girieca lorusso

Brain metastasis is a complication of increasing incidence in patients with breast cancer at advanced disease stage. It is a severe condition characterized by a rapid decline in quality of life and poor prognosis. There is a critical clinical need to develop effective therapies to prevent and treat brain metastases. Here, we describe a unique and robust spontaneous preclinical model of breast cancer metastasis to the brain (4T1-BM 2 ) in mice that has been instrumental in uncovering molecular mechanisms guiding metastatic dissemination and colonization of the brain. Key experimental findings were validated in the additional murine D2A1-BM 2 model and in human MDA231-BrM 2 model. Gene expression analyses and functional studies, coupled with clinical transcriptomic and histopathological investigations, identified connexins (Cxs) and focal adhesion kinase (FAK) as master molecules orchestrating breast cancer colonization of the brain. Cx31 promoted homotypic tumor cell adhesion, hetero...

Early detection and adjuvant therapies have significantly improved survival of patients with breast cancer over the past three decades. In contrast, management of metastatic disease remains unresolved. Brain metastasis is a late complication frequently observed among patients with metastatic breast cancer, whose poor prognosis calls for novel and more effective therapies. Here, we report that active hypoxia inducible factor-1 (HIF1) signaling and loss of the miRNA let-7d concur to promote brain metastasis in a recently established model of spontaneous breast cancer metastasis from the primary site to the brain (4T1-BM2), and additionally in murine and human experimental models of breast cancer brain metastasis (D2A1-BM2 and MDA231-BrM2). Active HIF1 and let-7d loss upregulated expression of platelet-derived growth factor (PDGF) B/A in murine and human brain metastatic cells, respectively, while either individual silencing of HIF1α and PDGF-A/B or let-7d overexpression suppressed bra...

In recent years, immunotherapy has become the most promising therapy for a variety of cancer types. The development of immune checkpoint blockade (ICB) therapies, the adoptive transfer of tumor-specific T cells (adoptive cell therapy (ACT)) or the generation of T cells engineered with chimeric antigen receptors (CAR) have been successfully applied to elicit durable immunological responses in cancer patients. However, not all the patients respond to these therapies, leaving a consistent gap of therapeutic improvement that still needs to be filled. The innate immune components of the tumor microenvironment play a pivotal role in the activation and modulation of the adaptive immune response against the tumor. Indeed, several efforts are made to develop strategies aimed to harness innate immune cells in the context of cancer immunotherapy. In this review, we describe the contribution of innate immune cells in T-cell-based cancer immunotherapy and the therapeutic approaches implemented t...

Matricellular proteins play multiple roles in primary tumor growth, local invasion and tumor angiogenesis. However, their contribution to metastasis and the putative mechanisms involved are less well characterized. In ER-negative human breast cancer, elevated expression levels of the matricellular protein Cysteine-rich angiogenic inducer 61 (CYR61) are associated with more aggressive progression. Here, we investigated the role of CYR61 in breast cancer lung metastasis using the triple negative human breast cancer cell lines MDA-MB-231 and SUM159. Silencing of CYR61 significantly decreased lung metastasis from tumors orthotopically implanted in pre-irradiated or naive mammary tissue and upon tail vein injection. Constitutive CYR61 silencing impaired cancer cell extravasation to the lung during the first 24 hours after tail vein injection. In contrast, CYR61 inducible silencing starting 24 hours after cancer cell injection had no impact on lung metastasis formation. In vitro experimen...

The numerous studies on Angiostatin (AST) mechanisms of angiogenesis inhibition have focused on the potential receptor-mediated interactions with endothelial cells. Although these studies have provided potential mechanisms, no single receptor system has been definitively shown to mediate AST activity. Recent studies have shown that inflammatory cells, in particular macrophages and neutrophils, are also targets of AST. These cells are part of a network that generates one of the most powerful anti-angiogenic cytokines: interleukin-12 (IL-12). We show that in in vivo preclinical studies, IL-12 is an essential mediator of AST antiangiogenic activity. Function blocking antibodies to IL-12 revert angiogenesis inhibition induced by AST. Further, AST is unable to exert angiogenesis inhibition in mice with gene targeted deletions of the IL-12 receptor IL-12R\uf0622, specific for IL-12, or in mice gene targeted for the IL- 12 p40 subunit. However, AST retains anti-angiogenic activity in IFN\uf067\uf020gene targeted mice, suggesting an IFN\uf067\uf020independent angiogenesis inhibition mechanism as previously observed. We also identified a short synthetic peptide localized to the lysine binding domain that completely reproduces the IL-12 dependent anti-angiogenic properties of AST. Since endothelial cells do not express the IL-12 receptor, nor do they essentially change gene expression patterns when treated with AST in vitro, our data show that the immune system forms an integral part of the anti-angiogenic effects of AST, by both responding to AST and providing downstream signals to the endothelium. AST induced IL-12 synthesis by human macrophages polarized toward M1 phenotype in vitro. We also identified a short synthetic peptide with potential application in oncological therapy, that reproduced the IL-12 dependent anti-angiogenic properties of angiostatin

A77 Hyperforin (Hyp) is the major lipophilic anti-depressant compound of St. John’s wort (Hypericum perforatum), here we investigated its potential anti-inflammatory and anti-angiogenic properties as a stable salt (Hyp-DHCA) in vitro and in vivo. Hyperforin was able to control in a dose-dependent manner the chemotaxis of polymorphonuclear (PNM) leukocytes, with no collateral effects on their viability. In particular, non-cytotoxic micromolar concentration of hyperforin restrained in vitro the capacity of PMNs and monocytes to migrate towards relevant chemotactic stimuli such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1, CCL2), respectively. The anti-angiogenic properties of Hyperforin was confirmed in vitro in the Matrigel morphogenesis assay, where it limited the formation of capillary-like structures. Moreover, in the in vivo matrigel-sponge model assay Hyperforin exhibited anti-inflammatory and anti-angiogenic activities. The evaluation of haemoglobin content in matrigel pellets as an indicator of angiogenesis shows a strong anti-angiogenic activity of Hyperforin, both mixed in the matrigel solution at the same concentration used in vitro or when injected i.p. (1mM) twice a day, with a pre-treatment starting two days before matrigel injection. Micromolar Hyperforin was strongly effective in inhibiting chemokine-triggered angiogenesis in a dose dependent manner. Furthermore, in the highly angiogenic Kaposi’s sarcoma xenograft model Hyperforin inhibited tumor growth and vascularization.
 >We demonstrate that Hyperforin is a potent inhibitor of inflammation-triggered angiogenesis induced by chemokines of tumor angiogenesis.

A78 Angiogenesis, the formation of new blood vessels from the preexisting network, is now recognized to be a necessary process for tumor growth and dissemination. We have observed that the anti-tumor effect after treatment with chemopreventive agents is often due to their anti-angiogenic activity, a concept that we coined as ‘angioprevention’. We previously observed that various molecules are able to act within the tumor micro-environment inhibiting the recruitment and/or the activation of endothelial cells and phagocytes of the innate immunity.
 >Here we examined the angiopreventive action of the retinoic acid derivative Rexinoid LG100268. Rexinoids selectively bind the nuclear receptor transcription factors known as retinoid X receptors and modulate the activity of entire regulatory networks. In particular we investigated the activity of Rexinoid LG100268 both in vitro and in vivo. In the in vitro matrigel morphogenesis assay 500nM LG100268 succeed in preventing HUVECs from forming capillary-like structures. However, LG100268 did not affect cell growth or show toxicity or apoptosis induction in HUVECs in vitro. In vivo in the matrigel sponge assay 20nM LG100268 mixed with matrigel turns out to reduce vessel formation. Systemic administration of 1uM LG100268 by i.p. injection also significantly inhibited angiogenesis in vivo.
 >The data suggest that LG100268 is a promising agent for chemoprevention and therapy of cancer.

Angiogenesis is the base for solid tumour growth and dissemination, and anti-angiogenic drugs have been demonstrated to be active in clinical trials. In addition, it has become increasingly clear that inflammation is a key component in tumour insurgence. Chemoprevention focuses on the primary or secondary prevention of cancer using natural or synthetic agents that usually show mild or no collateral effects. We have noted that angiogenesis, particularly 'inflammatory angiogenesis', is a common target of many chemopreventive molecules, where they most likely suppress the angiogenic switch in pre-malignant tumours, a concept we have termed 'angioprevention'. We have shown that various molecules, such as flavonoids, antioxidants and retinoids, act in the tumour microenvironment inhibiting the recruitment and/or activation of endothelial cells and phagocytes of the innate immunity. We have recently assessed the activity of novel compounds derived from the oleanolic acid triterpenoid, called CDDO-Me and CDDO-Imm. These compounds show a potent anti-angiogenic activity at low dosages. In vivo they inhibit angiogenesis in the Matrigel sponge assay and in KS-Imm (an immortalized Kaposi's sarcoma cell line) tumour growth. In vitro they are able to prevent endothelial cell tubulogenesis when cultured on Matrigel. In human umbilical vein endothelial (HUVE) cells these compounds can inhibit the activation of the extracellular signal-regulated kinase ERK1/2 pathway after stimulation with vascular endothelial growth factor (VEGF). Moreover, from immunofluorescence experiments we observed that treatment with these triterpenoids prevents nuclear factor NF-kappaB translocation into the nucleus and thereby the activation of downstream pathways. The particularly potent anti-angiogenic activity seen in vivo suggest that CDDO-Me may be interacting with an important network of molecular and cellular targets, on endothelial cells, and could be employed for 'angioprevention'. These substances are being assessed in phase I trials in humans in the United States.

We show that the synthetic oleanane triterpenoid, CDDO-methyl ester (CDDO-Me; methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate) is an effective agent for suppressing angiogenesis, both in cell culture and in vivo. The potency of CDDO-Me is particularly striking when dosed in vivo to inhibit the angiogenic effects of vascular endothelial growth factor and tumor necrosis factor-α in Matrigel sponge assays; activity is seen at i.p. doses of CDDO-Me as low as 0.003 mg/kg of body weight. If the Matrigel sponges are impregnated with CDDO-Me just before implantation in the mice, picomolar doses of CDDO-Me will suppress angiogenesis. CDDO-Me also inhibits growth of endothelial cells in monolayer cultures and suppresses neovascular morphogenesis in three-dimensional cultures, but significantly higher doses (50-200 nmol/L) are required. We also show antiangiogenic effects of CDDO-Me on xenografts of Kaposi's sarcoma cells in immunocompromised mice, using CD31 as a marker. Several known ind...

Endothelial cell senescence and apoptosis are features of numerous human pathologies including atherosclerosis, allograft vasculopathy, heart failure, diabetic retinopathy and scleroderma. In contrast, endothelial cell activation and replication associated with vessel proliferation and angiogenesis are now therapeutic targets in other diseases such as cancer and macular dystrophy. Finally, preventive medicine, in particular cardiovascular and cancer chemoprevention, commonly involve the endothelium. Here we discuss several aspects of the interplay between endothelial cell aging, apoptosis and senescence. Further, we show novel microarray data on endothelial cells "aged" in culture, and note that many genes regulated by the aging process are also modulated by a chemopreventive anti-angiogenic and anti-apoptotic drug, N-acetyl-cysteine (NAC). Focusing on one of these genes, the leukocyte adhesion protein E-selectin, we show that E-selectin is down-modulated with time in culture and upon treatment with NAC at mRNA and protein levels. This correlates with reduced adhesion of breast cancer cells and NF-kB activation in NAC treated endothelial cells. These data underscore the effects of a chemoprevention agent in modulating parameters associated with endothelial cell aging.

Prostate cancer is the most common cancer in men and one of the leading causes of cancer-related deaths in Western countries. The extraordinary biological heterogeneity, the increasing incidence of this disease, and the presence of putative premalignant conditions make prostate cancer a crucial pathology to study and test pharmacological or nutritional chemopreventive strategies. It has been demonstrated that the incidence of prostate cancer is lower in Asian people, and that it increases in Asian men living in Western countries; these data point to a pivotal role of diet in the onset of prostate cancer. A large amount of work has been done in investigating chemopreventive properties of dietary compounds widely used in Asian countries (i.e. soy, soybeans, green tea, fish) in respect of the oxidants- and meat-rich diet typical of Western people, particularly of central and northern Europe. Some dietary products appear promising as chemo-preventive agents for prostate cancer, because they display both anti-oxidant and anti-inflammatory activity - and inflammation is crucial for the aetiology of adeno-carcinoma of the prostate. There is increasing evidence for close correlation between inflammation, the microenvironment and tumour-associated neo-angiogenesis causing the adverse outcomes of prostate cancer. It may thus be useful to develop new strategies to couple the treatment of inflammation-related prostate cancer and the generation of angiopreventive or antiinflammatory molecules to prevent this disease. The search for compounds with few or no adverse effects - particularly cardiovascular - as compared with the agents currently in use is therefore of greatest relevance.

Purpose: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. Experimental Design: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. Results: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b+c-Kit+Ly6GhighLy6Clow(Gr1+) myeloid cells through the HIF1-dependent expression of Kit ligan...

Tumor-mobilized bone marrow–derived CD11b+ myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b+ myelomonocytic cells develop proangiogenic properties during their differentiation from CD34+ hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34+ progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b+ cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti–Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b+ myelomonocytes. In a mouse model of b...