Background:Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated.Methods:Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice.Results:Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation.Conclusions:This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

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To assess the therapeutic outcome of selective block of VEGFR1, we have evaluated the activity of a new specific antagonist of VEGFR1, named iVR1 (inhibitor of VEGFR1), in syngenic and xenograft colorectal cancer models, in an artificial model of metastatization, and in laser-induced choroid neovascularization. iVR1 inhibited tumor growth and neoangiogenesis in both models of colorectal cancer, with an extent similar to that of bevacizumab, a monoclonal antibody anti-VEGF-A. It potently inhibited VEGFR1 phosphorylation in vivo, determining a strong inhibition of the recruitment of monocyte-macrophages and of mural cells as confirmed, in vitro, by the ability to inhibit macrophages migration. iVR1 was able to synergize with irinotecan determining a shrinkage of tumors that became undetectable after three weeks of combined treatment. Such treatment induced a significant prolongation of survival similar to that observed with bevacizumab and irinotecan combination. iVR1 also fully prevented lung invasion by HCT-116 cells injected in mouse tail vein. Also, iVR1 impressively inhibited choroid neovascularization after a single intravitreal injection. Collectively, data showed the strong potential of iVR1 peptide as a new anti-tumor and anti-metastatic agent and demonstrate the high flexibility of VEGFR1 antagonists as therapeutic anti-angiogenic agents in different pathological contexts.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward beta-amyloid, whereas minimizing the risks of a cell-mediated adverse reaction. We describe here two novel anti-beta-amyloid vaccines that consist of 'virus like particles' formed by a domain of the bacterial protein E2 that is able to self-assemble into a 60-mer peptide. Peptides 1-11 and 2-6 of beta-amyloid were displayed as N terminal fusions on the surface of the E2 particles. E2-based vaccines induced a fast-rising, robust and persistent antibody response to beta-amyloid in all vaccinated mice. The immune memory induced by a single administration of vaccine (1-11) E2 can be rapidly mobilized by a single booster injection, leading to a very high serum concentration of anti-beta-amyloid antibodies (above 1 mg ml À1 ). E2 vaccination polarizes the immune response toward the production of the anti-inflammatory cytokine interleukin-4 and does not induce a T cell response to beta-amyloid. Thus, E2-based vaccines are promising candidates for the development of immunotherapy protocols for AD. Alzheimer's disease (AD) is characterized by deposition of insoluble protein aggregates in the brain, known as amyloid plaques, which are mainly composed of beta-amyloid peptide. Active and passive immunization studies performed in transgenic mouse models of beta-amyloid deposition have demonstrated that antibodies against beta-amyloid are able to reduce plaques and improve cognition (reviewed in 1-3 ). In mouse models of AD, induction of a high titer of anti-beta-amyloid antibodies correlated with the therapeutic efficacy of vaccination [4][5][6] A clinical trial of immunization of AD patients with whole preaggregated beta-amyloid peptide has shown that immunization has the potential to reduce amyloid plaques in the brains of AD patients and delay disease progression. 7,8 However, the trial was interrupted when 6% of patients developed an adverse inflammatory reaction involving infiltration of T cells into the brain; moreover, only 59 of 300 individuals who received the vaccine mounted a humoral anti-beta-amyloid response with a titer higher than 1:2200. 9 Individuals with the highest titers of anti-beta-amyloid antibodies demonstrated the most pronounced depletion of plaques. 10 The development of a Th1-type response was found to correlate with an adverse inflammatory reaction. 11 The development of an effective and safe immunotherapy protocol faces two challenges, namely, overcoming the low immunogenicity of the beta-amyloid peptide and avoiding detrimental inflammatory reactions in the brain.The N-terminus of beta-amyloid is considered the most promising antibody target for inclusion in recombinant vaccines. 1 We have previously observed reduced deposition of beta-amyloid plaques in mice that received monthly injections of phage-based vaccine fdAD (2-6), a bacteriophage that displays the epitope AEFRH of betaamyloid as an N terminal fusion to the major capsid p...

Major histocompatibility complex class II mRNAs encode heterodimeric proteins involved in the presentation of exogenous antigens during an immune response. Their 3′UTRs bind a protein complex in which we identified two factors: EBP1, an ErbB3 receptor-binding protein and DRBP76, a double-stranded RNA binding nuclear protein, also known as nuclear factor 90 (NF90). Both are well-characterized regulatory factors of several mRNA molecules processing. Using either EBP1 or DRBP76/NF90-specific knockdown experiments, we established that the two proteins play a role in regulating the expression of HLA-DRA, HLA-DRB1 and HLA-DQA1 mRNAs levels. Our study represents the first indication of the existence of a functional unit that includes different transcripts involved in the adaptive immune response. We propose that the concept of ‘RNA operon’ may be suitable for our system in which MHCII mRNAs are modulated via interaction of their 3′UTR with same proteins.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

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