Új módszertani lehetőségek és ezek alkalmazása a hormonális rendszer daganatainak genetikai kivizsgálásában

The technical developments leading to revolution in clinical genetic testing offer new approaches for patients with cancer. From one mutation or one gene approach the scale of genetic testing moved to whole exome or whole genome scale. It is well known that many tumours are genetically determined ans they are part of familial tumour syndromes. In addition, some mutations indicate specific molecular targeted therapies. Although sampling and sample preparation are different for testing germline and somatic mutations, the technical background of the analysis is the same. The aim of clinical genetic testing is to identify patients who are carriers of disease-causing mutations or to test tumour tissue for the presence of genetic alterations which may be targets for therapeutic approaches. In this review the authors summarize novel possibilities offered by next-generation sequencing in clinical genetic testing of patients with endocrine tumours. In addition, the authors review recent guidelines on technical and ethical issues related to these novel methods. Orv. Hetil., 2015, 156(51), 2063-2069

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression

Background: Comparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors. Preliminary densitometry analysis of laminin-1, α -smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts. Methods: In vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored. Results: While normal fibroblasts produced components of interstitial matrix and TGF- β 1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α 6 β 4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells. Conclusions: Our results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix

Lack of Matrilin-2 Favors Liver Tumor Development via Erk1/2 and GSK-3 beta Pathways In Vivo

Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2(-/-) mice comparing them with wild-type (WT) mice in a diethylnitrosamine (DEN) model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2(-/-) mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2(-/-) mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 mu g/g body weight DEN treatment, the liver of Matn2(-/-) mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3 alpha/beta and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in beta-Catenin protein expression were detected in Matn2(-/-) livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2(-/-) livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3 beta, play strategic roles

Chronic hyperglycemia induces trans-differentiation of human pancreatic stellate cells and enhances the malignant molecular communication with human pancreatic cancer cells

BACKGROUND: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment. METHODOLOGY/PRINCIPAL FINDINGS: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-β1 treatment (3.09-fold with a 2.73-fold without TGF-β1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour. CONCLUSIONS: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration

β-defensin 1 expression in HCV infected liver/liver cancer: an important role in protecting HCV progression and liver cancer development

Abstract β-defensin family plays a role in host defense against viral infection, however its role in HCV infection is still unknown. In this study, we demonstrated that β-defensin 1 was significantly reduced in HCV-infected liver specimens. Treatment with interferon and ribavirin upregulated β-defensin-1, but not other β-defensin tested, with the extent and duration of upregulation associated with treatment response. We investigated β-defensin family expression in liver cancer in publicly available datasets and found that among all the β-defensins tested, only β-defensin 1 was significantly downregulated, suggesting β-defensin 1 plays a crucial role in liver cancer development. Further analysis identified E-cadherin as the top positive correlated gene, while hepatocyte growth factor-regulated tyrosine kinase substrate as the top negative correlated gene. Expression of two proteoglycans were also positively correlated with that of β-defensin 1. We have also identified small molecules as potential therapeutic agents to reverse β-defensin 1-associated gene signature. Furthermore, the downregulation of β-defensin 1 and E-cadherin, and upregulation of hepatocyte growth factor-regulated tyrosine kinase substrate, were further confirmed in liver cancer and adjacent normal tissue collected from in-house Chinese liver cancer patients. Together, our results suggest β-defensin 1 plays an important role in protecting HCV progression and liver cancer development

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas

Hungarian Resarch Fund (OTKA PD100648) National Development Agency (KTIA_AIK-2-2012-0010)

Inhibition of Pediatric Glioblastoma Tumor Growth by the Anti-Cancer Agent OKN-007 in Orthotopic Mouse Xenografts

We thank the Peggy and Charles Stephenson Cancer Center at the University of Oklahoma, Oklahoma City, OK, for funding, who received an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103639 for the use of the Histology and Immunohistochemistry Core for providing immunohistochemistry and photographic services. This work was also supported by Oklahoma State University, Center of Veterinary Health Science (Support Grant AE-1-50060 to P.C.S.), the Musella Foundation (R.A.T.), and the Childhood Brain Tumor Foundation (R.A.T.).Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. Here, we describe the use of conventional and advanced in vivo magnetic resonance imaging (MRI) techniques to assess a novel orthotopic xenograft pGBM mouse (IC-3752GBM patient-derived culture) model, and to monitor the effects of the anti-cancer agent OKN-007 as an inhibitor of pGBM tumor growth. Immunohistochemistry support data is also presented for cell proliferation and tumor growth signaling. OKN-007 was found to significantly decrease tumor volumes (p<0.05) and increase animal survival (p<0.05) in all OKN-007-treated mice compared to untreated animals. In a responsive cohort of treated animals, OKN-007 was able to significantly decrease tumor volumes (p<0.0001), increase survival (p<0.001), and increase diffusion (p<0.01) and perfusion rates (p<0.05). OKN-007 also significantly reduced lipid tumor metabolism in responsive animals (Lip1.3 and Lip0.9)-to-creatine ratio (p<0.05), as well as significantly decrease tumor cell proliferation (p<0.05) and microvessel density (p<0.05). Furthermore, in relationship to the PDGFRα pathway, OKN-007 was able to significantly decrease SULF2 (p<0.05) and PDGFR-α (platelet-derived growth factor receptor-α) (p<0.05) immunoexpression, and significantly increase decorin expression (p<0.05) in responsive mice. This study indicates that OKN-007 may be an effective anti-cancer agent for some patients with pGBMs by inhibiting cell proliferation and angiogenesis, possibly via the PDGFRα pathway, and could be considered as an additional therapy for pediatric brain tumor patients.Yeshttp://www.plosone.org/static/editorial#pee