Cristina Gallo

Cancer stem cells and the tumor microenvironment: interplay in tumor heterogeneity.

Abstract Tumor cells able to recapitulate tumor heterogeneity have been tracked, isolated and characterized in different tumor types, and are commonly named Cancer Stem Cells or Cancer Initiating Cells (CSC/CIC). CSC/CIC are disseminated in the tumor mass and are resistant to anti-cancer therapies and adverse conditions. They are able to divide into another stem cell and a “proliferating” cancer cell. They appear to be responsible for disease recurrence and metastatic dissemination even after apparent eradication of the primary tumor. The modulation of CSC/CIC activities by the tumor microenvironment (TUMIC) is still poorly known. CSC/CIC may mutually interact with the TUMIC in a special and unique manner depending on the TUMIC cells or proteins encountered. The TUMIC consists of extracellular matrix components as well as cellular players among which endothelial, stromal and immune cells, providing and responding to signals to/from the CSC/CIC. This interplay can contribute to the mechanisms through which CSC/CIC may reside in a dormant state in a tissue for years, later giving rise to tumor recurrence or metastasis in patients. Different TUMIC components, including the connective tissue, can differentially activate CIC/CSC in different areas of a tumor and contribute to the generation of cancer heterogeneity. Here, we review possible networking activities between the different components of the tumor microenvironment and CSC/CIC, with a focus on its role in tumor heterogeneity and progression. We also summarize novel therapeutic options that could target both CSC/CIC and the microenvironment to elude resistance mechanisms activated by CSC/CIC, responsible for disease recurrence and metastases.

N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase Inhibitors: Hit Selection and in Vivo Antiangiogenic Activity.

Matrix metalloproteinases (MMPs) have been shown to be involved in tumor-induced angiogenesis. In particular, MMP-2, MMP-9, and MMP-14 have been reported to be crucial for tumor angiogenesis and the formation of metastasis, thus becoming attractive targets in cancer therapy. Here, we report our optimization effort to identify novel N-isopropoxy-arylsulfonamide hydroxamates with improved inhibitory activity toward MMP-2, MMP-9, and MMP-14 with respect to the previously discovered compound 1. A new series of hydroxamates was designed, synthesized, and tested for their antiangiogenic activity using in vitro assays with human umbilical vein endothelial cells (HUVECs). A nanomolar MMP-2, MMP-9, and MMP-14 inhibitor was identified, compound 3, able to potently inhibit angiogenesis in vitro and also in vivo in the matrigel sponge assay in mice. Finally, X-ray crystallographic and docking studies were conducted for compound 3 in order to investigate its binding mode to MMP-9 and MMP-14.

Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation

Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named “angioprevention”. Several natural compounds exert their anti-tumor properties by activating 5′ adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKβ, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer.

Effect of a Purified Extract of Olive Mill Waste water on Endothelial CellProliferation, Apoptosis, Migration and Capillary-Like Structure in vitroand in vivo

Olive oil, a major feature of the Mediterranean diet, is an important source of phenolic compounds. Poliphenols are associated with inhibition of several pathological processes, including cancer. Soluble phenols are contained in the aqueous part of olive and are discarded during oil production in the ‘olive mill wastewaters’. Here we investigated the properties of a purified extract of olive mill wastewaters, named A009, as potential anti-angiogenic compound. While the strong anti-oxidant activity of olive derived phenolic compounds has been well characterized, little is known about their anti-angiogenic properties. We investigated effects of A009 on endothelial cell morphogenesis, proliferation, migration and apoptosis, comparing the results obtained with the activity of a well-characterized olive oil phenol, Hydroxytyrosol (HT). Further, we tested the effects of A009 and HT in an in vivo angiogenesis assay. We found that A009 exerted strong anti-angiogenic effects both in vitro and in vivo, and that the complex natural purified extract has stronger anti-angiogenic potential when compared to the same concentrations of HT in most of the assays performed. These data demonstrate that a novel purified, phenols enriched, extract with anti-angiogenic, and angiopreventive potential can be obtained from olive oil mill waste material, recovering useful products from an agricultural waste.

Biomarkers of cancer angioprevention for clinical studies

With the great advances made in the treatment and prevention of infectious diseases over the last century, chronic degenerative diseases—cardiovascular, cerebrovascular, and cancer—represent the major causes of death in the developed world. Although massive efforts and investments have been made in cancer therapy, the progress made towards reducing mortality has been more successful for cardiovascular disease than for tumours. This can be attributable largely to an active prevention approach implemented for cardiovascular disease. Cardiologists treat their patients before the overt disease becomes life threatening, performing early interventions in phenotypically healthy patients, by using several markers that predict risk. If the concept of prevention could be applied to cancer in a more extensive way, a significant number of tumours could be avoided through preventive measures. Prevention approaches range from avoiding tobacco exposure to dietary strategies to active pharmacological approaches in higher risk groups. Host targets rather than the tumour cells themselves are attractive for chemoprevention, in particular endothelial and immune cells. Angioprevention i.e. preventing cancer angiogenesis is a key concept that we introduced; yet one of the major current challenges for anti-angiogenesis in therapy and prevention is finding the right biomarkers. Here we discuss the importance of angioprevention and the potential use of VEGF, PlGF, CD31, Ang and Tie, circulating vascular cell precursors, and microRNA as potential biomarkers.

The bHLH transcription factor DEC1 promotes thyroid cancer aggressiveness by the interplay with NOTCH1

Aberrant re-activation of transcription factors occurs frequently in cancer. Recently, we found the basic helix-loop-helix transcription factors DEC1 and DEC2 significantly up-regulated in a model of highly aggressive thyroid cancer, raising the hypothesis that these factors might be part of the program driving progression of these tumors. Here, we investigated for the first time the function of DEC1 and DEC2 in thyroid cancer. Using both gain- and loss-of-function approaches, we showed that DEC1 more than DEC2 sustains progression of thyroid cancer by promoting cell growth and invasiveness. We demonstrated that DEC1 controls NOTCH1 expression and that the interplay with the NOTCH pathway is relevant for DEC1 function in thyroid cancer. We confirmed this observation in vivo showing that DEC1 expression is a specific feature of tumor cells, that this transcription factor is significantly over-expressed in all major thyroid cancer histotypes and that its expression correlated with NOTCH1 in these tumors. Finally, we performed RNA-sequencing to define the DEC1-associated gene expression profile in thyroid cancer cells and we discovered that DEC1 drives the expression of many cell cycle-related genes, uncovering a potential new function for this transcription factor in cancer.

Abstract 4652: Wide antiproliferative and proapoptotic activity of the natural prenyl-flavonoid Xanthohumol and its derivatives on cancer cells, peripheral blood mononuclear cells, and human primary endothelial cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Chemo-preventive drugs are molecules able to prevent or retard cancer development and progression. Recently, diet flavonoids have been recognized as chemo- and angio-preventive agents. Among these, Xanthohumol (XN), is the principal prenylated chalcone of the female inflorescence of the hop plant (Humulus lupulus L.) with chemo-/angio-preventive properties. The biological effects of the chalcones mostly depend on their chemical structure, whose variations influence their anti-tumor effects. In order to identify novel potential chemo-/angio-preventive agents, we analyzed the effects of seven synthetic derivatives of XN on tumor and normal cells. We used different tumor cell lines (lymphoma, colon and prostate cancer cell lines) and healthy human primary cells (mononuclear cells isolated from peripheral blood (PBMCs) and umbilical vein endothelial cells (HUVEC)). XN derivatives have a lower IC50 on both cancer and normal cells as compared to XN. XN10 exerts the highest anti-proliferative activity in cancer and normal primary cells and we therefore focused our experiments on this compound in comparison with XN master molecule. Interestingly, the IC50 values for XN10 are two-three fold higher in primary cells than tumor cells, suggesting an anti-tumor effect of XN10 which spares healthy cells both from peripheral blood and the microenvironment. The treatment with XN10 leads to apoptosis as demonstrated in lymphoma cell lines by PARP-1 cleavage and dose-dependent caspase-3 activation. Preliminary data also show that XN downregulates the enzyme cyclooxygenase-1 while inducing a DNA damage as demonstrated by the increase of the specific marker γ-H2AX in the cell lysates treated with the indicated drugs. We then treated both PBMCs and HUVECs with XN and XN10 in a range of 1-20μM for 48 hr. XN10 affects PBMCs and HUVEC cells viability starting at lower doses (1 μM) than the parental molecule. 20μM XN mainly decreases monocyte population whereas XN10 affects both CD19+/CD20+ and CD14+/CD33+ cells in parallel with the increase of apoptosis and caspase activation. Further, in HUVECs, XN activates AMPK in a time and dose-dependent manner with a peak of activation after 5 minutes of exposure up to 1h, independently from LKB1 activation. Activation of AMPK signalling pathway by XN was confirmed by ACC phosphorylation at Ser-79 at the same timepoints. In this cellular context, XN modulates AMPK downstream target, mTOR and eNOS, particularly inhibiting eNOS phosphorylation. Collectively, our data show that XN and its novel derivative XN10 exert a strong antiproliferative activity on a variety of tumor cell lines and normal cells. XN10 features the strongest pro-apoptotic activity yet causing the most dramatic cytotoxic effect on normal cells. Moreover, XN activity seems to involve AMPK activation, in HUVEC cells. Citation Format: Katiuscia Dallaglio, Valentina Fragliasso, Cristina Gallo, Raffaele Frazzi, Clara Maccari, Armando Rossello, Adriana Albini. Wide antiproliferative and proapoptotic activity of the natural prenyl-flavonoid Xanthohumol and its derivatives on cancer cells, peripheral blood mononuclear cells, and human primary endothelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4652. doi:10.1158/1538-7445.AM2015-4652