Arianna Pagani

miR181b is induced by the chemopreventive polyphenol curcumin and inhibits breast cancer metastasis via down-regulation of the inflammatory cytokines CXCL1 and -2.

Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and ‐2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti‐tumorigenic properties of this natural occurring polyphenol.

Orchestration of angiogenesis by immune cells.

It is widely accepted that the tumor microenvironment (TUMIC) plays a major role in cancer and is indispensable for tumor progression. The TUMIC involves many “players” going well beyond the malignant-transformed cells, including stromal, immune, and endothelial cells (ECs). The non-malignant cells can acquire tumor-promoting functions during carcinogenesis. In particular, these cells can “orchestrate” the “symphony” of the angiogenic switch, permitting the creation of new blood vessels that allows rapid expansion and progression toward malignancy. Considerable attention within the context of tumor angiogenesis should focus not only on the ECs, representing a fundamental unit, but also on immune cells and on the inflammatory tumor infiltrate. Immune cells infiltrating tumors typically show a tumor-induced polarization associated with attenuation of anti-tumor functions and generation of pro-tumor activities, among these angiogenesis. Here, we propose a scenario suggesting that the angiogenic switch is an immune switch arising from the pro-angiogenic polarization of immune cells. This view links immunity, inflammation, and angiogenesis to tumor progression. Here, we review the data in the literature and seek to identify the “conductors” of this “orchestra.” We also suggest that interrupting the immune → inflammation → angiogenesis → tumor progression process can delay or prevent tumor insurgence and malignant disease.

Inflammatory Angiogenesis and the Tumor Microenvironment as Targets for Cancer Therapy and Prevention

In addition to aberrant transformed cells, tumors are tissues that contain host components, including stromal cells, vascular cells (ECs) and their precursors, and immune cells. All these constituents interact with each other at the cellular and molecular levels, resulting in the production of an intricate and heterogeneous complex of cells and matrix defined as the tumor microenvironment. Several pathways involved in these interactions have been investigated both in pathological and physiological scenarios, and diverse molecules are currently targets of chemotherapeutic and preventive drugs. Many phytochemicals and their derivatives show the ability to inhibit tumor progression, angiogenesis, and metastasis, exerting effects on the tumor microenvironment. In this review, we will outline the principal players and mechanisms involved in the tumor microenvironment network and we will discuss some interesting compounds aimed at interrupting these interactions and blocking tumor insurgence and progression. The considerations provided will be crucial for the design of new preventive approaches to the reduction in cancer risk that need to be applied to large populations composed of apparently healthy individuals.

Environmental impact of multi-wall carbon nanotubes in a novel model of exposure: systemic distribution, macrophage accumulation, and amyloid deposition.

Carbon nanotubes (CNTs) have been extensively investigated and employed for industrial use because of their peculiar physical properties, which make them ideal for many industrial applications. However, rapid growth of CNT employment raises concerns about the potential risks and toxicities for public health, environment, and workers associated with the manufacture and use of these new materials. Here we investigate the main routes of entry following environmental exposure to multi-wall CNTs (MWCNTs; currently the most widely used in industry). We developed a novel murine model that could represent a surrogate of a workplace exposure to MWCNTs. We traced the localization of MWCNTs and their possible role in inducing an innate immune response, inflammation, macrophage recruitment, and inflammatory conditions. Following environmental exposure of CD1 mice, we observed that MWCNTs rapidly enter and disseminate in the organism, initially accumulating in lungs and brain and later reaching the liver and kidney via the bloodstream. Since recent experimental studies show that CNTs are associated with the aggregation process of proteins associated with neurodegenerative diseases, we investigated whether MWCNTs are able to induce amyloid fibril production and accumulation. Amyloid deposits in spatial association with macrophages and MWCNT aggregates were found in the brain, liver, lungs, and kidneys of exposed animals. Our data suggest that accumulation of MWCNTs in different organs is associated with inflammation and amyloid accumulation. In the brain, where we observed rapid accumulation and amyloid fibril deposition, exposure to MWCNTs might enhance progression of neurodegenerative and other amyloid-related diseases. Our data highlight the conclusion that, in a novel rodent model of exposure, MWCNTs may induce macrophage recruitment, activation, and amyloid deposition, causing potential damage to several organs.

Systemic distribution of single-walled carbon nanotubes in a novel model: alteration of biochemical parameters, metabolic functions, liver accumulation, and inflammation in vivo

The increasing use of carbon nanotubes (CNTs) in several industrial applications raises concerns on their potential toxicity due to factors such as tissue penetrance, small dimensions, and biopersistence. Using an in vivo model for CNT environmental exposure, mimicking CNT exposition at the workplace, we previously found that CNTs rapidly enter and disseminate in the organism, initially accumulating in the lungs and brain and later reaching the liver and kidneys via the bloodstream in CD1 mice. Here, we monitored and traced the accumulation of single-walled CNTs (SWCNTs), administered systemically in mice, in different organs and the subsequent biological responses. Using the novel in vivo model, MITO-Luc bioluminescence reporter mice, we found that SWCNTs induce systemic cell proliferation, indicating a dynamic response of cells of both bone marrow and the immune system. We then examined metabolic (water/food consumption and dejections), functional (serum enzymes), and morphological (organs and tissues) alterations in CD1 mice treated with SWCNTs, using metabolic cages, performing serum analyses, and applying histological, immunohistochemical, and ultrastructural (transmission electron microscopy) methods. We observed a transient accumulation of SWCNTs in the lungs, spleen, and kidneys of CD1 mice exposed to SWCNTs. A dose- and time-dependent accumulation was found in the liver, associated with increases in levels of aspartate aminotransferase, alanine aminotransferase and bilirubinemia, which are metabolic markers associated with liver damage. Our data suggest that hepatic accumulation of SWCNTs associated with liver damage results in an M1 macrophage-driven inflammation.

Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

The Angiogenic Switch: Role of Immune Cells

Angiogenesis is a frequent hallmark of inflammation; in acute inflammation angiogenesis occurs to revascularize damaged tissues, with subsequent vascular pruning and maturation, which results in restoration of an adequate blood supply to the tissue involved. Angiogenesis is also a characteristic of chronic inflammation, and may be one of the links between inflammation and cancer (Colotta et al. 2009; Kobayashi and Lin 2009).

Abstract 2303: Innate immunity driving tumor angiogenesis: the role of natural killer cells in non small cell lung cancer (NSCLC) .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Immune cells infiltrating tumors often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity that can potentially control tumors by their cytotoxic activity. Nevetherless the role of NK cells in angiogenesis remains to be defined. Here we investigate the subset distribution of tumor infiltrating NKs and the correlation with pro-angiogenic factor production, like VEGF, PlGF and IL-8, on clinical samples belonging from patient with non-small cell lung cancer (NSCLC). Samples (tumor, adjacent normal tissue and peripheral blood), solid tissues are mechanically dispersed, while blood is processed using a Ficoll gradient. Flow cytometry (FC) analyses were performed to evaluate specific markers (CD56, CD16, CD335, CD3) for NK cells. The CD56brightCD16−NK phenotype predominated in all NSCLC samples while the CD56dimCD16+ cytotoxic NK phenotype prevailed in adjacent normal tissues and in non-oncologic lung tissues, independent of smoking status. The CD56+CD16− NK subset was associated with angiogenic cytokines production like VEGF, PlGF and IL-8 (CXCL8) Further, patients with the SQK histotype showed significantly higher angiogenic factor production by CD56+CD16−NK cells infiltrating tumors, adjacent tissues and peripheral blood, compared to those from adenocarcinoma (ADK) and control tissues. Supernatants derived from NSCLC infiltrating CD56+CD16−NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SQK derived NK cell supernatants and absent in controls As TGFβ1 expression has been associated with poorer survival in non-small cell lung cancer (NSCLC) squamous cell carcinoma (SQK) and polarization of peripheral NK cells towards a decidual NK phenotype, a subset associated with angiogenesis, we evaluated whether TGFb culd be implicated in these polarization phenomena. We demonstrated as exposure to TGFβ1 up-regulated VEGF and PlGF production by peripheral blood CD56+CD16−NK cells from healthy subjects. Our data suggest that, like other immune cells, NK cells in NSCLC can be switched to an angiogenic phenotype, particularly evident for SQK and likely mediated by TGFβ1, which could contribute to angiogenesis in SQK NSCLC development and progression. Citation Format: Adriana Albini, Antonino Bruno, Arianna Pagani, Andrea Imperatori, Marco Spagnoletti, Nicola Rotolo, AnnaRita Cantelmo, Francesca Franzi, Carlo Capella, Guido Ferlazzo, Lorenzo Mortara, Douglas M. Noonan. Innate immunity driving tumor angiogenesis: the role of natural killer cells in non small cell lung cancer (NSCLC) . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2303. doi:10.1158/1538-7445.AM2013-2303