Nora Meggyeshazi

Correlations of Differentially Expressed Gap Junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with Breast Cancer Progression and Prognosis

Background and Aims Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. Materials and Methods Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. Results The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. Conclusion Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

MEK1 is associated with carboplatin resistance and is a prognostic biomarker in epithelial ovarian cancer

BackgroundPrimary systemic treatment for ovarian cancer is surgery, followed by platinum based chemotherapy. Platinum resistant cancers progress/recur in approximately 25% of cases within six months. We aimed to identify clinically useful biomarkers of platinum resistance.MethodsA database of ovarian cancer transcriptomic datasets including treatment and response information was set up by mining the GEO and TCGA repositories. Receiver operator characteristics (ROC) analysis was performed in R for each gene and these were then ranked using their achieved area under the curve (AUC) values. The most significant candidates were selected and in vitro functionally evaluated in four epithelial ovarian cancer cell lines (SKOV-3-, CAOV-3, ES-2 and OVCAR-3), using gene silencing combined with drug treatment in viability and apoptosis assays. We collected 94 tumor samples and the strongest candidate was validated by IHC and qRT-PCR in these.ResultsAll together 1,452 eligible patients were identified. Based on the ROC analysis the eight most significant genes were JRK, CNOT8, RTF1, CCT3, NFAT2CIP, MEK1, FUBP1 and CSDE1. Silencing of MEK1, CSDE1, CNOT8 and RTF1, and pharmacological inhibition of MEK1 caused significant sensitization in the cell lines. Of the eight genes, JRK (p = 3.2E-05), MEK1 (p = 0.0078), FUBP1 (p = 0.014) and CNOT8 (p = 0.00022) also correlated to progression free survival. The correlation between the best biomarker candidate MEK1 and survival was validated in two independent cohorts by qRT-PCR (n = 34, HR = 5.8, p = 0.003) and IHC (n = 59, HR = 4.3, p = 0.033).ConclusionWe identified MEK1 as a promising prognostic biomarker candidate correlated to response to platinum based chemotherapy in ovarian cancer.

Expression of proliferation markers Ki67, cyclin A, geminin and aurora-kinase A in primary breast carcinomas and corresponding distant metastases

Aims To assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and aurora-kinase A (aurkA); and to compare the expression of these markers in early MBC (EMBC) and late MBC separated into groups according to median time point on metastatic event occurred (28 months). Methods The expression of the above mentioned markers was analysed in a total of 47 primary MBCs and 59 DMs (out of which 37 were pairs) by immunohistochemistry. Fourteen breast carcinomas with no relapse over a 10-year follow-up period were utilised as control cases (CBC). Results Among the MBCs, 22 metastasised to the bone, 4 to the lung and 21 to the central nervous system (CNS). Geminin (p<0.001) and Ki67 (p=0.001) were increased in the MBCs while aurkA and cyclin A showed no difference when compared with CBCs. There were no differences between aurkA, cyclin A and geminin expression in MBCs and DMs in general. Expression of Ki67 was, however, elevated (p=0.027) in DMs. In CNS metastases all markers showed elevated expression as compared to MBCs. In bone metastases, geminin was lower (p<0.001) compared with primary MBCs. In the metastases of the lung, the evaluated markers did not show different expression. According to the median follow-up until the metastatic event, Ki67 was found to be significantly elevated in EMBC (p=0.018). Conclusions Ki67 index and geminin distinguish a fraction of MBC with worse prognosis, showing increased levels in the latter in comparison to CBC being tumour-free over a 10-year follow-up period. Ki67 could possibly identify a group of MBCs that develop early DMs.

Changes in the Distribution of Type II Transmembrane Serine Protease, TMPRSS2 and in Paracellular Permeability in IPEC-J2 Cells Exposed to Oxidative Stress

The effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2 (TMPRSS2) were studied using porcine epithelial IPEC-J2 cells on membrane inserts. Increased paracellular permeability of FITC-dextran 4 kDa (fluorescence intensity 43,508 ± 2,391 versus 3,550 ± 759) and that of gentamicin (3.41 ± 0.06 % increase to controls) were measured parallel with the reduced transepithelial electrical resistance (23.3 ± 4.06 % decrease) of cell layers 6 h after 1 h 1 mM H2O2 treatment. The immunohistochemical localization of adherens junctional β-catenin was not affected by reactive oxygen species (ROS) up to 4 mM H2O2. Peroxide-triggered enhanced paracellular permeability of IPEC-J2 cell layer was accompanied by predominantly cytoplasmic occurrence of TMPRSS2 embedded in cell membrane under physiological conditions. These results support that ROS can influence paracellular gate opening via multifaceted mode of action without involvement of β-catenin redistribution in adherens junction. Altered distribution pattern of TMPRSS2 and relocalized transmembrane serine protease activity may contribute to weakening of epithelial barrier integrity under acute oxidative stress.

Bystander Effect of Oncothermia

Metastatic form of malignant tumor diseases is the most serious problem in oncology and the greatest challenge in tumor therapy. Conventional therapeutical approaches (surgery, irradiation, and chemotherapy) cannot manage this challenge in oncological practice. According to our theory, oncothermia treatment-induced immunogenic tumor cell death can be a very good basis for immunotherapy combination to make systemic tumor control from a local tumor destruction effect. We summarize the molecular basis of the oncothermia treatment-induced immunogenic cell death as a necessary basic condition to achieve the bystander effect.

Modulation Effect in Oncothermia

Conventional hyperthermia is based on the local or systemic heating, which is measured by the realized temperature in the process. Oncothermia applies nanoheating, which means high energy absorption in the nanoscopic range of the malignant cell membrane selectively. This high temperature and its consequent stress create special effects: it evolves the possibility for chaperone proteins to be expressed on the outer membrane by softening the membrane and starts various excitations for programmed cell death of the targeted malignant cell. The process needs special delivery of the energy which selects as desired. A strict 13.56 MHz sinusoidal carrier frequency is amplitude modulated by time-fractal signals. The modulation is far from any sinus or other periodic patterns; it is a 1/f spectrum having definite templates for its construction. In some personalized cases, a definite template is used for the fractal pattern, which is copied from the actual character of the tumor pathology or any other specialty of the target.

Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro

Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.

Selective in situ protein expression profiles correlate with distinct phenotypes of basal cell carcinoma and squamous cell carcinoma of the skin

Non-melanoma skin cancer is the most common malignancy that shows increasing incidence due to our cumulative exposure to ultraviolet irradiation. Its major subtypes, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) differ in pathobiology, phenotype and clinical behavior, which must be reflected at the molecular level. In this study, protein expression profiles of BCC and SCC were tested in tissue microarrays and correlated with that of actinic keratosis, Bowens disease, seborrheic keratosis and normal epidermis by detecting 22 proteins involved in cell interactions, growth, cell cycle regulation or apoptosis. The significantly more reduced collagen XVII, CD44v6, pan-Desmoglein levels and more evident E-Cadherin delocalization in BCC compared to SCC correlated with the de novo dermal invasion of BCC against the progressive invasion from in situ lesions in SCC development. EGFR was also expressed at a significantly higher level in SCC than in BCC. The upregulated cell communication protein connexin43 in BCC could contribute to the protection of BCC from metastatic invasion. Elevated cell replication in BCC was underlined by the increased topoisomerase IIα and reduced p21(waf1) and p27(kip1) positive cells fractions compared to SCC. Compared to differentiated keratinocytes, caspase-8 and -9 were equally upregulated in skin carcinoma subtypes for either mediating apoptosis induction or immune escape of tumor cells. Hierarchical cluster analysis grouped SCC and actinic keratosis cases exclusively together in support of their common origin and malignant phenotype. BCC cases were also clustered fully together. Differentially expressed proteins reflect the distinct pathobiology of skin carcinoma subtypes and can serve as surrogate markers in doubtful cases.

Connexin 43 Communication Channels in Follicular Dendritic Cell Development and in Follicular Lymphomas

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ex vivo” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ex vivo” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.

Deep Temperature Measurements in Oncothermia Processes

Temperature in depth of various model systems was measured, starting with muscle and other phantoms. It was shown that the temperature can be selectively increased in the target. In water-protein phantom, the protein coagulation (>60°C) was observed selectively while the water temperature around it was a little higher than room temperature.