma, with almost complete penetrance, thus hampering the study of other tumor histotypes related to p53. To ablate ...lymphoma development, we crossed p53 knockout mice with Rag2-/-;Il2rg-/- (RGKO) mice, which lack T, B and NK cells. We then compared the tumor spectrum of homozygous (p53-/-) and heterozygous (p53+/-) mice with those of mice lacking lymphocytes (RGKO-p53-/- and RGKO-p53+/-, respectively). All mice were of BALB/c genetic background. Lymphoma incidence in p53-/- mice exceeded 80%, whereas in RGKO-p53-/- it was near zero. The prevalent tumor of RGKO-p53-/- mice was hemangiosarcoma (incidence was about 60%in both sexes, median latency 19 weeks), other tumors included soft tissue sarcomas (incidence ∼10%), osteosarcomas and mammary carcinomas. Changes in tumor spectrum occurred also in p53 heterozygotes, in which lymphomas were relatively rare (<20%). Male RGKO-p53+/- had an increased incidence of hemangiosarcomas, reaching ∼25%, whereas females had an increased incidence of osteosarcomas, reaching ∼20%. The latter shared with human osteosarcoma the involvement of limbs and a high metastatic ability, in particular to the lungs. Molecular studies of tumors, in vitro cultured tumor cells and established cell lines showed specific alterations in the expression of p53-related genes (p16Ink4a, p19Arf, p15Ink4b, p21Cip1) and high expression levels of insulin-like growth factors and their receptors. In conclusion, genetic ablation of lymphoma in p53 knockout mice led to the development of new models of sarcoma development that can be now explored to study the development and metastatic spread of hemangiosarcoma and osteosarcoma.
Citation Format: Lorena Landuzzi, Giordano Nicoletti, Arianna Palladini, Marianna Ianzano, Valentina Grosso, Dario Ranieri, Carla De Giovanni, Pier-Luigi Lollini, Patrizia Nanni. Genetic ablation of lymphoma development inp53 knockout mice reveals a novel model of osteosarcoma and hemangiosarcoma. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2013-1409
Abstract
HER-2 gene products found in human breast cancer include the full-length p185 oncoprotein and various shorter isoforms that lack C-terminal, N-terminal or internal portions.
Delta16 isoform ...lacks exon 16 and displays the properties of an activated oncogene. Transgenic mice expressing Delta16 in the mammary gland develop more mammary carcinomas, and at a younger age than mice transgenic for full-length HER-2.
Human breast cancers, unlike transgenic mice, co-express Delta16 and full-length HER-2. To study mammary carcinogenesis in a mouse model that mimics the human situation, we obtained hybrid mice bearing heterozygous copies of both human transgenes (Delta16/HER-2 mice), and we compared them to parental mice (referred to as Delta16 and HER-2 transgenic mice, respectively).
In Delta16/HER2 hybrid mice, mammary carcinogenesis was similar to that of Delta16 mice, with a median latency time of 17 weeks and a mean of 7 tumors per mouse, thus indicating a dominant expression of Delta16 over HER-2.
However, after tumor onset, tumor growth rate and metastatic spread were similar in the three mouse lines, thus suggesting that Delta16 was mainly involved in neoplastic transformation and in the early phases of mammary carcinogenesis, rather than in advanced tumor progression. This could result from cancer cell intrinsic activity of the oncogene or from interactions with the microenvironment, in particular with vasculogenesis.
Using isoform-specific PCR analysis we found that most tumors expressed only Delta16, some expressed both Delta16 and HER-2, and some expressed only HER-2. The level of Delta16 surface protein expression, as detected by FACS analysis with cross-reactive antibodies, was generally lower than that of HER-2.
We established representative cell lines for in vitro studies. Exposure of these cells to trastuzumab, lapatinib, or their combination showed that Delta16 did not confer resistance to HER-2-targeted drugs in comparison to full-length HER-2.
In conclusion, the study of Delta16/HER-2 double transgenic mice suggests that the activated isoform Delta16 plays a dominant role in the early phases of mammary carcinogenesis.
Supported by grants from the Italian Association for Cancer Research (AIRC)
Citation Format: Pier-Luigi Lollini, Valentina Grosso, Dario Ranieri, Arianna Palladini, Marianna Ianzano, Massimiliano Dall'Ora, Lorena Landuzzi, Giordano Nicoletti, Tania Balboni, Roberta Laranga, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni. Coexpression of Delta16 isoform and full-length HER-2 in F1 hybrid transgenic mice: effects on tumor growth and malignancy. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2774. doi:10.1158/1538-7445.AM2014-2774
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