[show abstract][hide abstract] ABSTRACT: Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of 5 patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term "immune response". Human Leukocyte Antigen E (HLA-E) was ranked 19(th) among melanoma-overexpressed genes, and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than melanocytes, and protected the former (6/6 cell lines) from lysis by Natural Killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis. This article is protected by copyright. All rights reserved.
Pigment Cell & Melanoma Research 09/2013; · 5.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aim: Gastric cancer is among the most common causes of cancer-related death worldwide. Since microRNAs (miRNAs) represent an emerging field of cancer research, there is an increasing interest regarding the miRNA responses to environmental and lifestyle exposures. The aim of our study was to analyze whether social status, living conditions and lifestyle behaviours, such as cigarette smoking and alcohol consumption, are associated with specific miRNA expression patterns of gastric adenocarcinoma.
Thirty-nine formalin-fixed paraffin-embedded primary gastric adenocarcinoma and nine non-tumourous samples were analyzed by real-time polymerase chain reaction. The expression levels of miR-21, miR-34a, miR-93, miR-143, miR-203, miR-205, miR-223 were compared in gastric adenocarcinoma samples of patients with different demographical characteristics, social status, drinking and smoking habits.
Overexpression of miR-21, miR-143 and underexpression of miR-34a were observed in gastric cancer samples relative to the controls. Elevated expression of miR-21 was detected in patients with low social status. Smokers showed higher expression of miR-21 and lower expression of miR-143. Up-regulation of miR-203, miR-205 and miR-223 was identified in patients with regular alcohol consumption. Patients living in an urban environment had elevated expression levels of miR-143 and miR-34a. Discussion: Differential miRNA expression patterns of gastric adenocarcinoma of the same histopathology from a small geographic region's population show homogenous correlations with the existence of common risk factors.
Anticancer research 08/2013; 33(8):3195-200. · 1.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Despite the fundamental pathophysiological importance of β-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that β-arrestin-1 (β-arr1) is an epigenetic regulator of endothelin (ET)-1-induced β-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ET(A)R) activation by ET-1, β-arr1 increases its nuclear translocation and direct binding to β-catenin. This in turn enhanced β-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant β-arr1 incapable of nuclear distribution. β-arr1-β-catenin interaction controls β-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by β-arr1 silencing or by mutant β-arr1, as well as by β-catenin or p300 silencing, confirming that nuclear β-arr1 forms a functional complex capable of regulating epigenetic changes in β-catenin-driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of β-arr1 or mutant β-arr1 expression, as well as ET(A)R blockade, inhibits metastasis. In human EOC tissues, β-arr1-β-catenin nuclear complexes are selectively enriched at β-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo β-arr1-β-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a β-arr1-mediated epigenetic mechanism controls β-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.Oncogene advance online publication, 3 December 2012; doi:10.1038/onc.2012.527.
[show abstract][hide abstract] ABSTRACT: Endothelin receptor B (ET(B)R) is a G-protein-coupled receptor overexpressed in melanoma, blood, and lymphatic endothelial cells. Given that aberrant signal transduction can be mediated through cross talk between receptors, here, we explore the functional relationship between ET(B)R and the vascular endothelial growth factor receptor (VEGFR)-3 system and how this cross talk might influence the aggressive behavior of melanoma cells. The expression of VEGFR-3 and its ligands, VEGF-C and VEGF-D, significantly increased after activating ET(B)R by ET-1 in primary and metastatic melanoma cell lines. These effects, similarly to those induced by hypoxia, were mediated by hypoxia-inducible factor (HIF)-1α and HIF-2α. ET-1 caused the phosphorylation of VEGFR-3, which was accompanied by the activation of the downstream signaling molecules, such as MAPK and AKT. Inhibition of c-Src activity or silencing of the scaffold protein β-arrestin-1 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that, upon ET-1 stimulus, β-arrestin-1 is involved with c-Src in the ET(B)R-mediated VEGFR-3 transactivation. Moreover, ET-1 in combination with VEGF-C further increased VEGFR-3, MAPK, and AKT phosphorylation and markedly promoted cell migration and vasculogenic mimicry. Dual inhibition of ET(B)R and VEGFR-3 was required for the effective inhibition of these effects, as well as for VEGFR-3 phosphorylation, demonstrating that ET(B)R cross talk with VEGFR-3 enhances cell plasticity and motility. Finally, in melanoma xenografts, ET(B)R antagonist inhibited tumor growth and the activation of the VEGF-C/VEGFR-3 axis, indicating that targeting ET(B)R may improve melanoma treatment acting directly or indirectly by impairing ET(B)R cross talk with VEGFR-3.
Journal of Molecular Medicine 09/2012; · 4.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Cancer 07/2012; · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene expression profiling is a powerful method to classify human tumours on the basis of biological aggressiveness, response to therapy, and outcome for the patient, but its application in melanoma lags behind that of other cancers. From more than 100 articles available on the topic, we selected 14 focusing on patients' outcome. We review and briefly discuss salient findings, and list ten reasons why melanoma molecular classes are not yet used in clinical diagnosis and prognosis. The available evidence suggests that we are on the verge of creating a framework for the use of melanoma molecular classes in prognosis, but so far there is little consensus to put together informative diagnostic and prognostic gene sets.
The lancet oncology 05/2012; 13(5):e205-11. · 14.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.
PLoS ONE 01/2012; 7(2):e31467. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.
Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.
We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.
PLoS ONE 01/2012; 7(9):e45513. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: No treatment prolongs the survival of malignant mesothelioma (MM) patients. Since MM elicits anti-tumor host's immune responses, immunotherapy represents a promising strategy for its control. Immunomodulatory antibodies against components of the B7 family of immunomodulatory molecules that regulate T cell activation are being investigated in human malignancies including MM. The expression of B7-H3, a new component of the B7 family was investigated in primary cultures of human mesothelial cells (HMC) and in MM cell lines by flow cytometry and molecular analyses, and in MM tissues by immunohistochemistry. The role of DNA hypomethylating agents in modulating levels of B7-H3 expression in MM cells was also studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that B7-H3 mRNA was consistently detectable in mesothelial and MM cells investigated; however, real-time quantitative RT-PCR analyses showed highly heterogeneous levels of B7-H3 mRNA among investigated MM cells. The analysis of B7-H3 protein expression indicated that comparable levels of B7-H3 were expressed on both cell types. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine did not significantly affect the expression of B7-H3 mRNA in MM cells. In vivo, while B7-H3 was expressed in all 13 tumor biopsies of the epithelial variant, with high levels in 54% of cases, it was rarely detectable in spindle type MM in which 1/5 biopsies weakly expressed B7-H3. These findings suggest that B7-H3 is a promising target for new immunotherapeutic strategies in MM, with particular emphasis in the epithelial variant.
Journal of Cellular Physiology 10/2011; 226(10):2595-600. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.
Neoplasia (New York, N.Y.) 09/2011; 13(9):822-30. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [125I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.Oncogene advance online publication, 8 August 2011; doi:10.1038/onc.2011.322.
[show abstract][hide abstract] ABSTRACT: An unknown primary tumor (UPT) is defined by the presence of a metastatic cancer without a known primary site of origin despite a standardized diagnostic workup. Clinically, UPTs show rapid progression and early dissemination, with signs and symptoms related to the metastatic site. The molecular bases of their biology remain largely unknown, with no evidence as to whether they represent a distinct biological entity. Immunohistochemistry remain the best diagnostic tool in term of cost-effectiveness, but the time-consuming "algorithmic process" it relies on has led to the application of new molecular techniques for the identification of the primary site of UPTs. For example, several microarray or miRNA classifications of UPTs have been used, with an accuracy in the prediction of the primary site as high as 90%. It should be noted that validating a prediction of tissue origin is challenging in these patients, since most of them will never have a primary site identified. Moreover, prospective studies to determine whether selection of treatment options based on such profiling methods actually improves patient outcome are still missing. In the last few years functional imaging (i.e. FDG-PET/CT) has gained a main role in the detection of the site of origin of UPTs and is currently recommended by the European Association of Nuclear Medicine. However, despite recent refinements in the diagnostic workup, the site of origin of UPT often remains elusive. As a consequence, treatment of patients with UPT is still empirical and inadequate.
Biochimica et Biophysica Acta 02/2011; 1816(1):13-24. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial-mesenchymal transition (EMT) in cancer. Endothelin-1 (ET-1)/endothelin A receptor (ET(A)R) axis is implicated in the pathobiology of epithelial ovarian cancer (EOC) by driving tumor-promoting effects, including EMT. Here, we analyzed how ET(A)R regulates chemoresistance and EMT in EOC.
The effects of ET-1 axis on cell proliferation, drug-induced apoptosis, invasiveness, and EMT were analyzed in cultured EOC cells sensitive and resistant to cisplatinum and taxol. Tumor growth in response to ET(A)R antagonist was examined in EOC xenografts. ET(A)R expression was examined in 60 human EOC tumors by immunohistochemistry and correlated with chemoresistance and EMT.
In resistant EOC cells ET-1 and ET(A)R are upregulated, paralleled by enhanced mitogen activated protein kinase (MAPK) and Akt phosphorylation and cell proliferation. Moreover, in these cells the expression of E-cadherin transcriptional repressors, including Snail, Slug, and Twist, as well as of mesenchymal markers, such as vimentin and N-cadherin, were upregulated and linked with enhanced invasive behavior. Interestingly, ET(A)R blockade with zibotentan, a specific ET(A)R antagonist, or its silencing, downregulated Snail activity, restored drug sensitivity to cytotoxic-induced apoptosis, and inhibited the invasiveness of resistant cells. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, and sensitized to chemotherapy. Analysis of EOC human tissues revealed that ET(A)R is overexpressed in resistant tumors and is associated with EMT phenotype.
Our data provide the first evidence that blockade of ET(A)R-driven EMT can overcome chemoresistance and inhibit tumor progression, improving the outcome of EOC patients' treatment.
Clinical Cancer Research 01/2011; 17(8):2350-60. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Anti-CD40 antibodies are in clinical development in patients with metastatic melanoma, a cancer that has been reported earlier to express CD40. The antitumor activity of anti-CD40 antibodies may be mediated by direct cytotoxic effects on CD40-positive melanoma cells or indirectly through modulation of host cells. In these studies, biopsies of patients with primary and metastatic melanoma, short-term cultures, and established melanoma cell lines were analyzed for CD40 expression using a combination of methods including immunohistochemistry, flow cytometry, and gene expression profiling, and the cytotoxic effects of the anti-CD40 antibody CP-870,893 on melanoma cell lines were tested using cell viability assays. CD40 was expressed at a higher frequency in metastatic melanoma lesions compared with primary melanomas. There was a variable expression of CD40 in synchronous and metachronous melanoma metastases. Expression of CD40 was present in slightly over half of a large panel of short-term primary melanoma cultures, with a wide range of expression levels by flow cytometry. Similar results were obtained in established melanoma cell lines when analyzed at the mRNA level or by surface protein staining. In approximately one-third of cell lines, the expression of CD40 could be up-regulated with a histone deacetylase inhibitor. Treatment with increasing concentrations of CP-870,893 had no antitumor activity against either CD40-positive or negative melanoma cell lines. In conclusion, approximately one-third to one-half of melanomas expresses CD40 at variable levels. Direct exposure to a CD40-activating antibody does not lead to antitumor activity in melanoma cell lines, suggesting that the antitumor effects of these antibodies in the clinic may be indirectly mediated.