Helen C. Owen

Distinct MicroRNA Alterations Characterize High- and Low-Grade Bladder Cancer

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy.

Hypermethylation of CpG Islands and Shores around Specific MicroRNAs and Mirtrons Is Associated with the Phenotype and Presence of Bladder Cancer

Purpose: To analyze the role and translational potential for hypermethylation of CpG islands and shores in the regulation of small RNAs within urothelial cell carcinoma (UCC). To examine microRNAs (miR) and mirtrons, a new class of RNA located within gene introns and processed in a Drosha-independent manner. Experimental design: The methylation status of 865 small RNAs was evaluated in normal and malignant cell lines by using 5-azacytidine and microarrays. Bisulfite sequencing was used for CpG regions around selected RNAs. Prognostic and diagnostic associations for epigenetically regulated RNAs were examined by using material from 359 patients, including 216 tumors and 121 urinary samples (68 cases and 53 controls). Functional analyses examined the effect of silencing susceptible RNAs in normal urothelial cells. Results: Exonic/UTR-located miRs and mirtons are most susceptible to epigenetic regulation. We identified 4 mirtrons and 16 miRs with CpG hypermethylation across 35 regions in normal and malignant urothelium. For several miRs, hypermethylation was more frequent and dense in CpG shores than islands (e.g., miRs-9/149/210/212/328/503/1224/1227/1229), and was associated with tumor grade, stage, and prognosis (e.g., miR-1224 multivariate analysis OR = 2.5; 95% CI, 1.3–5.0; P = 0.006). The urinary expression of epigenetically silenced RNAs (miRs-152/328/1224) was associated with the presence of UCC (concordance index, 0.86; 95% CI, 0.80–0.93; ANOVA P < 0.016). Conclusions: Hypermethylation of mirtrons and miRs is common in UCC. Mirtrons appear particularly susceptible to epigenetic regulation. Aberrant hypermethylation of small RNAs is associated with the presence and behavior of UCC, suggesting potential roles as diagnostic and prognostic biomarkers. Clin Cancer Res; 17(6); 1287–96. ©2010 AACR.

The growth plate sparing effects of the selective glucocorticoid receptor modulator, AL-438

Long-term use of glucocorticoids (GC) can cause growth retardation in children due to their actions on growth plate chondrocytes. AL-438, a non-steroidal anti-inflammatory agent that acts through the glucocorticoid receptor (GR) retains full anti-inflammatory efficacy but has reduced negative effects on osteoblasts compared to those elicited by prednisolone (Pred) or dexamethasone (Dex). We have used the murine chondrogenic ATDC5 cell line to compare the effects of AL-438 with those of Dex and Pred on chondrocyte dynamics. Dex and Pred caused a reduction in cell proliferation and proteoglycan synthesis, whereas exposure to AL-438 had no effect. LPS-induced IL-6 production in ATDC5 cells was reduced by Dex or AL-438, showing that AL-438 has similar anti-inflammatory efficacy to Dex in these cells. Fetal mouse metatarsals grown in the presence of Dex were shorter than control bones whereas AL-438 treated metatarsals paralleled control bone growth. These results indicate that the adverse effects Dex or Pred have on chondrocyte proliferation and bone growth were attenuated following AL-438 exposure, suggesting that AL-438 has a reduced side effect profile on chondrocytes compared to other GCs. This could prove important in the search for new anti-inflammatory treatments for children.

Low Frequency of Epigenetic Events in Urothelial Tumors in Young Patients

PURPOSE Bladder urothelial cell carcinoma is uncommon in young patients. We recently reported a series of tumors in patients younger than 20 years at diagnosis and performed exhaustive genetic screening for molecular alterations. Few events typical of bladder urothelial cell carcinoma were detected. Since many carcinogenic events occur at the epigenetic rather than the genetic level, we analyzed the same tumors for alterations in DNA hypermethylation. We compared our findings with those in tumors in older patients with similar pathological profiles. MATERIALS AND METHODS We analyzed 76 bladder urothelial cell carcinomas from 3 groups stratified by age at diagnosis, including less than 19, 20 to 45 and greater than 46 years (median 78), and matched for low grade and nonmuscle invasive stage. We used quantified methyl specific polymerase chain reaction to investigate promoter methylation for 8 tumor suppressor genes implicated in urothelial carcinogenesis. RESULTS Tumors in the youngest age group had the lowest incidence of global hypermethylation compared to the other tumors with a methyl index of 37.5% vs 62.5% and 50%, respectively (ANOVA p = 0.009). When individual loci were analyzed, younger patients had a significantly lower rate and concentration of methylation at APC, Bcl2, MGMT and E-cadherin promoters than in the older groups (p <0.05). Few differences were present between the 2 older cohorts but the APC and MGMT methylation concentration increased with age. CONCLUSIONS Urothelial tumors in patients younger than 19 years have a low rate of epigenetic alteration. Tumors in patients older than 20 years have epigenetic profiles similar to those of tumors in patients within the typical bladder urothelial cell carcinoma age range.

Perioperative blood transfusion is associated with a gene transcription profile characteristic of immunosuppression: a prospective cohort study

IntroductionBlood transfusion in the perioperative period has frequently been associated with an excess of nosocomial infections. Whilst transfused whole blood induces specific host immune alteration that may predispose to nosocomial infections, the immunomodulating properties associated with leukodepleted blood remain incompletely understood. In this study, we explore the hypothesis that the transfusion of leukodepleted allogeneic blood during or following major gastrointestinal surgery is associated with an immunosuppressed phenotype, which may in turn predispose to postoperative infectious complications.MethodsPatients aged over 45 years undergoing scheduled inpatient major gastrointestinal surgery were recruited. Gene expression profiles of specific inflammatory genes were assayed from blood collected preoperatively, at 24 and at 48 hours after surgery. Genes were selected based on their ability to represent specific immune pathways. Gene expression was quantified using quantitative real-time polymerase chain reaction (qRT-PCR) to measure messenger RNA (mRNA) levels. Postoperative infections were documented using predefined criteria.ResultsOne hundred and nineteen patients were recruited. Fifteen (13%) patients required blood transfusion within 24 hours of surgery, 44 (37%) patients developed infections and 3 (2%) patients died prior to discharge. Patients receiving a blood transfusion were more likely to develop postoperative infections (P =0.02) and to have lower tumour necrosis factor alpha (TNFα), interleukin (IL)-12, IL-23 and RAR-related orphan receptor gamma T (RORγt) gene expression in the postoperative period (P <0.05). The TNFα/IL-10 mRNA ratio at 24 hours (P =0.0006) and at 48 hours (P =0.01) was lower in patients receiving a blood transfusion over this period. Multivariable analysis confirmed that these observations were independent of the severity of the surgical insult.ConclusionsAn association between an immunosuppressive pattern of gene expression and blood transfusion following major elective gastrointestinal surgery is described. This gene expression profile includes a reduction in the activity of innate immunity and T helper cell type 1 (Th1) and T helper cell type 17 (Th17) pathways in those patients receiving a blood transfusion. Blood transfusion was also associated with an excess of infectious complications in this cohort. A mechanistic link is suggested but not proven.

The perioperative immune response.

Purpose of reviewA host of immune modulators are now available in clinical practice. The perioperative period is characterized by profound alterations in host immunity, which can result in poor outcomes, which include infection, cancer recurrence and organ failure. Manipulation of the perioperative immune response has the potential to improve outcomes. A complete understanding of the mechanisms and clinical consequences of altered immune function in this setting is therefore imperative. Recent findingsRecent in-vivo data have emerged which further our understanding of the interaction between tissue damage, immune modulation and clinical outcomes by utilizing novel laboratory techniques capable of monitoring single-cell immune signatures. Traditional gene expression assays have continued to demonstrate their utility and have been instrumental in defining the host response to perioperative allogeneic blood transfusion. These mechanistic studies are complemented by large clinical studies describing associations between anaesthetic modalities and immune-related outcomes. SummaryLaboratory techniques are now available that can monitor the perioperative immune response and could be further developed to introduce personalized care pathways. Consideration must also be given to anaesthesia techniques and perioperative treatments that, although not immediately harmful, may be associated with poor outcomes temporally distant from the treatment, secondary to induced immunosuppression.

Systemic inflammatory response syndrome after major abdominal surgery predicted by early upregulation of TLR4 and TLR5

Objectives:To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. Background:Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. Methods:Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. Results:Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-&kgr;B/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon &agr;-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14++CD16+ monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). Conclusions:These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Changes in gene expression following trauma are related to the age of transfused packed red blood cells.

BACKGROUND Transfusion of packed red blood cells (PRBCs) is associated with an increased incidence of nosocomial infections and an increased risk of death. The duration of storage before transfusion may influence these outcomes. Here, we explore the association between the age of transfused PRBCs and specific patterns of inflammatory gene expression in severely injured trauma patients. METHODS Severely injured trauma patients requiring intensive care unit treatment and receiving transfusion of PRBCs within 24 hours of the injury were recruited. Blood samples were obtained within 2 hours of the trauma, at 24 hours, and at 72 hours. Messenger RNA was extracted from whole blood, and gene expression was quantified using quantitative polymerase chain reaction. The median age of the units of PRBCs transfused to each patient was recorded. The primary outcome measure was the change in candidate gene expression over the initial 72 hours. RESULTS Sixty-four patients were studied. Fifty-three patients (83%) were male, and the median age was 40.5 years (interquartile range [IQR], 31–59). Median Injury Severity Score (ISS) was 31.5 (IQR, 23–43), and 55 patients (86%) experienced a blunt injury. Forty-one patients (64%) developed a nosocomial infection, and 15 patients (23%) died before hospital discharge. Each patient received a median of 5 U of PRBCs (IQR, 4–9.8 U) during the first 24 hours of hospital admission. The median age of the units of PRBCs transfused in each patient was 20 days (IQR, 17–22 days). Older blood was associated with greater decreases in interleukin 12 (IL-12), IL-23, and ROR&ggr;t (all p’s < 0.05) gene expression over the initial 24 hours, greater decreases in IL-12 gene expression over 72 hours, and a rise in transforming growth factor &bgr; gene expression over the first 72 hours. A multivariate analysis confirmed the independence of these associations. CONCLUSION Increasing the duration of storage of PRBCs before transfusion is associated with a pattern of gene expression consistent with more severe immunosuppression. LEVEL OF EVIDENCE Epidemiologic study, level III.

Humanized Culture of Periosteal Progenitors in Allogeneic Serum Enhances Osteogenic Differentiation and In Vivo Bone Formation

The translation of stem cell‐based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno‐free culturing practices. In an attempt to apply this approach to a bone‐forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon‐mediated signaling was downregulated, whereas JAK‐STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS‐cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS‐cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo‐formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue‐formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.

Critical illness-induced bone loss is related to deficient autophagy and histone hypomethylation

BackgroundSurvivors of critical illness are at increased risk of fractures. This may be due to increased osteoclast formation during critical illness, leading to trabecular bone loss. Such bone loss has also been observed in Paget’s disease, and has been related to deficient autophagy. Deficient autophagy has also been documented in vital organs and skeletal muscle of critically ill patients. The objective of this study was to investigate whether deficient autophagy can be linked to critical illness-induced bone loss.MethodsOsteoclasts grown in vitro and their precursor cells isolated from peripheral blood of critically ill patients and from matched healthy volunteers were analysed for the expression of autophagy genes (SQSTM1, Atg3 and Atg7), and proteins (p62, Atg–5, and microtubule-associated protein light chain 3–II (LC3–II)) and for autophagy and epigenetic signalling factors via PCR arrays and were treated with the autophagy inducer rapamycin. The effect of rapamycin was also investigated at the tissue level in an in vivo rabbit model of critical illness.ResultsMany more osteoclasts formed in vitro from the blood precursor cells isolated from critically ill patients, which accumulated p62, and displayed reduced expression of Atg5, Atg7, and LC3–II compared to healthy controls, suggesting deficient autophagy, whilst addition of rapamycin reduced osteoclast formation. PCR arrays revealed a down-regulation of histone methyltransferases coupled with an up-regulation of negative regulators of autophagy. Critically ill rabbits displayed a reduction in trabecular and cortical bone, which was rescued with rapamycin.ConclusionsDeficient autophagy in osteoclasts and their blood precursor cells at least partially explained aberrant osteoclast formation during critical illness and was linked to global histone hypomethylation. Treatment with the autophagy activator Rapamycin reduced patient osteoclast formation in vitro and reduced the amount of bone loss in critically ill rabbits in vivo. These findings may help to develop novel therapeutic targets to prevent critical illness-induced bone loss.