Ondřej Slabý

MicroRNA-206: a Promising Theranostic Marker

MicroRNAs (miRs) are small non-coding RNAs that negatively regulate gene expression by binding to the 3` untranslated regions (3`UTR) of their target mRNAs. MiRs were shown to play pivotal roles in tissue development and function and are also involved in the pathogenesis of various diseases including cancer. MicroRNA-206, which belongs to the group of so-called “myomiRs”, is one of the most studied miRs thus far. In addition to being involved in skeletal muscle development and pathology, it has also been established that it is involved in the pathogenesis of numerous diseases including heart failure, chronic obstructive pulmonary disease, Alzheimers disease and various types of cancers. The aim of this review is to provide a complex overview of microRNA-206, including regulating its expression, a brief description of its known functions in skeletal muscle and a complex overview of its roles in the biology and pathology of other tissues, emphasizing its significant diagnostic and therapeutic potential.

MicroRNAs involved in skeletal muscle development and their roles in rhabdomyosarcoma pathogenesis

MicroRNAs (miRs) are small non‐coding RNAs known to fulfill various functions in tissue development, function, and pathogenesis of various diseases, including cancer. Rhabdomyosarcoma (RMS) represents the most common soft tissue tumor in the pediatric population. miRs have been shown to play important roles in RMS pathogenesis and some of the studies suggest their potential as diagnostic, prognostic, and even therapeutic tools facilitating better management of this disease. This review summarizes current information about the role of miRs in the development of normal skeletal muscle and their deregulation in RMS. Pediatr Blood Cancer 2013;60:1739–1746.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

Abstract Background: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. Methods: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. Results: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient’s age at the time of diagnosis. Conclusions: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Association of Glutathione S-Transferase Polymorphisms with Dietary Composition but Not Anthropometry in Obese as Well as Nonobese Individuals

ABSTRACT Objectives: Glutathione S-transferases (GSTs) are detoxifying enzymes for a number of substrates, including some food compounds. Selected GST polymorphisms have been proven to significantly affect enzymatic activity; however, it is unclear whether this altered metabolism influences dietary composition. The objective of this study was to locate the correlation between GST polymorphisms and selected nutritional parameters, namely, fiber and vitamin C intake. Methods: This study was conducted on a cohort of 472 individuals (mean age 45.26 years; mean body mass index [BMI] 32.36) from the South Moravian region of the Czech Republic. Basic anthropometrical parameters were measured and no association was found for the selected polymorphisms. Polymorphisms in GSTA1, GSTM1, and GSTT1 were genotyped using a polymerase chain reaction (PCR)-based methodology. Food intake was monitored using a self-administered 7-day questionnaire that was subsequently analyzed with a special focus on vitamin C intake, fiber intake, and total energy intake. Results: For GSTA1 and GSTM1 polymorphisms, an association was observed with fiber intake. Though no association was found with vitamin C intake, mean vitamin C intake was found to be higher than recommended daily values. No association was found with either daily energy intake or anthropometric parameters. Conclusion: Based on our results, GST polymorphisms seem to affect dietary composition; however, they have no effect on total energy intake or any association with obesity.

Current Possibilities for Predicting Responses to EGFR Blockade in Metastatic Colorectal Cancer

BACKGROUND The combination of modern systemic chemotherapy and anti-EGFR monoclonal antibodies improves overall survival and quality of life for patients with metastatic colorecal cancer. By contrast, the addition of anti-EGFR therapy to the treatment regime of resistant patients may lead to worse progression-free survival and overall survival. Therefore, identifying sensitive and resistant patients prior to targeted therapy of metastatic colorecal cancer is a key point during the initial decision making process. Previous research shows that primary resistance to EGFR blockade is in most cases caused by constitutive activation of signaling pathways downstream of EGFR. Of all relevant factors (mutation of KRAS, NRAS, BRAF, and PIK3CA oncogenes, inactivation of tumor suppressors PTEN and TP53, amplification of EGFR and HER2, and expression of epiregulin and amphiregulin, mikroRNA miR-31-3p, and miR-31-5p), only evaluation of KRAS and NRAS mutations has entered routine clinical practice. The role of the other markers still needs to be validated. The ongoing benefit of anti-EGFR therapy could be indicated by specific clinical parameters measured after the initiation of targeted therapy, including early tumor shrinkage, the deepness of the response, or hypomagnesemia. The accuracy of predictive dia-gnostic tools could be also increased by examining a combination of predictive markers using next generation sequencing methods. However, unjustified investigation of many molecular markers should be resisted as this may complicate interpretation of the results, particularly in terms of their specific clinical relevance. AIM The aim of this review is to describe current possibilities with respect to predicting responses to EGFR blockade in the context of the EGFR pathway, and the utilization of such results in routine clinical practice.

Abstract 3979: Urinary cell-free microRNAs as potential biomarkers of urothelial carcinoma of the urinary bladder

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Urothelial carcinoma of the urinary bladder (UCUB) is the most common malignancy of the urinary system. UCUB is divided into muscle invasive and non-muscle invasive bladder cancer (superficial), which represents approximately 80% of cases. Despite the relatively high degree of superficial tumors is UCUB associated with high local recurrence rate and approximately 20% of non-muscle invasive tumors progress to invasive form. Although cystoscopy remains a fundamental investigative tool in the detection and surveillance of urothelial bladder cancer, carcinoma in situ (CIS) or small papillary tumors can be with this method easily missed. This has led to the development of newer technologies and several molecular urinary tests, but currently there is no sensitive biomarker enabling early detection of relapse, which occurs in almost 70% of cases of superficial UCUB or biomarker with ability to predict the risk of progression of non-invasive to invasive form of UCUB. Such requirements could fit with diagnostic approach based on the detection of microRNAs (miRNAs) in urine, where have already showed remarkably high stability and good analytical properties. Patients and methods: Using Affymetrix miRNA microarrays we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 UCUB patients (6 invasive, 5 high-grade non-invasive, 5 low-grade non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI). Ability of selected miRNAs to identify UCUB from urine was confirmed in the validation phase based on independent cohort of 80 UCUB patients using qRT-PCR method. Results: Global expression profiling revealed set of 76 miRNAs significantly differentially expressed in urine of UCUB patients (P < 0,01) compared to healthy controls, thereof 64 highly up-regulated and 12 down-regulated. These miRNAs were specific for UCUB also when compared to other examined cohorts of patients (RCC, UTI). Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P < 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p < 0,01). Subsequent validation on larger independent cohort of UCUB patients lead to definition of urinary miRNA panel enabling sensitive and highly specific diagnosis of UCUB from urine. Conclusion: Our data have shown that urinary miRNAs could serve as sensitive and specific biomarkers of UCUB and after further independent validations could be useful tool to increase sensitivity of standard cytological examination potentially decreasing high costs for long-term follow-up of UCUB patients. This work has been supported by IGA MZCR: NT/13860-4/2012. Citation Format: Jaroslav Juracek, Hana Mlcochova, Michal Stanik, Barbora Peltanova, Robert Iliev, Taňa Machackova, Jitka Mlcochova, Renata Hežova, Jan Doležel, Ondřej Slabý. Urinary cell-free microRNAs as potential biomarkers of urothelial carcinoma of the urinary bladder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2015-3979

[The role of MicroRNAs in the pathophysiology of neuroblastoma and their possible use in diagnosis, prognosis and therapy].

Neuroblastoma (NBL) is a typical childhood tumor developing from the precursor cells of the sympathetic nervous tissue and accounting for approximately 7% of total malignancies in pediatrics and 15% of deaths associated with this malignancy. MicroRNAs (miRNAs) are small single-stranded RNA molecules that are involved in posttranscriptional regulation of gene expression, whereas the pathophysiology of neuroblastoma tumor growth involves both upregulation of the protooncogenic miRNAs as well as downregulation of the tumor-suppresor ones. Comparison of the expression profiles of miRNAs in specific subtypes of neuroblastoma seems to be a useful tool adding to the classification of the diseases, and the assessment of the levels of specific miRNAs may be useful for estimation of the individual treatment response as well as prognosis of the patient. This paper provides the basic review of the studies focused on the role of miRNAs in pathogenesis of neuroblastoma and provides a survey of current/ possible use of these miRNAs in diagnostics, therapy or prognosis estimation in the neuroblastoma patients.

Genes of the microRNA biogenesis pathway are deregulated in Colorectal cancer

B cancer is one of the most common cancers affecting women. Our lab has previously shown that arginine methylation by arginine methyltransferases (PRMTs) is upregulated in breast cancer. We have further identified a novel modular protein, Tudor domain containing protein 3, TDRD3, and shown its role in reading/sensing of arginine methylation. Furthermore, TDRD3 was among the top hits in the genes that are strongly correlated with poor prognosis of estrogen receptor negative breast cancer (Nagahata et al., 2004). Moreover in the CancerGenome Atlas’ Data Portal, TDRD3 was overexpressed in 58/158 of breast cancer patients. This suggests a potent role of this that this novel gene in the implication of breast cancer. TDRD3 was further shown to re-localzie to stress granules (SGs) upon various environmental stresses. SGs have implications in cancer as they been shown to promote tumor cell survival and mediate resistance to cancer therapy. Therefore, we hypothesize that TDRD3 contributes to late hallmarks of breast cancer pathogenesis through regulating SGs. In this project, we elucidate the role of TDRD3 in assembly/disassembly and dynamic of stress granules. Gaining insight into this novel gene in the light of its role in SGs may eventually have implication on elucidating further, the pathology of breast cancer.MicroRNAs (miRNAs) are small non-coding RNAs 18-25 nucleotides long that regulate gene expression on post-transcriptional level. Therefore, their production and maturation have to be strictly regulated. Their biogenesis starts with the transcription, which is followed by several steps that lead to processing of primary transcripts to mature miRNAs. Each step of this pathway is sophistically regulated and any disruption of control mechanisms may leads to the cancer occurrence. The aim of this study was to analyse the expression of the crucial genes involved in the biogenesis of miRNAs in colorectal cancer (CRC). Expression of 19 selected genes (EIF2C1-4, GEMIN4, DDX20, TARBP2, DICER1, XPO5, DROSHA, DGCR8, POLR2A, DDX5, DDX17, ADAR, ADARB1, TNRC6A, LIN28A/B) has been analysed in tumour tissues of 120 clinically characterized patients with CRC and in 120 parallel healthy tissues by qRT-PCR. Using Wilcoxon test, genes with different expression between tumour tissue and healthy tissue have been identified. Subsequently, Kruskal-Wallis test has been used to find any correlation with the clinical-pathological features of the patients. We have found significantly higher expression of POLRIIA, ADAR, ADARB1, DGCR8, DDX5, DDX17, DROSHA, XPO5, EIF2C1-C4, TARBP2, TNRC6A, GEMIN3, DDX20 and DICER1 (P < 0.0001) and significantly lower expression of LIN28A in tumour tissue of CRC patients. Moreover, negative correlation between the expression of AGO3 and clinical stage of patients (P = 0.0017) and grade (P = 0.0151) and positive correlation between the expression of DICER1 (P = 0.0230) and DROSHA (P = 0.0212) and grade of patients has been observed. Our results show that changes in the expression of genes associated with biogenesis of miRNAs may be associated not only with the origin, but also to progression of CRC and therefore, these molecules could serve as potential new biomarkers or therapeutic targets.Background: Bromelain is the extract of pineapple stem primarily comprised of sulfhydryl containing proteolytic enzymes. N-acetylcysteine (NAC) is also a sulfhydryl-containing compound found in allium plants, asparagus, and red pepper. Biological effects of bromelain and NAC have been studied in some pathologic conditions including cancer. In the present study, we investigated the cytotoxic effects of bromelain and/or NAC on the gastric carcinoma cell line KATO-III in vitro. Methods: KATO-III gastric carcinoma cells were treated with a range of concentrations of bromelain and/or NAC. The effect of the single agent or combination therapy on the growth and proliferation of the cancer cells was determined after 72 hours of treatment using sulforhodamine B assay. A similar method was employed to assess any possible synergism when the treatment was combined with chemotherapy. Results: We observed that bromelain, at the concentration of 200 μg/ml, and NAC, at the concentration of 25 mM, significantly inhibited the cell proliferation (p-values of <0.0001 and 0.0095, respectively). When combined together, synergistic effects of the combination therapy was evident. In addition, we examined if the treatment was capable of potentiating the cytotoxic effects of chemotherapy. Cytotoxicity of single agent cisplatin, paclitaxel, 5-fluorouracil and vincristine was compared against that of their combination with bromelain/NAC. Our results indicated a higher potency of each drug when used in combination with bromelain/NAC. Conclusion: In the present study, we showed that the utility of bromelain and N-acetylcysteine, as single agent or in combination, yields cytotoxic effects in the gastric carcinoma cell line KATO-III and potentiates chemotherapy. Afshin Amini et al., J Cancer Sci Ther 2013, 5:10 http://dx.doi.org/10.4172/1948-5956.S1.029

MicroRNA and Glial Tumors: Tiny Relation with Great Potential

MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that act as post-transcriptional regulators of gene expression. Dysregulation of these molecules has been observed in many types of cancers. Altered expression levels of several miRNAs were identified also in gliomas. It was many times showed that miRNAs are involved in core signaling pathways, which play roles in crucial cellular processes, such as proliferation, apoptosis, cell cycle regulation, invasion, angiogenesis and stem cell behaviour. Therefore, miRNAs have a great potential for oncodiagnostic as well as could be promising therapeutic targets in gliomas.