TGFα-EGFR pathway in breast carcinogenesis, association with WWOX expression and estrogen activation.

WWOX is a tumor-suppressive steroid dehydrogenase, which relationship with hormone receptors was shown both in animal models and breast cancer patients. Herein, through nAnT-iCAGE high-throughput gene expression profiling, we studied the interplay of estrogen receptors and the WWOX in breast cancer cell lines (MCF7, T47D, MDA-MB-231, BT20) under estrogen stimulation and either introduction of the WWOX gene by retroviral transfection (MDA-MB-231, T47D) or silenced with shRNA (MCF7, BT20). Additionally, we evaluated the consequent biological characteristics by proliferation, apoptosis, invasion, and adhesion assays. TGFα-EGFR signaling was found to be significantly affected in all examined breast cancer cell lines in response to estrogen and strongly associated with the level of WWOX expression, especially in ER-positive MCF7 cells. Under the influence of 17ß-estradiol presence, biological characteristics of the cell lines were also delineated. The study revealed modulation of adhesion, invasion, and apoptosis. The obtained results point at a complex role of the WWOX gene in the carcinogenesis of the breast tissue, which seems to be closely related to the presence of estrogen α and/or ß receptors.

Safety assessment of chronic hepatitis C treatment using h-FABP and 24-hour ECG Holter monitoring.

Every year, 3-4 million people become infected with HCV, most of them are asymptomatic. In more than 20-30 years from infection, it leads to 10-20% of patients with cirrhosis, followed by hepatocellular carcinoma. Cardiological complications of the antiviral treatment are relatively rare, but force us to take additional diagnostic or discontinuation of therapy. AIM: The aim of study was to assess the cardiovascular safety of chronic hepatitis C treatment of genotype 1 in a triple regimen containing pegylated interferon-α in combination with ribavirin and boceprevir based on analysis of 24-hour ECG Holer monitoring, as well as changes in the concentration of cardiac fraction of fatty acid binding proteins (h-FABP). MATERIALS AND METHODS: 14 hepatitis C patients and 15 healthy people were included. The participants had an ambulatory 24-hour ECG-Holter recording at home condition and the determined level of h-FABP at baseline, after 4 and 12-16 weeks of treatment and 2 weeks after the end of therapy. The HRV parameters, AC/DC and QTc was calculated. RESULTS: At baseline there were no statistically significant differences in the HRV parameters, DC/AC, and QTc-interval. Absolute DC/AC values, HRV parameters: SDNN-ix, rMSDD, TP, HF, VLF and ULF were significantly lower in the treated group. LF/HF ratio was higher in this group (p=0.047). These changes persisted during the follow-up and disappeared after treatment. QTc was the shortest in the 4th week and withdrew during further follow-up. H-FABP levels did not differ statistically significantly between any subsequent determinations. CONCLUSIONS: At baseline there were no statistically significant differences in the HRV parameters, DC/AC, and QTc-interval. Absolute DC/AC values, HRV parameters: SDNN-ix, rMSDD, TP, HF, VLF and ULF were significantly lower in the treated group. LF/HF ratio was higher in this group (p=0.047). These changes persisted during the follow-up and disappeared after treatment. QTc was the shortest in the 4th week and withdrew during further follow-up. H-FABP levels did not differ statistically significantly between any subsequent determinations.

WWOX Tumor Suppressor Gene in Breast Cancer, a Historical Perspective and Future Directions.

The WWOX tumor suppressor gene is located at 16q23. 1-23.2, which covers the region of FRA16D-a common fragile sites. Deletions within the WWOX coding sequence are observed in up to 80% of breast cancer cases, which makes it one of the most common genetic alterations in this tumor type. The WWOX gene is known to play a role in breast cancer: increased expression of WWOX inhibits cell proliferation in suspension, reduces tumor growth rates in xenographic transplants, but also enhances cell migration through the basal membrane and contributes to morphological changes in 3D matrix-based cell cultures. The WWOX protein may act in several ways, as it has three functional domains-two WW domains, responsible for protein-protein interactions and an SDR domain (short dehydrogenase/reductase domain) which catalyzes conversions of low molecular weight ligands, most likely steroids. In epithelial cells, WWOX modulates gene transcription through interaction with p73, AP-2γ, and ERBB4 proteins. In steroid hormone-regulated tissues like mammary gland epithelium, the WWOX SDR domain acts as a steroid dehydrogenase. The relationship between WWOX and hormone receptors was shown in an animal model, where WWOX(C3H)+/-mice exhibited loss of both ER and PR receptors. Moreover, in breast cancer specimens, a positive correlation was observed between WWOX expression and ER status. On the other hand, decreased WWOX expression was associated with worse prognosis, namely higher relapse and mortality rates in BC patients. Recently, it was shown that genomic instability might be driven by the loss of WWOX expression. It was reported that WWOX plays role in DNA damage response (DDR) and DNA repair by regulating ATM activation through physical interaction. A genome caretaker function has also been proposed for WWOX, as it was found that WWOX sufficiency decreases homology directed repair (HDR) and supports non-homologous end-joining (NHEJ) repair as the dominant DSB repair pathway by Brca1-Wwox interaction. In breast cancer cells, WWOX was also found to modulate the expression of glycolysis pathway genes, through hypoxia-inducible transcription factor 1α (HIF1α) regulation. The paper presents the current state of knowledge regarding the WWOX tumor suppressor gene in breast cancer, as well as future research perspectives.

Finding NEMO in preeclampsia.

BACKGROUND: The mechanism of preeclampsia and its way of inheritance are still a mystery. Biochemical and immunochemical studies reveal a substantial increase in tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 concentrations in the blood of women with preeclampsia. The level of these factors is regulated by nuclear facxtor-kappa B, whose activation in a classical pathway requires inhibitory kappa B kinase gamma (known as NEMO or IKBKG). Moreover, NEMO can schedule between cytoplasma and the nucleus. In the nucleus, IKBKG interacts with other proteins, and thus, it is implicated in the regulation of different gene expressions, which are related to cell cycle progression, proliferation, differentiation, and apoptosis. OBJECTIVE: This is the first study investigating the association between the level of NEMO gene expression and the presence of preeclampsia. We tested the hypothesis that the simultaneous increase in NEMO gene expression both in the mother and her fetus may be responsible for the preeclampsia development. Moreover, the relationships between clinical risk factors of preeclampsia and the levels of NEMO gene expression in blood, umbilical cord blood, and placentas were investigated. STUDY DESIGN: A total of 91 women (43 preeclamptic women and 48 controls) and their children were examined. Real-time reverse transcription-polymerase chain reaction was used to assess the amount total NEMO messenger ribonucleic acid (mRNA) content and the mRNA level of each NEMO transcript from exons 1A, 1B, and 1C in maternal blood, umbilical cord blood, and placentas. Univariate analyses and correlation tests were performed to examine the association between NEMO gene expression and preeclampsia. RESULTS: Newborn weight and height, maternal platelet number, and gestational age (week of delivery) were lower in the group of women with preeclampsia than controls. NEMO gene expression level was found to be almost 7 times higher in the group of women with preeclampsia than healthy controls. The correlation analysis found that a simultaneous increase in the expression level of total NEMO mRNA in maternal blood and the mRNA for total NEMO (Rs = 0.311, P < .05), transcripts 1A (Rs = 0.463, P < .01), 1B (Rs = 0.454, P < .01), and 1C (Rs = 0.563, P < .001) in fetal blood was observed in preeclamptic pregnancies. In addition, the mRNA levels for total NEMO and transcripts 1A, 1B, and 1C were lower in placentas derived from pregnancies complicated by preeclampsia. CONCLUSION: Simultaneous increase of NEMO gene expression in maternal and fetal blood seems to be relevant for preeclampsia development. The results of our study also suggest that a decreased NEMO gene expression level in preeclamptic placentas may be the main reason for their intensified apoptosis.

The whole-genome expression analysis of peripheral blood mononuclear cells from aspirin sensitive asthmatics versus aspirin tolerant patients and healthy donors after in vitro aspirin challenge.

BACKGROUND: Up to 30% of adults with severe asthma are hypersensitive to aspirin and no unambiguous theory exists which provides a satisfactory explanation for the occurrence of aspirin-induced asthma (AIA) in some asthmatic patients. Therefore, the aim of this study was to compare the AIA expression profile against aspirin tolerant asthma (ATA) and healthy volunteers (HV) profile in peripheral blood mononuclear cells (PBMCs) after in vitro aspirin challenge in Caucasian population. METHODS: PBMCs were separated from blood of three groups of subjects--11 AIA, 7 ATA and 15 HV and then stimulated by either 2 µM lysine aspirin or 20 µM lysine as a control. Subsequently, RNA was isolated, transcribed into cDNA and subjected to microarray and qPCR studies. Simultaneously, protein was extracted from PBMCs and used in further immunoblotting analysis. RESULTS: The validation of results at mRNA level has shown only three genes, whose expression was significantly altered between comprising groups. mRNA expression of CNPY3 in PBMCs in AIA was significantly lower (-0.41 ± 2.67) than in HV (1.04 ± 2.69), (p = 0.02); mRNA expression of FOSL1 in PBMCs in AIA was also significantly decreased (-0.66 ± 2.97) as opposed to HV (0.31 ± 4.83), (p = 0.02). While mRNA expression of ERAS in PBMCs was increased (1.15 ± 0.23) in AIA in comparison to HV (-1.32 ± 0.41), (p = 0.03). At protein level the changed expression of one protein was confirmed. Protein expression of FOSL1 in PBMCs in AIA was both significantly lower (-0.86 ± 0.08) than in ATA (0.39 ± 0.42), (p = 0.046) and in HV (0.9 ± 0.27), (p = 0.007). CONCLUSIONS: This pilot study implies a positive association between CNPY3, ERAS, FOSL1 and aspirin-intolerant asthma, suggesting that these findings would be useful for further investigations of NSAIDs mechanism.

The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer.

This study defines the role of WWOX in the regulation of epithelial to mesenchymal transition. A group of 164 endometrial adenocarcinoma patients was studied as well as an ECC1 well-differentiated steroid-responsive endometrial cell line, which was transducted with WWOX cDNA by a retroviral system. The relationship between WWOX gene and EMT marker (CDH1, VIM, ZEB1, SNAI1) expression on mRNA (RT-qPCR) and protein levels (western blotting) was evaluated. The EMT processes were also analysed in vitro by adhesion of cells to extracellular matrix proteins, migration through a basement membrane, anchorage-independent growth and MMP activity assay. DNA microarrays (HumanOneArray™) were used to determine WWOX-dependent pathways in an ECC1 cell line. A positive correlation was observed between WWOX and ZEB1, and a negative correlation between CDH1 and VIM. WWOX expression was found to inversely correlate with the risk of recurrence of tumors in patients. However, in the WWOX-expressing ECC1 cell line, WWOX expression was found to be inversely related with VIM and positively with CDH1. The ECC1/WWOX cell line variant demonstrated increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. However, these cells were not able to form colonies in suspension and revealed decreased adhesion to fibronectin and fibrinogen. Microarray analysis demonstrated that WWOX has an impact on the variety of cellular pathways including the cadherin and integrin signalling pathways. Our results suggest that the WWOX gene plays a role in the regulation of EMT processes in endometrial cancer by controlling the expression of proteins associated with cell motility, thus influencing tissue remodeling, with the suppression of mesenchymal markers.

WWOX modulates the gene expression profile in the T98G glioblastoma cell line rendering its phenotype less malignant.

The aim of the present study was to assess the influence of WWOX gene upregulation on the transcriptome and phenotype of the T98G glioblastoma cell line. The cells with high WWOX expression demonstrated a significantly different transcription profile for approximately 3,000 genes. The main cellular pathways affected were Wnt, TGFß, Notch and Hedgehog. Moreover, the WWOX-transfected cells proliferated at less than half the rate, exhibited greatly lowered adhesion to ECM, increased apoptosis and impaired 3D culture formation. They also demonstrated an increased ability for crossing the basement membrane. Our results indicate that WWOX, apart from its tumor-suppressor function, appears to be a key regulator of the main cellular functions of the cell cycle and apoptosis. Furthermore, our results showed that WWOX may be involved in controlling metabolism, cytoskeletal structure and differentiation.

Diverse effect of WWOX overexpression in HT29 and SW480 colon cancer cell lines.

WW-domain-containing oxidoreductase (WWOX) is the tumour suppressor gene from the common fragile site FRA16D, whose altered expression has been observed in tumours of various origins. Its suppressive role and influence on basic cellular processes such as proliferation and apoptosis have been confirmed in many in vitro and in vivo studies. Moreover, its protein is thought to take part in the regulation of tissue morphogenesis and cell differentiation. However, its role in colon cancer formation remains unclear. The aim of this study was to characterize the influence of WWOX on the process of colon cancerogenesis, the basic features of the cancer cell and its expression profiles. Multiple biological tests, microarray experiments and quantitative reverse transcriptase (RT)-PCR were performed on two colon cancer cell lines, HT29 and SW480, which differ in morphology, expression of differentiation markers, migratory characteristics and metastasis potential and which represent negative (HT29) and low (SW480) WWOX expression levels. The cell lines were subjected to retroviral transfection, inducting WWOX overexpression. WWOX was found to have diverse effects on proliferation, apoptosis and the adhesion potential of modified cell lines. Our observations suggest that in the HT29 colon cancer cell line, increased expression of WWOX may result in the transition of cancer cells into a more normal colon epithelium phenotype, while in SW480, WWOX demonstrated well-known tumour suppressor properties. Our results also suggest that WWOX does not behave as classical tumour suppressor gene, and its influence on cell functioning is more global and complicated.

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

PURPOSE: Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. METHODS: The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. RESULTS: We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). CONCLUSIONS: Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.