Rossana La Sorda

Effects of dietary supplementation of carnosine on mitochondrial dysfunction, amyloid pathology, and cognitive deficits in 3xTg-AD mice.

Background The pathogenic road map leading to Alzheimers disease (AD) is still not completely understood; however, a large body of studies in the last few years supports the idea that beside the classic hallmarks of the disease, namely the accumulation of amyloid-β (Aβ) and neurofibrillary tangles, other factors significantly contribute to the initiation and the progression of the disease. Among them, mitochondria failure, an unbalanced neuronal redox state, and the dyshomeostasis of endogenous metals like copper, iron, and zinc have all been reported to play an important role in exacerbating AD pathology. Given these factors, the endogenous peptide carnosine may be potentially beneficial in the treatment of AD because of its free-radical scavenger and metal chelating properties. Methodology In this study, we explored the effect of L-carnosine supplementation in the 3xTg-AD mouse, an animal model of AD that shows both Aβ- and tau-dependent pathology. Principal Findings We found that carnosine supplementation in 3xTg-AD mice promotes a strong reduction in the hippocampal intraneuronal accumulation of Aβ and completely rescues AD and aging-related mitochondrial dysfunctions. No effects were found on tau pathology and we only observed a trend toward the amelioration of cognitive deficits. Conclusions and Significance Our data indicate that carnosine can be part of a combined therapeutic approach for the treatment of AD.

Flavonoids inhibit melanoma lung metastasis by impairing tumor cells endothelium interactions

Flavonoids comprise a class of low molecular weight compounds displaying a variety of biological activities including inhibition of tumor growth and metastasis. To gain insight into the mechanisms underlying metastasis inhibition, we have employed the B16‐BL6 murine melanoma metastasis model. B57BL/6N mice were injected i.v. with tumor cells and Apigenin, Quercetin, or Tamoxifen, each at 50 mg/kg given i.p., and lung tumor cell colonies counted 14–6 days thereafter. Three different injection schedules were used for each drug: (a) daily injection, starting 24 h before injection of the tumor cells; (b) single dose, 24 h preceding tumor challenge; (c) daily injection, starting 24 h after the injection of the tumor cells. All three compounds significantly reduced tumor lung deposits (Apigenin = Quercetin > Tamoxifen). However, when treatment was delayed by 24 h after tumor cells (schedule c), multiple daily doses of Apigenin or Quercetin were less effective that a single dose of the same compound given 24 h before tumor challenge (schedule b). Apigenin and Quercetin, but not Tamoxifen, were found to inhibit VCAM‐1 expression in a dose‐dependent manner in HUVEC and in murine pulmonary endothelial cells. In ex vivo experiments, the number of tumor cells adhering to lung vessels was significantly diminished in animals treated with a single dose of Apigenin and Quercetin. These findings indicate that the inhibition of tumor cell metastasis by Apigenin or Quercetin may significantly depend on the ability of these compounds to alter the hosts microenvironment, further substantiating the role of the intravascular processes in the metastatic cascade. J. Cell. Physiol. 207: 23–29, 2006.

LGALS3BP, lectin galactoside-binding soluble 3 binding protein, induces vascular endothelial growth factor in human breast cancer cells and promotes angiogenesis

Elevated serum or tissue levels of lectin galactoside-binding soluble 3 binding protein (LGALS3BP) have been associated with short survival and development of metastasis in a variety of human cancers. However, the role of LGALS3BP, particularly in the context of tumor–host relationships, is still missing. Here, we show that LGALS3BP knockdown in MDA-MB-231 human breast cancer cells leads to a decreased adhesion to fibronectin, a reduced transendothelial migration and, more importantly, a reduced expression of vascular endothelial growth factor (VEGF). Production of VEGF, that was restored by exposure of silenced cells to recombinant LGALS3BP, required an intact PI3k/Akt signaling. Furthermore, we show that LGALS3BP was able to directly stimulate HUVEC tubulogenesis in a VEGF-independent, galectin-3-dependent manner. Immunohistochemical analysis of human breast cancer tissues revealed a correlation among LGALS3BP expression, VEGF expression, and blood vessel density. We propose that in addition to its prometastatic role, LGALS3BP secreted by breast cancer cells functions critically as a pro-angiogenic factor through a dual mechanism, i.e by induction of tumor VEGF and stimulation of endothelial cell tubulogenesis.

Trop-2 Is a Determinant of Breast Cancer Survival

Trop-2 is a calcium signal transducer that drives tumor growth. Anti-Trop-2 antibodies with selective reactivity versus Trop-2 maturation stages allowed to identify two different pools of Trop-2, one localized in the cell membrane and one in the cytoplasm. Of note, membrane-localized/functional Trop-2 was found to be differentially associated with determinants of tumor aggressiveness and distinct breast cancer subgroups. These findings candidated Trop-2 states to having an impact on cancer progression. We tested this model in breast cancer. A large, consecutive human breast cancer case series (702 cases; 8 years median follow-up) was analyzed by immunohistochemistry with anti-Trop-2 antibodies with selective reactivity for cytoplasmic-retained versus functional, membrane-associated Trop-2. We show that membrane localization of Trop-2 is an unfavorable prognostic factor for overall survival (1+ versus 0 for all deaths: hazard ratio, 1.63; P = 0.04), whereas intracellular Trop-2 has a favorable impact on prognosis, with an adjusted hazard ratio for all deaths of 0.48 (high versus low; P = 0.003). A corresponding impact of intracellular Trop-2 was found on disease relapse (high versus low: hazard ratio, 0.51; P = 0.004). Altogether, we demonstrate that the Trop-2 activation states are critical determinants of tumor progression and are powerful indicators of breast cancer patients survival.

mTrop1/Epcam Knockout Mice Develop Congenital Tufting Enteropathy through Dysregulation of Intestinal E-cadherin/β-catenin

Congenital tufting enteropathy (CTE) is a life-threatening hereditary disease that is characterized by enteric mucosa tufting degeneration and early onset, severe diarrhea. Loss-of-function mutations of the human EPCAM gene (TROP1, TACSTD1) have been indicated as the cause of CTE. However, loss of mTrop1/Epcam in mice appeared to lead to death in utero, due to placental malformation. This and indications of residual Trop-1/EpCAM expression in cases of CTE cast doubt on the role of mTrop1/Epcam in this disease. The aim of this study was to determine the role of TROP1/EPCAM in CTE and to generate an animal model of this disease for molecular investigation and therapy development. Using a rigorous gene-trapping approach, we obtained mTrop1/Epcam -null (knockout) mice. These were born alive, but failed to thrive, and died soon after birth because of hemorrhagic diarrhea. The intestine from the mTrop1/Epcam knockout mice showed intestinal tufts, villous atrophy and colon crypt hyperplasia, as in human CTE. No structural defects were detected in other organs. These results are consistent with TROP1/EPCAM loss being the cause of CTE, thus providing a viable animal model for this disease, and a benchmark for its pathogenetic course. In the affected enteric mucosa, E-cadherin and β-catenin were shown to be dysregulated, leading to disorganized transition from crypts to villi, with progressive loss of membrane localization and increasing intracellular accumulation, thus unraveling an essential role for Trop-1/EpCAM in the maintenance of intestinal architecture and functionality. Supporting information is available for this article.

Overexpression of activated phospholipase Cγ1 is a risk factor for distant metastases in T1‐T2, N0 breast cancer patients undergoing adjuvant chemotherapy

Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumors. We have previously reported that both stable and inducible PLCγ1 down‐regulation resulted in an almost complete inhibition of breast cancer‐derived experimental lung metastasis formation. The aim of our study is to evaluate the association between the expression of PLCγ1 and of PLCγ1 phosphorylated at Tyr1253 (PLCγ1‐pY1253) and at Tyr783 (PLCγ1‐pY783) with the clinical outcome of patients with node negative, T1/T2 breast cancers. The study groups consisted of 292 (training set) and 122 (validation set) patients presenting with primary unilateral breast carcinoma (T1‐T2), with no evidence of nodal involvement and distant metastases. PLCγ1, PLCγ1‐pY1253 and PLCγ1‐pY783 protein expression were assessed by immunohistochemistry on tissue microarrays and the results correlated with the clinical data using Kaplan–Meier curves and multivariate Cox regression analysis. Tumor cells while expressing variable proportions of cytoplasmic PLCγ1, express PLCγ1‐pY1253 and PLCγ1‐pY783 predominantly in the nucleus. High expression of PLCγ1, and of its activated forms, is associated with a worse clinical outcome in terms of incidence of distant metastases, and not of local relapse in T1‐T2, N0 breast cancer patients undergone adjuvant chemotherapy. PLCγ1 over‐expression appears to be a reliable predictive surrogate marker of development of metastases. Thus, targeting PLCγ1 pathways might represent a potential therapeutic approach for the prevention of metastatic disease in breast cancer.

Cytoplasmic Trop-1/Ep-CAM Overexpression is Associated with a Favorable Outcome in Node-positive Breast Cancer

OBJECTIVE Trop-1/Ep-CAM modulates growth and survival of transformed cells, and it is highly expressed in most carcinomas including breast cancer. Only membranous staining is typically considered in evaluating Trop-1/epithelial cell adhesion molecule (Ep-CAM) expression in tumor cells. However, there is evidence of retention of Trop-1/Ep-CAM, as functionally incompetent molecules, in intra-cytoplasmic vesicles. Hence, we investigated whether cytoplasmic immunostaining may have an independent clinical significance with respect to membranous staining. METHODS Membranous and cytoplasmic Trop-1/Ep-CAM expression was immunohistochemically investigated in 642 unilateral breast cancers from patients with a 99-month median follow-up. Multiple correspondence analysis was used to investigate the association between Trop-1/Ep-CAM and other biological variables. The impact of Trop-1/Ep-CAM expression on the patients outcome was evaluated as event-free survival by the Kaplan-Meier method and proportional hazard Cox model. RESULTS While tumors with intermediate/strong membranous staining were mostly associated with concomitant cytoplasmic Trop-1/Ep-CAM expression (97%), tumors with weak-to-nil membranous staining showed intermediate/high cytoplasmic expression in 23% of cases. Cytoplasmic overexpression was associated with a favorable outcome, especially in node-positive patients, regardless of the adjuvant therapy received. CONCLUSION Trop-1/Ep-CAM expression may have different clinical implications according to its subcellular localization.

Prognostic relevance of LGALS3BP in human colorectal carcinoma.

BackgroundA previous report has shown that LGALS3BP (also known as 90K or Mac-2 BP) has antitumor activity in colorectal cancer (CRC) via suppression of Wnt signalling with a novel mechanism of ISGylation-dependent ubiquitination of β-catenin. The role of LGALS3BP in CRC prognosis was investigated.MethodsThe role of LGALS3BP on CRC progression and clinical prognosis was analyzed by combining cell cultures, in vitro assays, and immunohistochemistry.ResultsSilencing of LGALS3BP in HCT-116 human colon cancer cells resulted in enhanced β-catenin expression that was reversed by addition of human recombinant LGALS3BP. Moreover, intra-tumor delivery of LGALS3BP reduced tumor growth of xenografts originating from LGALS3BP-silenced HCT-116 cells. Finally, in a series of 196 CRC patients, LGALS3BP expression in tumor tissue associated with clinical outcome. Patients with high LGALS3BP expression had lower risk of relapse and a longer overall survival time than those with low LGALS3BP expression. Multivariate analyses confirmed LGALS3BP expression status as the only independent prognostic factor of survival.ConclusionsThese results provide evidence that low expression of LGALS3BP participates in malignant progression of CRC and implicates poor prognosis, highlighting its augmentation as a potential therapeutic approach.

Thymic epithelial tumors express vascular endothelial growth factors and their receptors as potential targets of antiangiogenic therapy: A tissue micro array-based multicenter study

OBJECTIVES Tumor angiogenesis is an essential and complex process necessary for the growth of all tumors which represents a potential therapeutic target. Angiogenesis inhibitors targeting vascular endothelial growth factor (VEGF) or their receptor tyrosine kinases have been approved by the FDA. In thymic epithelial tumors (TET), targeted therapies have been sporadically applied due to their rarity. To ascertain the presence of potential therapeutic targets, we analyzed by immunohistochemistry the expression of angiogenesis-related biomarkers in a large series of TET arranged in Tissue Micro Arrays (TMA). MATERIALS AND METHODS We assessed by immunohistochemistry the expression of the possible molecular target of anti-angiogenic therapy, i.e. VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, and PDGFRβ, in a TMA series of 200 TET collected in the framework of a multi-institutional collaborative project for Rare Diseases. RESULTS When compared to the low-risk tumors, high-risk TET (B2, B3, carcinomas) contained higher proportion of cancer cells expressing VEGFA, VEGFC and VEGFD (P<0.001, P<0.001, and P<0.001) growth factors, and their receptors VEGFR1 (P=0.002), VEGFR2 (P=0.013), and VEGFR3 (P=0.041). No differences were observed in terms of PDGFRβ expression. CONCLUSIONS According to our data, it is possible to hypothesize the existence of multiple paracrine and/or autocrine loops in TET, particularly in the high-risk ones, involved in TET growth and progression. Anti-angiogenic agents, directed to inhibit these loops, are therefore to be considered as potential tools in advanced TET therapy.

Inhibition of Tumor Growth and Angiogenesis by SP-2, an Anti–Lectin, Galactoside-Binding Soluble 3 Binding Protein (LGALS3BP) Antibody

Accumulating evidence indicates that serum and tissue levels of lectin, galactoside-binding soluble 3 binding protein (LGALS3BP), a secreted glycoprotein, are elevated in human cancers. Recently, we have identified LGALS3BP as a factor capable of stimulating angiogenesis of microvascular endothelial cells in vitro as well as in vivo. However, the potential therapeutic implications of LGALS3BP function blockade have not been explored yet. Here, we tested the ability of an anti-LGALS3BP mouse monoclonal antibody, SP-2, to antagonize LGALS3BP-induced angiogenesis and tumor growth. The antibody was found to inhibit endothelial cell tubulogenesis induced by either conditioned medium of breast cancer and melanoma cells or human recombinant LGALS3BP. In addition, SP-2 inhibited phosphorylation of FAK and its recruitment to membrane sites as well as AKT and ERK phosphorylation promoted by LGALS3BP. When used in vivo, the antibody restrained LGALS3BP-stimulated angiogenesis and growth of tumor xenografts. Furthermore, the combination of SP-2 and low-dose bevacizumab was more effective than either agent alone. Taken together, these results lead to consideration of SP-2 as a promising candidate for LGALS3BP-targeted therapy. Mol Cancer Ther; 13(4); 916–25. ©2014 AACR.