Alessia Lamolinara

Multiorgan Metastasis of Human HER-2+ Breast Cancer in Rag2−/−;Il2rg−/− Mice and Treatment with PI3K Inhibitor

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.

Activated d16HER2 Homodimers and SRC Kinase Mediate Optimal Efficacy for Trastuzumab

A splice isoform of the HER2 receptor that lacks exon 16 (d16HER2) is expressed in many HER2-positive breast tumors, where it has been linked with resistance to the HER2-targeting antibody trastuzumab, but the impact of d16HER2 on tumor pathobiology and therapeutic response remains uncertain. Here, we provide genetic evidence in transgenic mice that expression of d16HER2 is sufficient to accelerate mammary tumorigenesis and improve the response to trastuzumab. A comparative analysis of effector signaling pathways activated by d16HER2 and wild-type HER2 revealed that d16HER2 was optimally functional through a link to SRC activation (pSRC). Clinically, HER2-positive breast cancers from patients who received trastuzumab exhibited a positive correlation in d16HER2 and pSRC abundance, consistent with the mouse genetic results. Moreover, patients expressing high pSRC or an activated d16HER2 metagene were found to derive the greatest benefit from trastuzumab treatment. Overall, our results establish the d16HER2 signaling axis as a signature for decreased risk of relapse after trastuzumab treatment.

Resolvin D1 enhances the resolution of lung inflammation caused by long-term Pseudomonas aeruginosa infection

Pseudomonas aeruginosa lung infection is a main cause of disability and mortality worldwide. Acute inflammation and its timely resolution are crucial for ensuring bacterial clearance and limiting tissue damage. Here, we investigated protective actions of resolvin (Rv) D1 in lung infection induced by the RP73 clinical strain of P. aeruginosa. RvD1 significantly diminished bacterial growth and neutrophil infiltration during acute pneumonia caused by RP73. Inoculum of RP73, immobilized in agar beads, resulted in persistent lung infection up to 21 days, leading to a non resolving inflammation reminiscent of human pathology. RvD1 significantly reduced bacterial titer, leukocyte infiltration, and lung tissue damage. In murine lung macrophages sorted during P. aeruginosa chronic infection, RvD1 regulated the expression of Toll-like receptors, downstream genes, and microRNA (miR)-21 and 155, resulting in reduced inflammatory signaling. In vitro, RvD1 enhanced phagocytosis of P. aeruginosa by neutrophils and macrophages, recapitulating its in vivo actions. These results unveil protective functions and mechanisms of action of RvD1 in acute and chronic P. aeruginosa pneumonia, providing evidence for its potent pro-resolution and tissue protective properties on airway mucosal tissue during infection.

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

The cyclic (c)AMP responsive element binding protein (CREB) plays a key role in many cellular processes, including differentiation, proliferation, and signal transduction. Furthermore, CREB overexpression was found in tumors of distinct origin and evidence suggests an association with tumorigenicity. To establish a mechanistic link between HER-2/neu–mediated transformation and CREB protein expression and function, in vitro models of HER-2/neu–overexpressing and HER-2/neu–negative/silenced counterparts as well as human mammary carcinoma lesions with defined HER-2/neu status were used. HER-2/neu overexpression resulted in the induction and activation of CREB protein in vitro and in vivo, whereas short hairpin RNA (shRNA)–mediated inhibition of HER-2/neu correlated with downregulated CREB activity. CREB activation in HER-2/neu–transformed cells enhanced distinct signal transduction pathways, whereas their inhibition negatively interfered with CREB expression and/or activation. CREB downregulation in HER-2/neu–transformed cells by shRNA and by the inhibitors KG-501 and lapatinib caused morphologic changes, reduced cell proliferation with G0–G1 cell-cycle arrest, which was rescued by CREB expression. This was accompanied by reduced cell migration, wound healing, an increased fibronectin adherence, invasion, and matrix metalloproteinase expression. In vivo shCREB-HER-2/neu+ cells, but not control cells, exerted a significantly decreased tumorgenicity that was associated with decreased proliferative capacity, enhanced apoptosis, and increased frequency of T lymphocytes in peripheral blood mononuclear cells. Thus, CREB plays an important role in the HER-2/neu–mediated transformation by altering in vitro and in vivo growth characteristics. Implications: These data suggest that CREB affects tumor immunogenicity and is a potential target for cancer therapy. Mol Cancer Res; 11(11); 1462–77. ©2013 AACR.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC.

Vitamin D3 improves the effects of low dose Der p 2 allergoid treatment in Der p 2 sensitized BALB/c mice.

BackgroundAirborne allergens can induce an immunological chronic disease characterized by airway hyper responsiveness and inflammation, mediated by exaggerated Th2 immune response. Allergen-specific immunotherapy (AIT) is effective for treating this condition because it is able to modify its natural course by opposing the underlying pathogenic mechanisms and determining immune suppression, immune deviation and tolerance. The rational for the present study was to investigate the possibility of improving allergoid-based IT in terms of efficacy and safety. Recently, 1α,25-dihydroxyvitamin D3 (VD3), the active metabolite of vitamin D3, was described to be a potent inducer of T regulatory cells and to be a good adjuvant in AIT settings.MethodsWe investigated whether the co-administration of VD3 could potentiate the effect of AIT even when added to a low dose of chemically-modified monomeric allergoid of Der p 2 (d2-OID), in a Derp p 2 (d2)-sensitized BALB/c mice model. Control groups where treated with sham, VD3 alone or d2-OID only.ResultsThe d2-OID alone was not fully successful, as expected for a low dose. VD3 administration was associated with some valuable, although limited, changes in the immunological parameters in the lung. On the contrary, the VD3 adjuvated allergoid vaccine induced the most prominent reduction of airway eosinophilia and Th2 cytokines and concomitant increase of T regulatory cells and IL-10 in the lung and Der p 2-specific IgG2a in the serum.ConclusionsThe addition of VD3 to a conventional AIT protocol would allow the reduction of allergoid dose needed and therefore, the production costs. Moreover, beneficial immunomodulatory effects have been achieved by the oral administration which might favour the management of the therapy by the patients and their adherence, possibly enhancing the efficacy of the treatment.

Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial–mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

Feasibility of Telomerase-Specific Adoptive T-cell Therapy for B-cell Chronic Lymphocytic Leukemia and Solid Malignancies

Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2-restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self-MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540-51. ©2016 AACR.

Intradermal DNA Electroporation Induces Cellular and Humoral Immune Response and Confers Protection against HER2/neu Tumor

Skin represents an attractive target for DNA vaccine delivery because of its natural richness in APCs, whose targeting may potentiate the effect of vaccination. Nevertheless, intramuscular electroporation is the most common delivery method for ECTM vaccination. In this study we assessed whether intradermal administration could deliver the vaccine into different cell types and we analyzed the evolution of tissue infiltrate elicited by the vaccination protocol. Intradermal electroporation (EP) vaccination resulted in transfection of different skin layers, as well as mononuclear cells. Additionally, we observed a marked recruitment of reactive infiltrates mainly 6–24 hours after treatment and inflammatory cells included CD11c+. Moreover, we tested the efficacy of intradermal vaccination against Her2/neu antigen in cellular and humoral response induction and consequent protection from a Her2/neu tumor challenge in Her2/neu nontolerant and tolerant mice. A significant delay in transplantable tumor onset was observed in both BALB/c (p ≤ 0,0003) and BALB-neuT mice (p = 0,003). Moreover, BALB-neuT mice displayed slow tumor growth as compared to control group (p < 0,0016). In addition, while in vivo cytotoxic response was observed only in BALB/c mice, a significant antibody response was achieved in both mouse models. Our results identify intradermal EP vaccination as a promising method for delivering Her2/neu DNA vaccine.

Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis

IntroductionWe 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.MethodsHER2/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.ResultsIL15KO/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.ConclusionsWe 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.