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(NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods: The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo. Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions: The ADK-VR2 cell line is a promising NSCLC preclinical model for the study

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.

BackgroundMetastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30.MethodsPC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings.ResultsHuman membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30’s ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test,p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30NegSOCS3PosPC, when compared to patients with IL30PosSOCS3NegPC.ConclusionsMembrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS–FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin–GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS–FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2–2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS–FLI1 targets, both in vitro and in vivo, even in the presence of EWS–FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

// Arianna Palladini 1, * , Giordano Nicoletti 2, * , Alessia Lamolinara 3, * , Massimiliano Dall’Ora 1, * , Tania Balboni 1 , Marianna L. Ianzano 1 , Roberta Laranga 1 , Lorena Landuzzi 2 , Veronica Giusti 1 , Claudio Ceccarelli 1, 4 , Donatella Santini 4 , Mario Taffurelli 5 , Enrico Di Oto 6 , Sofia Asioli 6 , Augusto Amici 7 , Serenella M. Pupa 8 , Carla De Giovanni 1 , Elda Tagliabue 8 , Manuela Iezzi 3 , Patrizia Nanni 1 and Pier-Luigi Lollini 1 1 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 2 Rizzoli Orthopedic Institute, Laboratory of Experimental Oncology, Bologna, Italy 3 Aging Research Centre, “Gabriele d’Annunzio” University, Chieti, Italy 4 Pathology Unit, Policlinico S.Orsola-Malpighi University Hospital, Bologna, Italy 5 Department of Medical and Surgical Sciences of Bologna, Bologna, Italy 6 Anatomic Pathology, Department of Biomedical and Neuromotor Sciences, Bellaria Hospital, University of Bologna, Bologna, Italy 7 University of Camerino, Camerino, Italy 8 Istituto Nazionale Tumori, Milano, Italy * These authors have contributed equally to this work Correspondence to: Patrizia Nanni, email: patrizia.nanni@unibo.it Keywords: HER2, Delta16, trastuzumab, breast cancer, model of host-tumor interactions Received: December 16, 2016 Accepted: March 22, 2017 Published: April 13, 2017 ABSTRACT Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae , whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy.

(1) Background: Human epidermal growth factor receptor 2 (HER2)/neu-driven carcinogenesis is delayed by preventive vaccines able to elicit autochthonous antibodies against HER2/neu. Since cooperation between different receptor tyrosine kinases (RTKs) can occur in human as well as in experimental tumors, we investigated the set-up of DNA and cell vaccines to elicit an antibody response co-targeting two RTKs: HER2/neu and the Insulin-like Growth Factor Receptor-1 (IGF1R). (2) Methods: Plasmid vectors carrying the murine optimized IGF1R sequence or the human IGF1R isoform were used as electroporated DNA vaccines. IGF1R plasmids were transfected in allogeneic HER2/neu-positive IL12-producing murine cancer cells to obtain adjuvanted cell vaccines co-expressing HER2/neu and IGF1R. Vaccination was administered in the preneoplastic stage to mice prone to develop HER2/neu-driven, IGF1R-dependent rhabdomyosarcoma. (3) Results: Electroporated DNA vaccines for murine IGF1R did not elicit anti-mIGF1R antibodies, even when combined with Treg-depletion and/or IL12, while DNA vaccines carrying the human IGF1R elicited antibodies recognizing only the human IGF1R isoform. Cell vaccines co-expressing HER2/neu and murine or human IGF1R succeeded in eliciting antibodies recognizing the murine IGF1R isoform. Cell vaccines co-targeting HER2/neu and murine IGF1R induced the highest level of anti-IGF1R antibodies and nearly significantly delayed the onset of spontaneous rhabdomyosarcomas. (4) Conclusions: Multi-engineered adjuvanted cancer cell vaccines can break the tolerance towards a highly tolerized RTK, such as IGF1R. Cell vaccines co-targeting HER2/neu and IGF1R elicited low levels of specific antibodies that slightly delayed onset of HER2/neu-driven, IGF1R-dependent tumors.

Background Insulin-like Growth Factor Receptor-1 (IGF1R) system sustains the genesis of rhabdomyosarcoma through IGF2 autocrine overexpression. While several IGF1R-targeted strategies have been investigated to interphere with rhabdomyosarcoma growth, no attempt to neutralize IGF2 has been reported. We therefore studied the possibility to hamper rhabdomyosarcoma growth with passive and active immune approaches targeting IGF2. Methods A murine model developing IGF2-overexpressing pelvic rhabdomyosarcoma, along with IGF2-independent salivary carcinoma, was used to investigate the efficacy and specificity of passive anti-IGFs antibody treatment. Active vaccinations with electroporated DNA plasmids encoding murine or human IGF2 were performed to elicit autochthonous anti-IGF2 antibodies. Vaccinated mice received the intravenous injection of rhabdomyosarcoma cells to study the effects of anti-IGF2 antibodies against developing metastases. Results Passive administration of antibodies neutralizing IGFs delayed the onset of IGF2-overexpressing rhabdomyosarcoma but not of IGF2-independent salivary carcinoma. A DNA vaccine against murine IGF2 did not elicit antibodies, even when combined with Treg-depletion, while a DNA vaccine encoding the human IGF2 gene elicited antibodies crossreacting with murine IGF2. Mice with anti-IGF2 antibodies were partially protected against the metastatic growth of IGF2-addicted rhabdomyosarcoma cells. Conclusions Immune targeting of autocrine IGF2 inhibited rhabdomyosarcoma genesis and metastatic growth.

Simple Summary Hyperprogressive disease (HPD) is a pattern of paradoxical tumor progression that has been reported in patients treated with immune checkpoint inhibitors (ICIs). Although a large number of studies have investigated HPD and several associated factors have been reported, the mechanisms that drive the acceleration of tumor growth remain unknown. In this review, we discuss the possible role of IFN-γ and CD38 in the development of HPD, and we report the main findings in the scientific literature on the signaling pathways in which these factors take part, and their involvement in response and resistance to ICI therapy. Abstract Immune checkpoint inhibitors (ICIs) improve the survival of patients with multiple types of cancer. However, low response rates and atypical responses limit their success in clinical applications. The paradoxical acceleration of tumor growth after treatment, defined as hyperprogressive disease (HPD), is the most difficult problem facing clinicians and patients alike. The mechanisms that underlie hyperprogression (HP) are still unclear and controversial, although different factors are associated with the phenomenon. In this review, we propose two factors that have not yet been demonstrated to be directly associated with HP, but upon which it is important to focus attention. IFN-γ is a key cytokine in antitumor response and its levels increase during ICI therapy, whereas CD38 is an alternative immune checkpoint that is involved in immunosuppressive responses. As both factors are associated with resistance to ICI therapy, we have discussed their possible involvement in HPD with the conclusion that IFN-γ may contribute to HP onset through the activation of the inflammasome pathway, immunosuppressive enzyme IDO1 and activation-induced cell death (AICD) in effector T cells, while the role of CD38 in HP may be associated with the activation of adenosine receptors, hypoxia pathways and AICD-dependent T-cell depletion.

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2–M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881–92. ©2018 AACR.

The TS/A cell line was established in 1983 from a spontaneous mammary tumor arisen in an inbred BALB/c female mouse. Its features (heterogeneity, low immunogenicity and metastatic ability) rendered the TS/A cell line suitable as a preclinical model for studies on tumor–host interactions and for gene therapy approaches. The integrated biological profile of TS/A resulting from the review of the literature could be a path towards the description of a precision experimental model of mammary cancer.

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.

Human tumors are dynamic entities that undergo variation, selection and progression within the patient. How well patient-derived xenografts (PDX) recapitulate tumor dynamics? To investigate these aspects we established a collection of breast cancer PDX, representative of the main intrinsic subtypes. From 66 primary breast cancer specimens, we obtained 5 transplantable PDXs (8%). The highest rate of PDX stabilization was obtained for HER2-positive (40%) followed by triple negative (17%) and luminal B (14%) subtypes. In 3/66 cases a human lymphoma developed without any further evidence of breast cancer. Two HER-2-positive and one non-amplified, HER-2++, luminal B PDXs were serially transplanted in mice for 7-25 passages over a period of 2-4 years. Morphological and immunohistochemical (ER, PR, Ki67, p53, HER2, HER1, HER3, IGFR) features were highly stable over serial passages of PDXs, which retained the histology and the expression pattern of the tumor of origin. After the second passage, one HER-2++ amplified PDX, named BO-HAT4, was split in six different sublines, which were then studied separately to analyze random and selective events in long-term evolution. Two sublines progressively acquired a marked acceleration of tumor growth rate, whereas the remaining four did not. Metastatic spread was absent in early passages and appeared sporadically in late passages. In vitro cultures derived from early in vivo passages showed a rapid onset of cell senescence, whereas late in vivo passages gave rise to long-term in vitro cultures. However, we have not yet been able to obtain continuous cell lines from breast cancer PDX. To study the onset of resistance to HER-2 targeted therapies, sequential in vivo passages of BO-HAT4 were treated with trastuzumab, leading to a progressive loss of sensitivity. After one year of treatment BO-HAT4 was completely resistant to trastuzumab. We are currently investigating the molecular changes underlying trastuzumab resistance. Interestingly, both trastuzumab-sensitive and -resistant tumors were highly inhibited by neratinib. In conclusion, the low take rate as PDX prevents the generalized analysis of human breast cancer patients. Individual PDX allow the analysis of target therapy response and onset of resistance, however long-term study of transplantable breast cancer PDX show that tumor progression in these model systems is a late and random event. Supported by grants from the Italian Association for Cancer Research (AIRC) and the University of Bologna, Italy. Citation Format: Lorena Landuzzi, Marianna L. Ianzano, Claudio Ceccarelli, Enrico Di Oto, Giordano Nicoletti, Veronica Giusti, Roberta Laranga, Tania Balboni, Carla De Giovanni, Massimiliano Dall'Ora, Sofia Asioli, Arianna Palladini, Donatella Santini, Maria Pia Foschini, Mario Taffurelli, Pier-Luigi Lollini, Patrizia Nanni. Functional stability, progression and evolution of targeted drug sensitivity of HER-2-positive breast cancer patient-derived xenografts [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 216.

This study evaluated the effects of combining an OX40 agonistic antibody (aOX40) with a cell vaccine targeting HER2/neu, called “Triplex”. Such HER2/neu cell vaccine included two biological adjuvants (interleukin 12 (IL12) and allogeneic histocompatibility antigens) and was previously found able to prevent autochthonous HER2/neu-driven mammary carcinogenesis. Timing of aOX40 administration, concomitantly or after cell vaccination, gave opposite results. Unexpectedly, vaccine efficacy was hampered by concomitant OX40 triggering. Such decreased immunoprevention was likely due to a reduced induction of anti-HER2/neu antibodies and to a higher level of Treg activation. On the contrary, aOX40 administration after the completion of vaccination slightly but significantly increased immunopreventive vaccine efficacy, and led to increased production of GM-CSF and IL10. In conclusion, OX40 triggering can either impair or ameliorate immunoprevention of HER2/neu-driven mammary carcinogenesis depending on the schedule of aOX40 administration.

The in vivo evaluation of three modified polyethylene glycol (PEG)-anilinoquinazoline derivatives labeled with (124)I, (18)F, and (11)C as potential positron emission tomography (PET) bioprobes for visualizing epidermal growth factor receptor (EGFR) in cancer using small animal PET. PROCEDURES: Xenograft mice with the human glioblastoma cell lines U138MG (lacking EGFR expression) and U87MG.wtEGFR (transfected with an overexpressing human wild-type EGFR gene) were used. Static and dynamic PET imaging was conducted for all three PEGylated compounds. Tumor necrosis, microvessel density, and EGFR levels were evaluated by histopathology and enzyme-linked immunosorbent assay. RESULTS: Nineteen animal models were generated (two U138MG, three U87MG, 14 with both U138MG and U87MG bilateral masses). In static images, a slight increase in tracer uptake was observed in tumors, but in general, there was no retention of tracer uptake over time and no difference in uptake between U138MG and U87MG masses. In addition, no significant uptake was demonstrated in dynamic scans of the (18)F-PEG tracer. No necrosis was present except in four animals. MVD was 9.6 and 48 microvessels/×400 field in the U138GM and U87GM masses, respectively (p = 0.00008). Similarly, the microvessel grades were generally higher in the U87GM group (p = 0.002). Total EGFR amount was higher in U87MG than U138MG masses (p = 0.001), but the ratio of activated (pY1068) to total EGFR did not differ (p = 0.95). CONCLUSIONS: PEGylated tracers labeled with (11)C, (124)I, and (18)F showed no significant difference in uptake between U138MG and U87MG glioblastoma xenograft mice. The tracer binding with EGFR could be influenced by activation of the tyrosine kinase portion of the receptor which was similar in U138MG and U87MG. Despite these results, these tracers should be investigated in animal models with mutant EGFR genes to determine whether aberrant receptor function plays a role in tumor uptake

Overexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20–30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccination may represent a safer, more effective and cheaper alternative, although the induction of strong and durable autoantibody responses is hampered by immune-tolerogenic mechanisms. Using a novel virus-like particle (VLP) based vaccine platform we show that directional, high-density display of human HER2 on the surface of VLPs, allows induction of therapeutically potent anti-HER2 autoantibody responses. Prophylactic vaccination reduced spontaneous development of mammary carcinomas by 50%-100% in human HER2 transgenic mice and inhibited the growth of HER2-positive tumors implanted in wild-type mice. The HER2-VLP vaccine shows promise as a new cost-effective modality for prevention and treatment of HER2-positive cancer. The VLP platform may represent an effective tool for development of vaccines against other non-communicable diseases.

Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell-to-cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c-MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event-free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross-validation and meta-analysis, which are indispensable tools for a rare tumor such as EWS.

Rhabdomyosarcoma is a group of soft-tissue sarcomas that share features of skeletal myogenesis, but show extensive heterogeneity in histology, age and site of onset, and prognosis. This review matches recent molecular data with biological features of rhabdomyosarcoma. Alterations in molecular pathways, animal models, cell of origin and potential new therapeutic targets are discussed.

Apc(Min) mice are widely used for mechanism and efficacy studies associated with the development of chemopreventive agents. APC10.1 cells have been derived from Apc(Min) mouse adenomas and retain the heterozygous Apc genotype. We tested the hypothesis that this cell type may provide an in vitro model to predict chemopreventive activity of agents in the Apc(Min) mouse in vivo. The growth inhibitory properties of 14 putative colorectal cancer chemopreventive agents, tricin, apigenin, 3',4',5',5,7-pentamethoxyflavone, resveratrol, curcumin, 3,4-methylenedioxy-3',4',5'-trimethoxychalcone (DMU135), 3,4,5,4'-tetramethoxystilbene (DMU212), celecoxib, aspirin, piroxicam, all-trans-retinoic acid, difluoromethylornithine (DFMO), quercetin and cyanidin-3-glucoside, were studied in this cell line, and the IC(50) values were calculated. The IC(50) values were plotted against previously published data of reduction of adenoma numbers caused by these agents in Apc(Min) mice. The correlation co-efficient was 0.678 (p

Several autocrine and paracrine growth factor circuits have been found in human rhabdomyosarcoma cells. In this study we show that endothelin-3 (ET-3), a vasoactive peptide, is produced by human rhabdomyosarcoma cell lines, whereas it is not expressed by human sarcoma cell lines of non-muscle origin. We did not find evidence of a significant autocrine loop; nevertheless ET-3 produced by rhabdomyosarcoma cells can act as a paracrine factor, since it promotes migration of endothelial cells. Moreover ET-3 is present in plasma of mice bearing xenografts of human rhabdomyosarcoma cells, and may be potential new marker of the human rhabdomyosarcoma to be studied further.

Purpose: Autocrine/paracrine stimulation of c-kit has been recently observed in Ewing’s sarcoma (ES) cell lines. In this study, we tested the prognostic and therapeutic role of the receptor in this tumor. Methods: One hundred one ES tumor biopsies were evaluated for the expression of c-kit by the avidin-biotin-peroxidase procedure. Effectiveness of STI-571 (Gleevec; Novartis, Basel, Switzerland), a selective inhibitor of specific tyrosine kinases, was analyzed with respect to in vitro growth and migration inhibition, as single agent or in combination with doxorubicin. Results: Approximately 30% of patients expressed c-kit in their primary tumors. No significant association between the expression of the receptor and the clinical outcome was observed. In vitro growth of ES cell lines showing high levels of c-kit demonstrated limited inhibition by exposure to STI-571 (10 μmol/L is required to obtain 40% to 50% of growth inhibition). A decrease of stem-cell factor–mediated ES cell migration was also found. The drug acted additively with doxorubicin in inhibiting ES cell growth. Conclusion: The negative prognostic findings and the limited in vitro therapeutic activity of STI-571 indicate that the putative aberrant signaling provided by c-kit overexpression may be dispensable for ES development and unlikely to constitute a critical therapeutic target. Accordingly, the dose of STI-571 required to give a significant ES growth inhibition is much higher than for those tumors in which mutations of c-kit constitute a relevant pathogenetic event. Nevertheless, in the subset of ES patients showing a high level of c-kit expression, the activity of the drug may be exploited in combination with standard therapy.

FVB-NeuN (N#202) female mice transgenic for the HER-2/neu protooncogene driven by the murine mammary tumor virus (MMTV) promoter develop mammary carcinomas with a progression from focal atypical hyperplasia to in situ carcinoma and to invasive carcinoma that closely resembles that of human neoplasia. Here we report that the combination of tamoxifen plus interleukin 12 (IL-12) results in a very effective prevention of mammary carcinogenesis, significantly higher than those obtained with either tamoxifen or IL-12 alone. At 1 year of age, 20% of control mice resulted tumor-free, whereas 80% of mice receiving the combined treatment were tumor-free. At 2 years of age, less than 5% of control mice were tumor-free, as opposed to 70% of mice treated with tamoxifen plus IL-12. The combined treatment inhibited mammary carcinogenesis mainly through a reduction in the number of mammary cells at risk of progression, a reduction in estrogen receptors (ERs) expression and a reduction in the angiogenic support to mammary development, likely due to cross-talk between tamoxifen and interferon-gamma (IFN-gamma) (the main downstream mediator elicited in vivo by IL-12). The addition of IL-12 to the tamoxifen treatment more than doubled mouse lifetime and did not exacerbate known side effects of tamoxifen.

IGF-IR plays an essential role in the establishment and maintenance of the transformed phenotype of ES cells and interference with the IGF-IR pathways causes reversal of the malignant potential of this neoplasm. In this report, we stably transfected a dominant negative IGF-IR expression plasmid in an ES cell line to determine the effectiveness of this strategy against the in vitro and in vivo growth of ES cells. DXR sensitivity of TC-71 cells expressing dominant negative mutants of IGF-IR was also examined. The mutated IGF-IR that we used carries a mutation in the ATP-binding domain of the intracellular β subunit, while the extracellular, ligand-binding α subunit remains unchanged. Cells carrying the dominant mutant IGF-IR had a marked decrease in proliferation, a significant increase in anoikis-induced apoptosis and a severely reduced ability to form colonies in soft agar. In vivo, when cells carrying dominant negative IGF-IR were injected into nude mice, the tumor formation and metastatic abilities of ES cells were reduced and survival increased. Furthermore, transfected clones showed significantly higher sensitivity to DXR, a major drug in the treatment of ES. These results indicate that the IGF/IGF-IR stimulation of ES cells may be inhibited by expression of mutated IGF-IR on their surfaces and that this strategy may be considered a possible alternative to impair this important target of ES cells, whose therapeutic potential was further confirmed. © 2002 Wiley-Liss, Inc.

Background Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. Methods Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. Results Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. Conclusions Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients.

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75NTR. Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.

Cancer immunoprevention posits that the enhancement of immune defenses in healthy individuals could control tumor onset. Immunoprevention of viral tumors is already implemented at the population level for human hepatocellular and cervical carcinomas. Altogether, viral vaccines could prevent more than 10% of all human tumors. The big question is whether immunoprevention can be applied to nonviral tumors, including breast cancer. Promising results were obtained in preclinical models, in particular in HER-2/neu transgenic mice, which are prone to mammary carcinoma development, using vaccines against HER-2/neu oncoprotein p185. The life expectancy of vaccinated mice was more than doubled. Protective immune mechanisms elicited by effective vaccines were mainly based on helper T cell cytokines, in particular γ-interferon, and anti-p185 antibodies. The term “oncoantigens” was coined to define those antigenic molecules that, like HER-2, are indispensable for tumor growth, thus representing the best class of targets for cancer immunoprevention. The study of immunopreventive vaccines against subsequent phases of neoplastic progression showed a dramatic loss of efficacy against established mammary carcinomas, whereas the prevention of micrometastasis growth was successful. Preclinical results provide useful indications for the translation of cancer immunoprevention to humans, and useful hints for cancer immunotherapy.

Transgenic FVB-NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu-encoded p185neu protein. In vitro expression of p185neu among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185neu expression, along with others with very high p185neu protein level. Upon in vivo injection, p185neu-positive cells gave rise to fast-growing tumors with a short latency, while p185neu-negative cells required a very long latency time, and the resulting tumors were invariably p185neu-positive. The lower growth ability of p185neu-negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage-independent growth in soft agar revealed colony formation from p185neu-positive but not p185neu-negative cells. The direct involvement of p185neu in clonogenicity was demonstrated by the inhibition of p185neu-positive colony growth in soft agar in the presence of an anti-p185neu monoclonal antibody. By contrast, a higher level of anchorage-dependent clonogenic growth and proliferation was observed in p185neu-negative cells as compared to p185neu-positive cells, thus explaining the relative ease with which p185neu-negative cell lines and clones were established in vitro. Together, the results indicate that p185neu expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage-independent growth ability rather than to proliferation or host-dependent mechanisms. Int. J. Cancer 87:186–194, 2000. © 2000 Wiley-Liss, Inc.

Five human rhabdomyosarcoma cell lines were used to investigate the surface expression of HER/erbB receptors, particularly of HER-2, HER-3 and HER-4, by flow cytometry. HER-2 was expressed in 3/5 rhabdomyosarcoma cell lines. HER-3 was expressed in all rhabdomyosarcoma cell lines. None of the rhabdomyosarcoma cell lines investigated showed HER-4 surface expression. To study the biological activity of HER/erbB receptors in rhabdomyosarcomas, cells were cultured in the presence of glial growth factor 2 (GGF-2), a specific ligand of HER-3 that is able to stimulate myogenesis in normal myoblasts. In 3 of 3 human rhabdomyosarcoma cell lines positive for both HER-2 and HER-3 receptors, the treatment with GGF-2 induced a significant increase in myogenic differentiation.

Transgenic mice carrying the HER-2/neu proto-oncogene under tissue-specific transcriptional control of a mammary tumor virus long terminal repeat (Tg-MMTVneu mice) spontaneously develop mammary carcinomas. HER-2/neu is a tumor antigen that can be recognized by cytotoxic T lymphocytes if tumor cells present the appropriate major histocompatibility complex (MHC) class I glycoproteins. The purpose of this work was to assess whether mammary carcinomas arising in Tg-MMTVneu mice correctly expressed MHC (H-2q) class I gene products. We analyzed by flow cytometry 51 primary tumors from 19 transgenic mice. About one-half of the tumors showed a reduced expression of class I antigens. All tumors were highly positive for membrane neu. Some mice had multiple mammary carcinomas with widely different MHC expression levels, and most mice had at least one tumor with a low expression. Treatment with γ-interferon of carcinoma cells cultured in vitro induced a strong re-expression of H-2q antigens. Our results suggest that the immune response activated in vivo by HER-2/neu-positive tumors can lead to the emergence of escape variants characterized by a down-regulation of MHC class I products. Int. J. Cancer 77:937–941, 1998.© 1998 Wiley-Liss, Inc.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon α1 (IFN-α). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-α transfectants was similar to that of IFN-γ transfectants previously obtained from TSA. Admixture of IFN-α and IFN-γ transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-α or IFN-γ transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.

Background: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. Methods: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. Results: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. Conclusion: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Oncolytic viruses aim to specifically kill tumor cells. A major challenge is the effective targeting of disseminated tumors in vivo. We retargeted herpes simplex virus (HSV) tropism to HER-2 oncoprotein p185, overexpressed in ovary and breast cancers. The HER-2-retargeted R-LM249 exclusively infects and kills tumor cells expressing high levels of human HER-2. Here, we assessed the efficacy of systemically i.p. delivered R-LM249 against disseminated tumors in mouse models that recapitulate tumor spread to the peritoneum in women. The human ovarian carcinoma SK-OV-3 cells implanted intraperitoneally (i.p.) in immunodeficient Rag2−/−;Il2rg−/− mice gave rise to a progressive peritoneal carcinomatosis which mimics the fatal condition in advanced human patients. I.p. administration of R-LM249 strongly inhibited carcinomatosis, resulting in 60% of mice free from peritoneal diffusion, and 95% reduction in the total weight of neoplastic nodules. Intraperitoneal metastases are a common outcome in breast cancer: i.p. administration of R-LM249 strongly inhibited the growth of ovarian metastases of HER-2+ MDA-MB-453 breast cells. Brain metastases were also reduced. Cumulatively, upon i.p. administration the HER-2-redirected oncolytic HSV effectively reduced the growth of ovarian and breast carcinoma disseminated to the peritoneal cavity., Author Summary In the genome of human herpes simplex virus (HSV) we have replaced part of the sequences encoding the receptor-binding glycoprotein with antibody fragments directed against the oncoprotein HER-2, overexpressed in human breast and ovarian cancers. The retargeted HSV only infects and kills human cancer cells expressing high levels of HER-2. Earlier experiments showed that the retargeted HSV injected inside localized tumors inhibits human tumor growth in immunodeficient mice. As tumor dissemination is the major cause of cancer mortality, we have now used the HER-2-retargeted HSV to treat disseminated HER-2+ ovarian and breast cancer in immunodeficient mice. Intraperitoneal treatments significantly inhibited the peritoneal spread (carcinomatosis and ascites formation) of ovarian cancer cells and the metastatic growth of breast cancer cells in the ovaries. Brain metastases were also inhibited. Our results showed that a HER-2-redirected oncolytic HSV is an effective therapeutic agent against metastatic HER-2+ cancers and, more generally, provide the first demonstration of the efficacy of a systemically-administered, retargeted oncolytic HSV.

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.

Attempts to obtain mice reconstituted with a human immune system (HIS) mice, also referred to as HIS mice, date back to the end of 1980s, when mice carrying the Prkdcscid mutation were first used as recipients of human progenitors, with limited success (Shultz et al, 2007). In the last decade, mice with engineered combined immunodeficiencies were obtained and a more efficient engraftment of human hematopoietic stem cells (HSC) was described (Shultz et al, 2007; Manz and Di Santo, 2009; Brehm et al, 2010). Even though the full human reconstitution is generally not achieved, HIS mice have already given valuable results for a better understanding of the differentiation potential of human immune populations (Huntington and Di Santo, 2008; Meek et al, 2010) and for the study of viral human pathogens (Legrand et al, 2009). Moreover, HIS mice are interesting models for human vaccine development (Koo et al, 2009; Becker et al, 2010). Human immune system mice, could provide new experimental models to study the relationships between human tumours and human immune effectors (Brehm et al, 2010; Wege et al, 2011). BALB/c Rag2−/−;Il2rg−/− (hereafter referred to as BRG mice), display a severe combined immunodeficiency, with absence of T, B and NK cells (Nomura et al, 2008). These mice can unveal growth and metastatic ability of human xenografts, otherwise not apparent in mice with partial immunodeficiencies (Nanni et al, 2010). BALB/c Rag2−/−;Il2rg−/− mice have been effectively used to obtain HIS mice (Traggiai et al, 2004; Brehm et al, 2010) and to test antitumour activity of human immune effectors (Thiel et al, 2011). We exploited these features to study the possibility to induce immune responses against human cancer cells by human lymphocytes and their ability to control tumour growth and metastasis. As the tumoural counterpart, we selected a HER-2-positive human cancer cell line. Therapeutic targeting of HER-2-overexpressing cancers with the humanised monoclonal antibody trastuzumab has already entered the clinical practice, but the search for active immunisation strategies against HER-2 is also pursued (Wei et al, 2008). In transgenic models of HER-2-driven mammary carcinogenesis, immune targeting of HER-2 with both active and passive approaches has shown preventive and therapeutic activity (Lollini et al, 2011). Transgenic models are suitable to study, at best, the murine immune response against the transgenic human HER-2 molecule, but cellular or soluble immune effectors elicited in transgenic mice are not of human origin. In the first part of this study we investigated the reconstituting ability of two types of HSC sorted from human cord blood: CD34+ and CD133+ cells. CD34+ progenitors have been extensively used to obtain HIS mice, see for BRG the pioneering study of Traggiai et al (2004). CD133+ HSC engraftment was reported after in vitro culture in growth factor-supplemented medium (Drake et al, 2011), whereas studies with freshly isolated CD133+ HSC in BRG mice and a direct comparison with CD34+ HSC engraftment ability have not been reported. Subsequently, we investigated the ability to elicit an immune response against HER-2-positive human cancer cells.

In vivo studies of the metastatic process are severely hampered by the fact that most human tumor cell lines derived from highly metastatic tumors fail to consistently metastasize in immunodeficient mice like nude mice. We describe a model system based on a highly immunodeficient double knockout mouse, Rag2−/−;Il2rg−/−, which lacks T, B and NK cell activity. In this model human metastatic HER-2+ breast cancer cells displayed their full multiorgan metastatic potential, without the need for selections or additional manipulations of the system. Human HER-2+ breast cancer cell lines MDA-MB-453 and BT-474 injected into Rag2−/−;Il2rg−/− mice faithfully reproduced human cancer dissemination, with multiple metastatic sites that included lungs, bones, brain, liver, ovaries, and others. Multiorgan metastatic spread was obtained both from local tumors, growing orthotopically or subcutaneously, and from cells injected intravenously. The problem of brain recurrencies is acutely felt in HER-2+ breast cancer, because monoclonal antibodies against HER-2 penetrate poorly the blood-brain barrier. We studied whether a novel oral small molecule inhibitor of downstream PI3K, selected for its penetration of the blood-brain barrier, could affect multiorgan metastatic spread in Rag2−/−; Il2rg−/− mice. NVP-BKM120 effectively controlled metastatic growth in multiple organs, and resulted in a significant proportion of mice free from brain and bone metastases. Human HER-2+ human breast cancer cells in Rag2−/−;Il2rg−/− mice faithfully reproduced the multiorgan metastatic pattern observed in patients, thus allowing the investigation of metastatic mechanisms and the preclinical study of novel antimetastatic agents.

Purpose: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma). Experimental Design: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewing's sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine. Results: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G1 phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti–insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects. Conclusions: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients. Clin Cancer Res; 16(2); 530–40

Cancer vaccine feasibility would benefit from reducing the number and duration of vaccinations without diminishing efficacy. However, the duration of in vivo studies and the huge number of possible variations in vaccination protocols have discouraged their optimization. In this study, we employed an established mouse model of preventive vaccination using HER-2/neu transgenic mice (BALB-neuT) to validate in silico–designed protocols that reduce the number of vaccinations and optimize efficacy. With biological training, the in silico model captured the overall in vivo behavior and highlighted certain critical issues. First, although vaccinations could be reduced in number without sacrificing efficacy, the intensity of early vaccinations was a key determinant of long-term tumor prevention needed for predictive utility in the model. Second, after vaccinations ended, older mice exhibited more rapid tumor onset and sharper decline in antibody levels than young mice, emphasizing immune aging as a key variable in models of vaccine protocols for elderly individuals. Long-term studies confirmed predictions of in silico modeling in which an immune plateau phase, once reached, could be maintained with a reduced number of vaccinations. Furthermore, that rapid priming in young mice is required for long-term antitumor protection, and that the accuracy of mathematical modeling of early immune responses is critical. Finally, that the design and modeling of cancer vaccines and vaccination protocols must take into account the progressive aging of the immune system, by striving to boost immune responses in elderly hosts. Our results show that an integrated in vivo–in silico approach could improve both mathematical and biological models of cancer immunoprevention. Cancer Res; 70(20); 7755–63. ©2010 AACR.

The relationship between autocrine interferon (IFN) production and the expression of class I Major Histocompatibility Complex (MHC) membrane glycoproteins in vitro was investigated in a panel of murine transformed cells of nonhaemopoietic origin. The panel included 11 cell lines of H-2Kb haplotype derived from fibrosarcomas, carcinomas and melanoma, and from transformed fibroblasts. IFN activity was detected in the conditioned medium of nine cell lines; fibrosarcomas were among the high IFN producers, while the non-producers were a melanoma clone and a lung carcinoma cell line. A significant correlation was found between IFN production and the expression of H-2K/D glycoproteins, thus suggesting that long-term maintainment of MHC glycoprotein expression in vitro could be mediated by self produced IFN. Two IFN producer cell lines, MN/MCA1 and R80/17, were cultured in the presence of a blocking antiserum against IFN-alpha/beta: a significant decrease in H-2b expression was observed, thus indicating the existence of an autocrine IFN circuit. Taken together these findings suggest that release of IFN is a frequent event among transformed nonhaemopoietic cells, and that self-produced IFN contributes to the regulation of MHC antigen levels in solid tumours.

In 2007, an Italian cancer research group proposed a specific concerted action aimed at the “analytical and clinical validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis.” The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators' meeting, “Analytical and clinical validation of new biomarkers for early diagnosis,” was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.

Immunodeficient animal models are invaluable tools to investigate the metastatic propensity of human tumours. However residual immune responses, in particular natural killer (NK) cells, severely hamper the traffic and growth of human tumour cells. We studied whether a genetically modified mouse host lacking T, B and NK immunity allowed an improved expression of the metastatic phenotype of malignant human tumours. Metastatic spread of a panel of human sarcoma cell lines was studied in double knockout Rag2(-/-);gammac(-/-) mice in comparison with NK-depleted nude mice. Rag2(-/-);gammac(-/-) mice receiving intravenous (i.v.) or subcutaneous (s.c.) human sarcoma cell lines developed extensive multiorgan metastases. Metastatic efficiency in Rag2(-/-);gammac(-/-) was superior than in nude mice in terms of both metastatic sites and metastasis number. Metastatic growth in Rag2(-/-);gammac(-/-) mice was faster than that in nude mice, thus allowing an earlier metastasis evaluation. Most human sarcomas metastasised in the liver of Rag2(-/-);gammac(-/-) mice, a kind of organ preference undetectable in nude mice and specific of sarcomas, as several carcinoma cell lines failed to colonise the liver of Rag2(-/-);gammac(-/-) mice, independently of their metastatic spread to other sites. In vitro analysis of the molecular mechanisms of liver metastasis of sarcomas implicated liver-produced growth and motility factors, in particular the insulin-like growth factor (IGF) axis. NVP-BEZ235, a specific inhibitor of downstream signal transduction targeting PI3K and mTOR, strongly inhibited liver metastasis of human sarcoma cells. In conclusion, the Rag2(-/-);gammac(-/-) mouse model allowed the expression of human metastatic phenotypes inapparent in conventional immunodeficient mice and the preclinical testing of appropriate targeted therapies.

Rhabdomyosarcoma is a tumor of striated muscle origin that displays defective myogenic differentiation. Terminal myogenesis switches off cell proliferation and migration, hence, the promotion of rhabdomyosarcoma differentiation should antagonize tumor growth and metastasis. Terminal myogenesis is controlled by cell-intrinsic myogenic transcription factors like myogenin and environmental mediators like interleukin 4 (IL-4). We studied whether the expression of myogenin or exposure to IL-4 could promote the myogenesis of poorly differentiating human rhabdomyosarcoma cells RD/12. Forced expression of myogenin amplified myosin expression and the formation of myotube-like elements, inhibited cell migration, and reduced the growth of local tumors and liver metastases in immunodepressed mice. In contrast, exposure to IL-4 promoted cell proliferation and survival, especially at high cell density, inhibited myogenin expression, and myogenesis. Moreover, IL-4 stimulated the directed migration of cells with low myogenin levels, but not of cells with higher (spontaneous or forced) levels. Thus, IL-4, which was known to promote late stages of normal myogenesis, favors growth and migration, and inhibits further differentiation of the myogenic stages attained by rhabdomyosarcoma cells. Strategies to increase myogenin expression and block IL-4 could simultaneously reduce growth and migration, and enhance terminal differentiation of rhabdomyosarcoma, thus contributing to the control of tumor growth and metastatic spread. [Mol Cancer Ther 2009;8(4):754–61]

The epidermal growth factor receptor (EGFr) is one of the most studied molecules as a target for cancer therapy. Over these last few years, several studies attempting to identify predictive biomarkers of treatment response, such as the receptor status or other molecules related to the downstream signalling pathway, have been conducted. However, from a clinical point of view, the information obtained from ex vivo analyses still has various limitations that may be overcome by the combination with molecular imaging technologies which may provide a noninvasive, global, in vivo evaluation of the molecular tumour background. The aim of this review is to report the preclinical results of all positron emission tomography (PET) tracers synthesized until now for in vivo detection of EGFr in cancer. Two classes of PET compounds have been developed: labelled small molecules such as tyrosine kinase inhibitors and labelled monoclonal antibodies. The in vitro and in vivo results of these PET tracers are very different depending on the chemical properties, positron emission radionuclide, or animal models. As a consequence, various critical questions are still open, and the implications of a translation in the clinical setting for EGFr imaging in cancer patients is discussed.

The development of prophylactic cancer vaccines that protect healthy hosts from tumor development leaves open the question whether such vaccines are also effective against established tumors and metastases. We tested the therapeutic activity of a proven prophylactic anti-HER-2/neu vaccine against successive stages of mammary carcinoma progression in HER-2/neu transgenic mice. The vaccine consisted of transgenic mammary carcinoma cells expressing HER-2/neu and two adjuvants: allogeneic class I histocompatibility antigens and interleukin (IL)-12. Vaccination of mice bearing lung micrometastases resulted in a 90% inhibition of metastasis development, whereas vaccination of mice with incipient local tumors was ineffective. The antimetastatic response was hampered by immune tolerance, as the protection of transgenic mice was lower than that of wild-type congenics not tolerant to HER-2/neu. A significant gain in immunotherapeutic activity in transgenic mice was obtained through the coadministration of anti-CD25 monoclonal antibody targeting regulatory T cells, which resulted in a >99% inhibition of metastasis. The immune responses elicited in transgenic mice comprised the activation of lung granulocytes and macrophages and of systemic adaptive responses based on helper T cells and their cytokines (IFN-γ and IL-4) and anti-HER-2/neu antibodies. Dissection of relevant antimetastatic mechanisms by means of knockout mice and of depleting antibodies revealed a major difference between tumor prevention, which was completely dependent on anti-HER-2/neu antibodies, and metastasis therapy, which was antibody independent. In conclusion, a vaccine successfully developed for cancer immunoprevention showed a strong therapeutic activity against lung metastases mediated by protective immune mechanisms distinct from those preventing the onset of primary mammary carcinoma. [Cancer Res 2007;67(22):11037–44]

The aim of this study is to investigate whether an active immunoprophylactic approach combining specific antigens and adjuvant stimuli would be able to inhibit prostate carcinogenesis in transgenic TRAMP mice. A vaccine consisting of allogeneic large T antigen (TAg)-positive SV40-transformed cells combined with systemic recombinant IL-12 was administered to TRAMP mice, starting from when they were still tumor-free at 5-6 weeks of age. The combined vaccine significantly inhibited prostate carcinogenesis, giving a more than doubled median latency time of prostatic tumors (53 weeks in comparison to 26 weeks in control mice). Vaccination with cells alone or IL-12 treatment alone was poorly effective (median latency of 30 and 39 weeks, respectively). The combined vaccine induced a very high CD4 response biased toward the Th1 pathway, with the induction of a humoral response that included TAg-specific antibodies. Therefore, such active immunoprophylactic approach based on the combination of allogeneic SV40 TAg-positive cells and systemic administration of recombinant IL-12 significantly delayed autochthonous urogenital carcinogenesis driven by SV40 TAg in TRAMP mice.

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular protein that belongs to the CCN (CYR61, CTGF, NOV) protein family. It is highly expressed by human rhabdomyosarcoma cells and sustains their survival. In this study we investigated CCN2 expression in a mouse model of spontaneous rhabdomyosarcomagenesis that combines HER-2/neu oncogene activation and p53 oncosuppressor gene inactivation (BALB-p53neu mice). Murine rhabdomyosarcoma cells showed a 4-26 fold increase in CCN2 mRNA expression regarding to normal thigh muscle. Moreover, they expressed CCN2 protein at levels comparable to human rhabdomyosarcoma cells. Therefore BALBp53neu mice might be useful for the evaluation of the role played by CCN2 in rhabdomyosarcoma in vivo.

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. In this system it has clearly been shown that the activation of immune defences in healthy individuals can effectively prevent the subsequent onset of highly aggressive mammary carcinomas. A complete prevention was obtained using a combination of three signals (the so called "triplex" vaccine) that included the specific antigen (p185, the product of HER-2/neu) and nonspecific signals like allogeneic histocompatibility antigens and interleukin 12. The analysis of protective immune responses in models of cancer immunoprevention revealed some unexpected features, in particular the central role of antibodies in immunoprevention, at variance with conventional immuno-therapy which is firmly based on cytotoxic T cells. In the HER-2/neu system anti-p185 antibodies, in addition to immunological functions leading to tumor cell lysis, inhibit p185 dimerization and induce its internalization, resulting in the inhibition of mitogenic signaling. Most current tumor antigens appear to be unsuitable targets for cancer immunoprevention. An ideal antigen should have a crucial pathogenetic role in tumor growth to avoid the selection of antigen loss variants. Downregulation of major histocompatibility complex (MHC) expression during tumor progression frequently limits antigen recognition by MHC-restricted T cells. Thus an ideal antigen for cancer immunoprevention should be recognized both by T cells and by antibodies. Antibody binding to cell surface oncogenic determinants, in addition to complement- and cell-mediated tumor cell lysis, can block mitogenic signaling and induce internalization, resulting in tumor growth arrest. A search for new tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response also in MHC loss variants. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention and will provide new targets also for cancer immunotherapy.

This study evaluated the ability of cytokine-engineered allogeneic (H-2q) HER-2/neu-positive cells to prevent tumor development in mammary cancer-prone virgin female BALB/c (H-2d) mice transgenic for the transforming rat HER-2/neu oncogene (BALB-neuT mice). Repeated vaccinations with cells engineered to release interleukin (IL)-2, IL-12, IL-15, or IFN-γ showed that IL-12-engineered cell vaccines had the most powerful immunopreventive activity, with >80% of 1-year-old BALB-neuT mice free of tumors. On the contrary all of the untreated mice and all of the mice vaccinated with IL-12-engineered cells lacking either HER-2/neu or allogeneic antigens developed mammary carcinomas within 22 or 33 weeks, respectively. Whole mount, histology, immunohistochemistry, and gene expression profile analysis showed that vaccination with IL-12-engineered cells maintained 26-week mammary glands free of neoplastic growth, with a gene expression profile that clustered with that of untreated preneoplastic glands. The IL-12-engineered cell vaccine elicited a high production of IFN-γ and IL-4 and a strong anti-HER-2/neu antibody response. Immune protection was lost or markedly impaired in BALB-neuT mice lacking IFN-γ or antibody production, respectively. The protection afforded by the IL-12-engineered cell vaccine was equal to that provided by the systemic administration of recombinant IL-12 in combination with HER-2/neu H-2q cell vaccine. However, IL-12-engineered cell vaccine induced much lower circulating IL-12 and IFN-γ, and therefore lower potential side effects and systemic toxicity.

APC10.1 is a new intestinal cell line derived from ApcMin/+ mice that retains both the heterozygous Apc genotype and a nonactivated Wnt signaling pathway and displays an early neoplastic phenotype. Although tumorigenic both in immunodepressed and in immunocompetent syngeneic mice, it requires a high cell dose and a long latency. Its epithelial/intestinal origin is shown, in a gene expression profile, by the expression of epithelial transcripts (such as cytokeratin and laminin isoforms) and of developmental regulatory genes (such as Tcf-4, Hnf3beta, p21, Ihh, Hes1) necessary for, or involved in, the maintenance of intestinal stem cells. The lack of activation of the Wnt cascade in APC10.1 cells is shown both by the expression profile of Wnt target genes and by the standard TCF reporter assay. APC10.1 cell line is a novel in vitro model that can contribute to a better understanding of the clinical evolution of familial adenomatous polyposis and to finding of new prophylactic and therapeutic approaches. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Vaccines effectively prevent the onset of tumors in transgenic mice carrying activated oncogenes; however, human tumors are caused by combined alterations in oncogenes and oncosuppressor genes. We evaluated the impact of prophylactic vaccines in HER-2/neu transgenic, p53 wild-type/null mice that succumb to an aggressive cancer syndrome comprising mammary and salivary gland carcinomas and rhabdomyosarcoma. A vaccine made of allogeneic mammary carcinoma cells expressing HER-2/neu and interleukin 12 afforded long-term protection from tumor onset. Tumor prevention was mediated by T cell–derived cytokines, in particular γ-interferon, and by anti–HER-2/neu antibodies. HER-2/neu expression was inhibited in target tissues of vaccinated mice, and somatic loss of the wild-type p53 allele did not occur. A highly effective vaccine against a single oncoprotein induced a powerful immune response that arrested multistep carcinogenesis in distinct target tissues.

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

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