Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3 using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor

Low spinophilin expression enhances aggressive biological behavior of breast cancer

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24-3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer

H19 Noncoding RNA, an Independent Prognostic Factor, Regulates Essential Rb-E2F and CDK8-β-Catenin Signaling in Colorectal Cancer

The clinical significance of long noncoding RNAs (lncRNAs) in colorectal cancer (CRC) remains largely unexplored. Here, we analyzed a large panel of lncRNA candidates with The Cancer Genome Atlas (TCGA) CRC dataset, and identified H19 as the most significant lncRNA associated with CRC patient survival. We further validated such association in two independent CRC cohorts. H19 silencing blocked G1-S transition, reduced cell proliferation, and inhibited cell migration. We profiled gene expression changes to gain mechanism insight of H19 function. Transcriptome data analysis revealed not only previously identified mechanisms such as Let-7 regulation by H19, but also RB1-E2F1 function and β-catenin activity as essential upstream regulators mediating H19 function. Our experimental data showed that H19 affects phosphorylation of RB1 protein by regulating gene expression of CDK4 and CCND1. We further demonstrated that reduced CDK8 expression underlies changes of β-catenin activity, and identified that H19 interacts with macroH2A, an essential regulator of CDK8 gene transcription. However, the relevance of H19-macroH2A interaction in CDK8 regulation remains to be experimentally determined. We further explored the clinical relevance of above mechanisms in clinical samples, and showed that combined analysis of H19 with its targets improved prognostic value of H19 in CRC

The role of HOXC6 in the pathogenesis of malignant renal neoplasms

Das Nephroblastom ist ein embryonaler Tumor der Niere, welcher histologisch aus epithelialen Strukturen, Blastem und Mesenchym besteht. Ein im Vorfeld dieser Arbeit durchgeführter CGH Array zeigte eine Amplifikation von HOXC6 in Nephroblastomen. Die im Zuge dieser Arbeit mittels qRT PCR analysierten Nephroblastome wiesen eine Überexpression von HOXC6 V1 & V2 in einem Drittel der Tumore auf. HOXC6 V1 & V2 zeigten im Vergleich zu normalem Nierenparenchym eine statistisch signifikant erhöhte mRNA Expression in den analysierten Nierenzellkarzinomen. Die immunhistologische Untersuchung wies eine Expression von HOXC6 in einem Drittel der analysierten Nephroblastome bzw. der Hälfte der klarzelligen Nierenzellkarzinome auf. Ein CNV Assay zeigte zudem eine Amplifikation von HOXC6 im Großteil der analysierten Nephroblastome. Wir identifizierten HOXC6 als potentielles Zielgen von miR-27a in einer in-silico Analyse, jedoch konnte dies nicht mittels Luziferase Assay bestätigt werden. Die durchgeführte Western Blot Analyse in HeLa Zellen zeigte jedoch eine Regulation der HOXC6 Proteinexpression durch die miR-27a. Im Vergleich zu normalen Nierenparenchym wies diese miRNA eine statistisch signifikant erniedrigte Expression in Nephroblastomen auf. Eine im Vorfeld dieser Arbeit durchgeführte Expressionsanalyse einer Nephroblastom Zelllinie, welche HOXC6 V1 & V2 überexprimiert, zeigte eine erhöhte Expression der Gene WBSCR17 und CDK6. WBSCR17 wies eine Überexpression in 60% der analysierten Nephroblastome und in 30% der Nierenzellkarzinome auf. Mittels Chromatin Immunopräzipitation-Analyse konnte die von uns vermutete Interaktion zwischen WBSCR17 und HOXC6 nicht bestätigt werden. Eine statistisch signifikant erhöhte CDK6 mRNA Expression wurde in den analysierten Nephroblastomen und klarzelligen Nierenzellkarzinomen festgestellt. Unsere Resultate lassen vermuten, dass HOXC6 V1 & V2, WBSCR17 und CDK6 eine Rolle in der Pathogenese von Nephroblastomen und Nierenzellkarzinomen spielen.Nephroblastoma is a triphasic embryonal neoplasm of the kidney consisting of blastema, stroma and epithelium. In the present study we focused on the gene HOXC6 because it was amplified in nephroblastomas and nephrogenic rests in a previously performed CGH array analysis. HOXC6 V1 and V2 mRNA expression levels were analysed in nephroblastomas and renal cell carcinomas by qRT PCR. Both variants were overexpressed in approximately one third of nephroblastomas investigated. Compared to mature kidney, HOXC6 V1 & V2 showed a statistically significantly increased mRNA expression in renal cell carcinomas. Immunohistological examination exhibited a protein expression of HOXC6 in approximately one third of nephroblastomas and more than half of clear cell carcinomas analysed. In addition, a CNV assay revealed that the HOXC6 gene is amplified in the majority of nephroblastomas. We identified HOXC6 as a new target of miR-27a by an in-silico analysis but the obtained result could not be confirmed by luciferase assay. In contrast, Western Blot analysis showed that HOXC6 is regulated by miR-27a via translational repression in HeLa cells. Accordingly, expression analysis in our nephroblastoma samples revealed a statistically significantly lower expression of miR-27a compared to mature kidney. A previously performed mRNA expression analysis of a nephroblastoma cell line (WTCLS-1) overexpressing HOXC6 V1 and V2 showed an upregulation of WBSCR17 and CDK6. In the current study an overexpression of WBSCR17 was noted in 60% of nephroblastomas and 30% of renal cell carcinomas analysed. A Chromatin Immunoprecipitation did not confirm an interaction between WBSCR17 and the transcription factor HOXC6. A statistically significantly increased mRNA expression of CDK6 was noted in nephroblastomas and clear cell renal cell carcinomas investigated. Our results indicate that HOXC6 V1 and V2, WBSCR17 and CDK6 might be involved in the pathogenesis of nephroblastomas and renal cell carcinomas.eingereicht von Verena StiegelbauerGraz, Univ., Masterarb., 2013(VLID)258140

The Development of High-Quality Multispecies Probiotic Formulations: From Bench to Market

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. To date, there is an increasing number of commercially available products containing probiotics on the market. Probiotics have been recommended by health care professionals for reasons ranging from their long-term immunomodulatory effects to proven benefits in the management of different health conditions. For probiotic products, there are several important aspects that determine the success rate of the development from bench to market. The aim of this review is to explore how the current knowledge on microbe&ndash;microbe and host&ndash;microbe interactions can be used to develop high-quality, evidence-based probiotic formulations, specifically probiotic dietary supplements, with a focus on the selection of safe strains with relevant functional properties. In addition, we will highlight aspects of the probiotic manufacturing process that need to be considered during the product development and the subsequent manufacturing process to guarantee consistent efficacy of a probiotic product. For each high-quality probiotic formulation, it is important to screen multiple strains, and select only those strains that show relevant functional properties and that can be considered safe for human consumption. In addition, it is imperative that attention is paid to the product development and manufacturing process, and that safety and quality properties are monitored. Importantly, the beneficial effects of probiotics should be evaluated in product efficacy studies and post-marketing surveys in order to demonstrate their clinical efficacy. All these aspects need to be evaluated and validated during the development of a successful high-quality and ready-to-market probiotic

MicroRNAs as novel predictive biomarkers and therapeutic targets in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in western countries. Despite significant improvement in available treatment options, CRC still remains the second leading cause of cancer-related death. Traditionally, 5-fluorouracil has been used as the main chemotherapy drug for treatment of metastatic CRC (mCRC). However, during the last two decades more effective chemotherapeutic agents such as oxaliplatin, irinotecan and the monoclonal antibodies cetuximab, panitumumab and bevacizumab have been used in clinical practice. More recently, the therapeutic armamentarium has been supplemented by the monoclonal antibodies bevacizumab, cetuximab and panitumumab as well as the protein-trap aflibercept and the small molecule multi-kinase inhibitor regorafenib. One of the major problems for the management of CRC is the inherent or acquired resistance to therapeutic approaches. The discovery of microRNAs (miRNAs), a class of small, endogenous, non-coding, single-stranded RNAs that play a role as post-transcriptional regulators, has added new dimensions to the diagnosis and treatment of cancer. Because miRNAs are important regulators of carcinogenesis, progression, invasion, angiogenesis and metastases in CRC, they might serve as potential predictive and prognostic factors and even as therapeutic targets themselves. Several miRNAs are already known to be dysregulated in CRCs and have been linked to biological processes involved in tumor progression and response to anti-cancer therapies. This review summarizes current therapeutic approaches for treating CRC and highlights the role of miRNAs as novel predictive biomarkers and potential drug targets in CRC patients