R Sorrentino

Thymosin α1 and cancer: action on immune effector and tumor target cells

Since it was first identified, thymosin alpha 1 (Tα1) has been characterized to have pleiotropic effects on several pathological conditions, in particular as a modulator of immune response and inflammation. Several properties exerted by Tα1 may be attributable to a direct action on lymphoid cells. Tα1 has been shown to exert an immune modulatory activity on both T cell and natural killer cell maturation and to have an effect on functions of mature lymphocytes, including stimulating cytokine production and cytotoxic T lymphocyte–mediated cytotoxic responses. In previous studies we have shown that Tα1 increases the expression of major histocompatibility complex class I surface molecules in murine and human tumor cell lines and in primary cultures of human macrophages. In the present paper, we describe preliminary data indicating that Tα1 is also capable of increasing the expression of tumor antigens in both experimental and human tumor cell lines. This effect, which is exerted at the level of the target tumor cells, represents an additional factor increasing the antitumor activity of Tα1.

Antitumor effects of the benzophenanthridine alkaloid sanguinarine in a rat syngeneic model of colorectal cancer.

To evaluate the in-vivo preclinical antitumor activity of sanguinarine in a rat syngeneic model of colorectal cancer. The effects of sanguinarine on DHD/K12/TRb colorectal adenocarcinoma cells were first evaluated in vitro by means of 3H-thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and terminal transferase dUTP nick end labeling (TUNEL) microscopy. For the in-vivo studies, DHD/K12/TRb cells (1.5×106 cells/0.3 ml of sterile saline/animal) were injected subcutaneously in syngeneic BDIX rats, which were chronically treated with sanguinarine (5 mg/kg/day per os) or control diluent. Tumor growth, body weight, hematologic, and clinical chemistry measurements were monitored in individual animals at defined time intervals. After killing, subcutaneous tumors were explanted from experimental animals for histopathological examination. In vitro, micromolar concentrations of sanguinarine inhibited dose-dependently DHD/K12/TRb cell proliferation and metabolism and induced cell death by apoptosis. In vivo, oral administration of sanguinarine induced a significant inhibition of tumor growth (P<0.01 vs. untreated controls), in the absence of any toxic or side effects. Marked apoptosis and reduced peritumoral vascularization were observed in tumors from sanguinarine-treated rats as compared with the controls. Additional basic studies are needed to fully characterize the mechanism/s underlying the inhibitory effects of sanguinarine on angiogenesis and tumor growth as well as the pharmacological and safety profile of this drug in experimental tumor models. Overall, findings from this study suggest that sanguinarine is a likely candidate for further evaluation in cancer therapy.

HERVs expression in Autism Spectrum Disorders.

Background Autistic Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder, resulting from complex interactions among genetic, genomic and environmental factors. Here we have studied the expression of Human Endogenous Retroviruses (HERVs), non-coding DNA elements with potential regulatory functions, and have tested their possible implication in autism. Methods The presence of retroviral mRNAs from four HERV families (E, H, K and W), widely implicated in complex diseases, was evaluated in peripheral blood mononuclear cells (PBMCs) from ASD patients and healthy controls (HCs) by qualitative RT-PCR. We also analyzed the expression of the env sequence from HERV-H, HERV-W and HERV-K families in PBMCs at the time of sampling and after stimulation in culture, in both ASD and HC groups, by quantitative Real-time PCR. Differences between groups were evaluated using statistical methods. Results The percentage of HERV-H and HERV-W positive samples was higher among ASD patients compared to HCs, while HERV-K was similarly represented and HERV-E virtually absent in both groups. The quantitative evaluation shows that HERV-H and HERV-W are differentially expressed in the two groups, with HERV-H being more abundantly expressed and, conversely, HERV-W, having lower abundance, in PBMCs from ASDs compared to healthy controls. PMBCs from ASDs also showed an increased potential to up-regulate HERV-H expression upon stimulation in culture, unlike HCs. Furthermore we report a negative correlation between expression levels of HERV-H and age among ASD patients and a statistically significant higher expression in ASD patients with Severe score in Communication and Motor Psychoeducational Profile-3. Conclusions Specific HERV families have a distinctive expression profile in ASD patients compared to HCs. We propose that HERV-H expression be explored in larger samples of individuals with autism spectrum in order to determine its utility as a novel biological trait of this complex disorder.

Human endogenous retroviruses and ADHD

Abstract Objectives. Several lines of evidences suggest that human endogenous retroviruses (HERVs) are implicated in the development of many complex diseases with a multifactorial aetiology and a strong heritability, such as neurological and psychiatric diseases. Attention deficit hyperactivity Disorder (ADHD) is a neurodevelopmental disorder that results from a complex interaction of environmental, biological and genetic factors. Our aim was to analyse the expression levels of three HERV families (HERV-H, K and W) in patients with ADHD. Methods. The expression of retroviral mRNAs from the three HERV families was evaluated in peripheral blood mononuclear cells (PBMCs) from 30 patients with ADHD and 30 healthy controls by quantitative RT-PCR. Results. The expression levels of HERV-H are significantly higher in patients with ADHD compared to healthy controls, while there are no differences in the expression levels of HERV-K and W. Conclusions. Since the ADHD aetiology is due to a complex interaction of environmental, biological and genetic factors, HERVs may represent one link among these factors and clinical phenotype of ADHD. A future confirmation of HERV-H overexpression in a larger number of ADHD patients will make possible to identify it as a new parameter for this clinical condition, also contributing to deepen the study on the role of HERVs in the neurodevelopment diseases.

HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features

BackgroundMelanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications.MethodsTVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression.ResultsMelanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells.ConclusionsHERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.

Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells

Human endogenous retroviruses (HERVs) have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E) was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individuals life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

Detection of high levels of Survivin–immunoglobulin M immune complex in sera from hepatitis C virus infected patients with cirrhosis

The identification and surveillance of patients with liver dysfunctions and the discovering of new disease biomarkers are needed in the clinical practice. The aim of this study was to investigate on Survivin–immunoglobulin (Ig)M immune complex (IC) as a potential biomarker of chronic liver diseases.

Activation of human endogenous retrovirus K (HERV-K) and cellular modifications in human melanoma cell line: transcriptional profiling analysis

Background Similarly to what shown for exogenous tumorigenic retroviruses, endogenous retrovitus (ERVs) have been implicated in the pathogenesis of cancer. ERVs may participate in the process of malignant transformation or promote tumor growth, e.g. through insertional mutagenesis or via counteracting tumor immunosurveillance. Growing evidences show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance.

Abstract C03: Role of human endogenous retrovirus-K in phenotype-switching of metastatic melanoma cells during microenvironment alterations

Background: Malignant melanoma is one of the most aggressive types of skin cancers and its etiology is not yet clear. Phenotypic-switching has been associated with melanoma tumor aggressiveness and metastasis. Microenvironmental factors such as estrogen, cytokines and other stress conditions determine epigenetic events, as hypo-methylation, which are involved in cell transformation. Interestingly, endogenous retroviruses contribute to melanoma progression and are transcriptionally regulated by epigenetic events. Previously, we demonstrated that the aggressiveness and immune evasion of metastatic melanoma is partly depends on human endogenous retrovirus-K (HERV-K) activation. Thus, studying the role of HERV-K activation in melanoma phenotypic-switching is important to understand melanomagenesis and find possible therapeutic targets. Objective: To investigate the potential role of HERV-K activation in melanoma cells phenotypic-switching in response to change in microenvironment conditions. Methods: the TVM-A12 cell line, isolated in our laboratory from a metastatic melanoma lesion, and other commercial melanoma cell lines (WM-266-4, WM-115 and A375) were cultured in standard, differentiation and serum-free cell culture media. RNA interference, flow cytometry, qRT-PCR, self-renewing assay, sphere-forming assay and migration/invasion assays were performed. Data were analyzed using SPSS software version 17. Results: TVM-A12 showed high cellular plasticity as capable to acquire different phenotypes depending on the modification of the microenvironment. Indeed, in differentiation media the TVM-A12 modified the morphology towards a more differentiated phenotype, but, surprisingly, the cells were not committed to a final stage of differentiation and were able to revert to the original phenotype. The exposure of TVM-A12 to a serum-free medium, induced changes in cell growth and morphology, from adherent towards sphere-like cellular aggregates, characterized by an increased activation of HERV-K expression and generation of a CD133 + subpopulation of melanoma cells. Under this stress-condition, the silencing of HERV-K expression in TVM-A12 cells by RNA interference, significantly abolished the generation of the CD133 + subpopulation, dysregulated the cellular aggregates phenotype and suppressed their proliferation. More appreciably, the induction of HERV-K expression and the stress-mediated generation of CD133 + subpopulation of melanoma cells were also seen in other primary and metastatic melanoma cell lines like WM-115, WM-266-4 and A375 with a considerable variability. Furthermore, theTVM-A12-CD133 + cells, sorted from TVM-A12, showed dynamic cell plasticity upon the modification of the microenvironments and displayed a significantly higher self-renewing, migration and invasion capacity than the parental TVM-A12 cells. Moreover, treatment of TVM-A12 and TVM-A12-CD133 + with the non-nucleoside reverse transcriptase inhibitors (NNRTIs), nevirapine and efavirenz, inhibited the expression of HERV-K and significantly induced high levels of apoptosis in TVM-A12-CD133 + cells. Conclusions: these results demonstrated for the first time that HERV-K has a decisive role on phenotype-switching in metastatic melanoma cells, and the generation of the more aggressive CD133 + subpopulation in stressful microenvironments. Moreover, NNRTIs treatments were able to inhibit the expression of HERV-K and affect the stress-mediated generation of the CD133 + subpopulation. Thus, the further understanding of the dynamics of HERV-K in melanoma will help to understand melanomagenesis and find possible new therapeutic targets. Citation Format: Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Roberta Sorrentino, Corrado Spadafora, Paola Sinibaldi-Vallebona, Claudia Matteucci. Role of human endogenous retrovirus-K in phenotype-switching of metastatic melanoma cells during microenvironment alterations. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C03.

Osteopontin-Igm as a Marker for the Diagnosis of Hepatocellular Carcinoma

Introduction Biomarkers for early hepatocellular carcinoma (HCC) detection are still a clinical need. Recent data indicate that cancer-associated antigens lead to the formation of circulating IgM-linked immune complexes as the result of natural IgM production by the innate immune response. The SCCA-IgM immune complex has already been described for primary liver cancer detection. Osteopontin (OPN) is a member of the SIBLING family of proteins recently shown to be related to tumorigenesis, progression and metastasis in various cancer types. Aim To evaluate the serum levels of the OPN-IgM complex in comparison to the SCCA-IgM complex in patients with HCC. Patients A total of 256 patients were analyzed including 151 patients with HCC (M/F 115/36; mean age ± SD: 67 ± 12 years) and 106 patients with cirrhosis (M/F 68/38; mean age ± SD: 62 ± 12 years). HCC, tested before any therapeutic approach, was mainly of viral etiology (58% HCV, 11% HBV), while alcohol abuse (22%) was the main risk factor for the remaining cases. A similar distribution was found in patients with cirrhosis (46% HCV, 15% HBV, 38% alcohol). Methods Serum levels of OPN-IgM were measured using a homemade ELISA assay with a polyclonal anti-human OPN antibody (Biodesign Int, USA). SCCA-IgM was detected in serum using an ELISA assay kit (Hepa-IC, Xeptagen). Results OPN-IgM was positive in 64/151 (42%) patients with HCC and in 45/106 (42%) patients with cirrhosis (specificity 58%), while the reference biomarker SCCA-IgM showed 35% sensitivity (49/139) and 71 % specificity. When patients were stratified on the basis of etiology, the sensitivity values for OPN-IgM and SCCA-IgM were 51% vs 44% in HCV patients, and 36% vs 23% in the other patients, while the specificity was lower for OPN-IgM (55% vs 69% in HCV patients; 62% vs 72% in the other patients). Combination of the two biomarkers resulted in an increase in sensitivity to 63% for viral etiology and to 40% for nonviral etiology. Conclusion Different IgM-linked biomarkers are detectable in primary liver cancer. OPN-IgM and SCCA-IgM showed a similar behavior, while the combination of these biomarkers increased the diagnostic performance for primary liver cancer.