The Prostate Cancer Androgen Receptor Cistrome in African American Men Associates with Upregulation of Lipid Metabolism and Immune Response.

African-American (AA) men are more likely to be diagnosed with and die from prostate cancer than European American (EA) men. Despite the central role of the androgen receptor (AR) transcription factor in prostate cancer, little is known about the contribution of epigenetics to observed racial disparities. We performed AR chromatin immunoprecipitation sequencing on primary prostate tumors from AA and EA men, finding that sites with greater AR binding intensity in AA relative to EA prostate cancer are enriched for lipid metabolism and immune response genes. Integration with transcriptomic and metabolomic data demonstrated coinciding upregulation of lipid metabolism gene expression and increased lipid levels in AA prostate cancer. In a metastatic prostate cancer cohort, upregulated lipid metabolism associated with poor prognosis. These findings offer the first insights into ancestry-specific differences in the prostate cancer AR cistrome. The data suggest a model whereby increased androgen signaling may contribute to higher levels of lipid metabolism, immune response, and cytokine signaling in AA prostate tumors. Given the association of upregulated lipogenesis with prostate cancer progression, our study provides a plausible biological explanation for the higher incidence and aggressiveness of prostate cancer observed in AA men. SIGNIFICANCE: With immunotherapies and inhibitors of metabolic enzymes in clinical development, the altered lipid metabolism and immune response in African-American men provides potential therapeutic opportunities to attenuate racial disparities in prostate cancer.

Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation.

Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson's trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.

Lack of UCP3 does not affect skeletal muscle mitochondrial function under lipid-challenged conditions, but leads to sudden cardiac death.

UCP3's exact physiological function in lipid handling in skeletal and cardiac muscle remains unknown. Interestingly, etomoxir, a fat oxidation inhibitor and strong inducer of UCP3, is proposed for treating both diabetes and heart failure. We hypothesize that the upregulation of UCP3 upon etomoxir serves to protect mitochondria against lipotoxicity. To evaluate UCP3's role in skeletal muscle (skm) and heart under lipid-challenged conditions, the effect of UCP3 ablation was examined in a state of dysbalance between fat availability and oxidative capacity. Wild type (WT) and UCP3(-/-) mice were subjected to high-fat feeding for 14 days. From day 6 onwards, they were given either saline or etomoxir. Etomoxir treatment induced an increase in markers of lipotoxicity in skm compared to saline. This increase upon etomoxir was similar for both, WT and UCP3(-/-) mice, suggesting that UCP3 does not play a role in protection against lipotoxicity. Interestingly, we observed 25 % mortality in UCP3(-/-)s upon etomoxir administration vs. 11 % in WTs. This increased mortality in UCP3(-/-) compared to WT mice could not be explained by differences in cardiac lipotoxicity, apoptosis, fibrosis (histology, immunohistochemistry), oxidative capacity (respirometry) or function (echocardiography). Electrophysiology demonstrated, however, prolonged QRS and QTc intervals and greater susceptibility to ventricular tachycardia upon programmed electrical stimulation in etomoxir-treated UCP3(-/-)s versus WTs. Isoproterenol administration after pacing resulted in 75 % mortality in UCP3(-/-)s vs. 14 % in WTs. Our results argue against a protective role for UCP3 on skm metabolism under lipid overload, but suggest UCP3 to be crucial in prevention of arrhythmias upon lipid-challenged conditions.

Long-term simulated microgravity causes cardiac RyR2 phosphorylation and arrhythmias in mice.

BACKGROUND: Long-term exposure to microgravity during space flight may lead to cardiac remodeling and rhythm disturbances. In mice, hindlimb unloading (HU) mimics the effects of microgravity and stimulates physiological adaptations, including cardiovascular deconditioning. Recent studies have demonstrated an important role played by changes in intracellular Ca handling in the pathogenesis of heart failure and arrhythmia. In this study, we tested the hypothesis that cardiac remodeling following HU in mice involves abnormal intracellular Ca regulation through the cardiac ryanodine receptor (RyR2). METHODS AND RESULTS: Mice were subjected to HU by tail suspension for 28 to 56 days in order to induce cardiac remodeling (n=15). Control mice (n=19) were treated equally, with the exception of tail suspension. Echocardiography revealed cardiac enlargement and depressed contractility starting at 28 days post-HU versus control. Moreover, mice were more susceptible to pacing-induced ventricular arrhythmias after HU. Ventricular myocytes isolated from HU mice exhibited an increased frequency of spontaneous sarcoplasmic reticulum (SR) Ca release events and enhanced SR Ca leak via RyR2. Western blotting revealed increased RyR2 phosphorylation at S2814, and increased CaMKII auto-phosphorylation at T287, suggesting that CaMKII activation of RyR2 might underlie enhanced SR Ca release in HU mice. CONCLUSION: These data suggest that abnormal intracellular Ca handling, likely due to increased CaMKII phosphorylation of RyR2, plays a role in cardiac remodeling following simulated microgravity in mice.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

Ryanodine receptor-mediated calcium leak drives progressive development of an atrial fibrillation substrate in a transgenic mouse model.

BACKGROUND: The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca(2+) leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca(2+) release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. METHODS AND RESULTS: CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca(2+) release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca(2+)/calmodulin-dependent protein kinase II-mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca(2+) release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca(2+)-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. CONCLUSIONS: RyR2-mediated SR Ca(2+) leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca(2+) release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

Junctophilin-2 is necessary for T-tubule maturation during mouse heart development.

AIMS: Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation-contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes. METHODS AND RESULTS: Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca(2+) handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8. CONCLUSION: Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

Role of RyR2 phosphorylation at S2814 during heart failure progression.

RATIONALE: Increased activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca(2+) leak is unclear. OBJECTIVE: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. METHODS AND RESULTS: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca(2+) leak and improved sarcoplasmic reticulum Ca(2+) loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. CONCLUSIONS: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca(2+) leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mice.

Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

BACKGROUND: Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. METHODS AND RESULTS: To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance. CONCLUSIONS: Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart.

Ryanodine receptor phosphorylation by calcium/calmodulin-dependent protein kinase II promotes life-threatening ventricular arrhythmias in mice with heart failure.

BACKGROUND: approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. METHODS AND RESULTS: mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. CONCLUSIONS: our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy.

Aberrant intracellular Ca(2+) regulation is believed to contribute to the development of cardiomyopathy in Duchenne muscular dystrophy. Here, we tested whether inhibition of protein kinase A (PKA) phosphorylation of ryanodine receptor type 2 (RyR2) prevents dystrophic cardiomyopathy by reducing SR Ca(2+) leak in the mdx mouse model of Duchenne muscular dystrophy. mdx mice were crossed with RyR2-S2808A mice, in which PKA phosphorylation site S2808 on RyR2 is inactivated by alanine substitution. Compared with mdx mice that developed age-dependent heart failure, mdx-S2808A mice exhibited improved fractional shortening and reduced cardiac dilation. Whereas application of isoproterenol severely depressed cardiac contractility and caused 95% mortality in mdx mice, contractility was preserved with only 19% mortality in mdx-S2808A mice. SR Ca(2+) leak was greater in ventricular myocytes from mdx than mdx-S2808A mice. Myocytes from mdx mice had a higher incidence of isoproterenol-induced diastolic Ca(2+) release events than myocytes from mdx-S2808A mice. Thus, inhibition of PKA phosphorylation of RyR2 reduced SR Ca(2+) leak and attenuated cardiomyopathy in mdx mice, suggesting that enhanced PKA phosphorylation of RyR2 at S2808 contributes to abnormal Ca(2+) homeostasis associated with dystrophic cardiomyopathy.

Accelerated development of pressure overload-induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.

In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although intracellular calcium (Ca(2+)) has a central role in hypertrophic signaling pathways, the Ca(2+) source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca(2+) leak through defective cardiac intracellular Ca(2+) release channels/ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca(2+)-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wild-type mice were subjected to transverse aortic constriction. Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after transverse aortic constriction in R176Q/+ compared with wild-type mice. R176Q/+ mice displayed an enhanced hypertrophic response compared with wild-type mice as assessed by heart weight:body weight ratios and cardiomyocyte cross-sectional areas after transverse aortic constriction. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after transverse aortic constriction. Moreover, pressure overload resulted in an increased sarcoplasmic reticulum Ca(2+) leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin 1, and a decrease in nuclear factor of activated T-cells phosphorylation in R176Q/+ mice compared with wild-type mice. Taken together, our results suggest that RyR2-dependent sarcoplasmic reticulum Ca(2+) leak activates the prohypertrophic calcineurin/nuclear factor of activated T-cells pathway under conditions of pressure overload.

Sudden infant death syndrome in mice with an inherited mutation in RyR2.

BACKGROUND: Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice. METHODS AND RESULTS: Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice. CONCLUSIONS: Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome.

Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Overexpressed TP73 induces apoptosis in medulloblastoma.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated DeltaNp73 to assess their effects on growth. METHODS: We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the DeltaNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and DeltaNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and DeltaNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. RESULTS: Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated DeltaNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or DeltaNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to chemotherapeutic agents. CONCLUSION: These results indicate that primary medulloblastomas express significant levels of TP73 isoforms, and suggest that they can modulate the survival and genotoxic responsiveness of medulloblastomas cells.

Patched haploinsufficient mouse rhabdomyosarcoma overexpress secreted phosphoprotein 1 and matrix metalloproteinases.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Improving the management of rhabdomyosarcoma requires a better understanding of growth regulation. Patched haploinsufficient (Ptch+/-) mice spontaneously develop soft tissue sarcomas that resemble human rhabdomyosarcomas. Using microarray profiling and quantitative real-time reverse transcriptase polymerase chain reaction, we identified candidate genes differentially expressed in Ptch+/- mouse rhabdomyosarcoma relative to mature muscle. Overexpressed genes include Secreted Phosphoprotein 1 (Spp1, Osteopontin), and Matrix Metalloproteinases-2 and -14 (Mmp2 and Mmp14). Spp1 is an integrin-binding phosphoglycoprotein upregulated in carcinomas, and Mmps regulate tumour invasion. Immunochemical analyses of murine and human rhabdomyosarcoma specimens confirmed increased expression of Spp1, Mmp2, Mmp14, nuclear factor-kappa B (NF-kappaB) p65 and its phosphorylated active isoform. Neutralising Spp1 antibody decreased Mmp14 RNA in murine rhabdomyosarcoma cultures, indicating a positive regulatory role for extracellular Spp1. Plasma from rhabdomyosarcoma patients display elevated levels of SPP1. These results implicate Spp1, NF-kappaB, and Mmp activation as a putative signalling pathway involved in rhabdomyosarcoma growth.