The Merkel Cell Polyomavirus T-Antigens and IL-33/ST2-IL1RAcP Axis: Possible Role in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is a causal factor in Merkel cell carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT) and small T-antigen (sT). Cytokines produced by tumor cells play an important role in cancer pathogenesis, and viruses affect their expression. Therefore, we compared human cytokine and receptor transcript levels in virus positive (V+) and virus negative (V-) MCC cell lines. Increased expression of IL-33, a potent modulator of tumor microenvironment, was observed in V+ MCC cell lines when compared to V- MCC-13 cells. Transient transfection studies with luciferase reporter plasmids demonstrated that LT and sT stimulated IL-33, ST2/IL1RL1 and IL1RAcP promoter activity. The induction of IL-33 expression was confirmed by transfecting MCC-13 cells with MCPyV LT. Furthermore, recombinant human cytokine domain IL-33 induced activation of MAP kinase and NF-κB pathways, which could be blocked by a ST2 receptor antibody. Immunohistochemical analysis demonstrated a significantly stronger IL-33, ST2, and IL1RAcP expression in MCC tissues compared to normal skin. Of interest, significantly higher IL-33 and IL1RAcP protein levels were observed in MCC patient plasma compared to plasma from healthy controls. Previous studies have demonstrated the implication of the IL-33/STL2 pathway in cancer. Because our results revealed a T-antigens-dependent induction of the IL-33/ST2 axis, IL-33/ST2 may play a role in the tumorigenesis of MCPyV-positive MCC. Therefore, neutralizing the IL-33/ST2 axis may present a novel therapeutic approach for MCC patients.

Direct interaction of the ATP-sensitive K+ channel by the tyrosine kinase inhibitors imatinib, sunitinib and nilotinib.

The ATP-regulated K+ channel (KATP) plays an essential role in the control of many physiological processes, and contains a ATP-binding site. Tyrosine kinase inhibitors (TKI) are commonly used drugs, that primarily target ATP-binding sites in tyrosine kinases. Herein, we used the patch-clamp technique to examine the effects of three clinically established TKIs on KATP channel activity in isolated membrane patches, using a pancreatic ß-cell line as a KATP channel source. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by imatinib with half-maximal concentration of approximately 9.4 µM. The blocking effect of imatinib was slow and reversible. No effect of imatinib was observed on either the large (KBK) or the small (KSK) conductance, Ca2+-regulated K+ channel. In the presence of ATP/ADP (ratio 1) addition of imatinib increased channel activity approximately 1.5-fold. Sunitinib and nilotinib were also found to decrease KATP channel activity. These findings are compatible with the view that TKIs, designed to interact at the ATP-binding pocket on the tyrosine receptor, also interact at the ATP-binding site on the KATP channel. Possibly, this might explain some of the side effects seen with TKIs.

Imatinib Regulates miR-483-3p and Mitochondrial Respiratory Complexes in Gastrointestinal Stromal Tumors.

Metabolic adaptation to increased oxidative phosphorylation (OXPHOS) has been found in gastrointestinal stromal tumor (GIST) upon imatinib treatment. However, the underlying mechanism of imatinib-induced OXPHOS is unknown. Discovering molecules that mediate imatinib-induced OXPHOS may lead to the development of therapeutic strategies synergizing the efficacy of imatinib. In this study, we explored the role of microRNAs in regulating OXPHOS in GIST upon imatinib treatment. Using a microarray approach, we found that miR-483-3p was one of the most downregulated miRNAs in imatinib-treated tumors compared to untreated tumors. Using an extended series of GIST samples, we further validated the downregulation of miR-483-3p in imatinib-treated GIST samples by RT-qPCR. Using both gain- and loss-of-function experiments, we showed that miR-483-3p could regulate mitochondrial respiratory Complex II expression, suggesting its role in OXPHOS regulation. Functionally, miR-483-3p overexpression could rescue imatinib-induced cell death. These findings provide the molecular link for imatinib-induced OXPHOS expression and the biological role of miR-483-3p in regulating cell viability upon imatinib treatment.

Merkel cell polyomavirus oncoproteins induce microRNAs that suppress multiple autophagy genes.

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T-antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV-positive tumors. Ectopic expression of MCPyV small T-antigen and truncated large T-antigen (LT), but not the wild-type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV-mediated tumorigenesis. Torin-1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan-caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV-mediated tumorigenesis and potential MCC treatment.

miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors.

The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells.

Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate-glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.

Secretome protein signature of human gastrointestinal stromal tumor cells.

Strategies for correct diagnosis, treatment evaluation and recurrence prediction are important for the prognosis and mortality rates among cancer patients. In spite of major improvements in clinical management, gastrointestinal stromal tumors (GISTs) can still be deadly due to metastasis and recurrences, which confirms the unmet need of reliable follow-up modalities. Tumor-specific secreted, shed or leaked proteins (collectively known as secretome) are considered promising sources for biomarkers, and suitable for detection in biofluids. Herein, we stimulated cell secretion in the imatinib-sensitive GIST882 cell line and profiled the secretome, collected as conditioned media, by using a shotgun proteomics approach. We identified 764 proteins from all conditions combined, 51.3% being predicted as classically/non-classically secreted. The protein subsets found were dependent on the stimulatory condition. The significant increase in protein release by the classical pathway was strongly associated with markers already found in other cancer types. Furthermore, most of the released proteins were non-classically released and overlapped to a high degree with proteins of exosomal origin. Imatinib pre-treatment radically changed these secretory patterns, which can have clinical implications when investigating biomarkers in imatinib-treated versus non-treated GIST patients. Our results show, for the first time, that GISTs contain a secretome signature. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics.

Novel functions and targets of miR-944 in human cervical cancer cells.

Altered expression of specific microRNAs (miRNAs) has been observed in human cervical cancer. However, the biological functions of many of these miRNAs are yet to be discovered. We previously showed that miR-944 is significantly more abundant in cervical cancer tissues than their normal counterparts. In this study, we investigated the functions and targets of miR-944 in human cervical cancer cells. MiR-944 is located in the intron of the tumor protein p63 (TP63) gene, which is frequently overexpressed in cervical carcinomas. Using gain- and loss-of-function experiments in vitro, we demonstrate that miR-944 promotes cell proliferation, migration and invasion, but has no effect on apoptosis, in human cervical cancer cells. To identify the targets of miR-944, we performed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation followed by deep sequencing. Among the candidate targets, we validated HECW2 (HECT domain ligase W2) and S100PBP (S100P binding protein) as direct targets of miR-944 using luciferase reporter assays and western blot analysis. Our findings reveal novel functions and targets of miR-944 in human cervical cancer cells, which may provide new insights of its role in cervical carcinogenesis.

Functional role of the Ca²âº-activated Cl⁻ channel DOG1/TMEM16A in gastrointestinal stromal tumor cells.

DOG1, a Ca(2+)-activated Cl(-) channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl(-) currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

MicroRNAs and Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is the most commonly diagnosed mesenchymal tumor in the gastrointestinal tract. This tumor type is driven by gain-of-function mutations in receptor tyrosine kinases (such as KIT, PDGFRA, and BRAF) or loss-of-function mutations in succinate dehydrogenase complex subunit genes (SDHx). Molecular studies on GIST have improved our understanding of the biology of the disease and have led to the use of targeted therapy approach, such as imatinib for KIT/PDGFRA-mutated GIST. Recently, microRNAs have emerged as important regulators of KIT expression, cancer cell behavior, and imatinib response in GIST. This chapter aims to provide an overview on current understanding of the biological roles of microRNAs in GIST and possible implications in prognosis and therapeutic response.

miR-128 regulates non-myocyte hyperplasia, deposition of extracellular matrix and Islet1 expression during newt cardiac regeneration.

Cardiovascular disease is a global scourge to society, with novel therapeutic approaches required in order to alleviate the suffering caused by sustained cardiac damage. MicroRNAs (miRNAs) are being touted as one such approach in the fight against heart disease, acting as possible post-transcriptional molecular triggers responsible for invoking cardiac regeneration. To further ones understanding of miRNAs and cardiac regeneration, it is prudent to learn from organisms that can intrinsically regenerate their hearts following injury. Using the red-spotted newt, an adult chordate capable of cardiac regeneration, we decided to delve deeper into the role miRNAs play during this process. RNA isolated from regenerating newt heart samples, was used in a microarray screen, to identify significantly expressed candidate miRNAs during newt cardiac regeneration. We performed quantitative qPCR analysis on several conserved miRNAs and found one in particular, miR-128, to be significantly elevated when cardiac hyperplasia is at its peak following injury. In-situ hybridisation techniques revealed a localised expression pattern for miR-128 in the cardiomyocytes and non-cardiomyocytes in close proximity to the regeneration zone and in vivo knockdown studies revealed a regulatory role for miR-128 in proliferating non-cardiomyocyte populations and extracellular matrix deposition. Finally, 3'UTR reporter assays revealed Islet1 as a biological target for miR-128, which was confirmed further through in vivo Islet1 transcriptional and translational expression analysis in regenerating newt hearts. From these studies we conclude that miR-128 regulates both cardiac hyperplasia and Islet1 expression during newt heart regeneration and that this information could be translated into future mammalian cardiac studies.

Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumor cells.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-of-function mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib levels in GIST patients.

Evidence for Ca(2+)-regulated ATP release in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca(2+)-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca(2+) influx since exclusion of extracellular Ca(2+) diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

miRNA-214 is related to invasiveness of human non-small cell lung cancer and directly regulates alpha protein kinase 2 expression.

The prognosis of non-small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA-214 is linked to a radioresistant phenotype of NSCLC. miRNA-214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA-214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA-214 increased invasive potential, and conversely, overexpression of miRNA-214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA-214, identified a number of metastasis-related target genes, including pregnancy-associated plasma protein A (PAPP-A), alpha protein kinase 2 (ALPK2), cyclin-dependent kinase 6 (CDK6) and tumor necrosis-factor alpha-induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA-214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA-214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate-high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane-localized PAPP-A had a higher expression level compared to the cytoplasm-localized. In conclusion, we show that low miRNA-214 expression is linked to a higher invasive potential of NSCLC cells.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

Modulation of E-Cadherin Function through the AmotL2 Isoforms Promotes Ameboid Cell Invasion.

The spread of tumor cells and the formation of distant metastasis remain the main causes of mortality in cancer patients. However, the mechanisms governing the release of cells from micro-environmental constraints remain unclear. E-cadherin negatively controls the invasion of epithelial cells by maintaining cell-cell contacts. Furthermore, the inactivation of E-cadherin triggers invasion in vitro. However, the role of E-cadherin is complex, as metastasizing cells maintain E-cadherin expression, which appears to have a positive role in the survival of tumor cells. In this report, we present a novel mechanism delineating how E-cadherin function is modulated to promote invasion. We have previously shown that E-cadherin is associated with p100AmotL2, which is required for radial actin formation and the transmission of mechanical force. Here, we present evidence that p60AmotL2, which is expressed in invading tumor cells, binds to the p100AmotL2 isoform and uncouples the mechanical constraint of radial actin filaments. We show for the first time that the coupling of E-cadherin to the actin cytoskeleton via p100AmotL2 is directly connected to the nuclear membrane. The expression of p60AmotL2 inactivates this connection and alters the properties of the nuclear lamina, potentiating the invasion of cells into micropores of the extracellular matrix. In summary, we propose that the balance of the two AmotL2 isoforms is important in the modulation of E-cadherin function and that an imbalance of this axis promotes ameboid cell invasion.

The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas.

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

Altered expression of the IGF2­H19 locus and mitochondrial respiratory complexes in adrenocortical carcinoma.

Abnormalities of the insulin­like growth factor 2 (IGF2)­H19 locus with the overexpression of IGF2 are frequent findings in adrenocortical carcinoma (ACC). The present study assessed the expression of RNAs and microRNAs (miRNAs/miRs) from the IGF2­H19 locus using PCR­based methods in ACC and adrenocortical adenoma (ACA). The results were associated with proteomics data. IGF2 was overexpressed in ACC, and its expression correlated with that of miR­483­3p and miR­483­5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR­675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR­483­3p, miR­483­5p or miR­675. Subsets of these proteins were differentially expressed between ACC and ACA. These included several proteins involved in mitochondrial metabolism. Among the mitochondrial respiratory complexes, complex I and IV were significantly decreased in ACC compared to ACA. The protein expression of NADH:ubiquinone oxidoreductase subunit C1 (NDUFC1), a subunit of mitochondrial respiratory complex I, was further validated as being lower in ACC compared to ACA and normal adrenals. The silencing of miR­483­5p increased NDUFC1 protein expression and reduced both oxygen consumption and glycolysis rates. On the whole, the findings of the present study reveal the dysregulation of the IGF2­H19 locus and mitochondrial respiration in ACC. These findings may provide a basis for the further understanding of the pathogenesis of ACC and may have potential values for diagnostics and treatment.

Synergistic effects of telomerase reverse transcriptase and regulator of telomere elongation helicase 1 on aggressiveness and outcomes in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is one of the deadliest endocrine malignancies and telomere maintenance by activated telomerase is critically required for ACC development and progression. Because telomerase reverse transcriptase (TERT) and regulator of telomere elongation helicase 1 (RTEL1) play key roles in telomere homeostasis, we determined their effect on ACC pathogenesis and outcomes. Analyses of TCGA and GEO datasets showed significantly higher expression of RTEL1 but not TERT in ACC tumors, compared to their benign or normal counterparts. Furthermore, gains/amplifications of both TERT and RTEL1 genes were widespread in ACC tumors and their expression correlated with their gene copy numbers. Higher expression of either TERT or RTEL1 was associated with shorter overall and progression-free survival (OS and PFS) in the TCGA ACC patient cohort, and higher levels of both TERT and RTEL1 mRNA predicted the shortest patient OS and PFS. However, multivariate analyses showed that only RTEL1 independently predicted patient OS and PFS. Gene set enrichment analysis further showed enrichments of wnt/ß-catenin, MYC, glycolysis, MTOR, and DNA repair signaling pathways in ACC tumors expressing high TERT and RTEL1 mRNA levels. Taken together, TERT and RTEL1 promote ACC aggressiveness synergistically and may serve as prognostic factors and therapeutic targets for ACC.

Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls.

BACKGROUND: Ultra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run. RESULTS: We prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001. CONCLUSIONS: This approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets.