Elucidating the role of microRNA-497 and WEE1 in neuroblastoma disease pathogenesis.

Neuroblastoma is a pediatric cancer that arises from precursor cells of the sympathetic nervous system and is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the 5 year overall survival rate remains poor in high-risk disease (20-30%). MiRNA profiling has emerged as a valuable tool in the diagnostic and prognostic evaluation of many cancers including neuroblastoma. Previously our laboratory identified two miRNA expression profiles; the first a 15 miRNA expression signature associated with survival and the second a miRNA expression profile associated with MYCN amplification. Based on this work, a number of individual miRNAs were chosen for further investigation. An evaluation of 4 selected miRNAs (miR-572, miR-30a*, miR-30e* and miR-497) revealed each was individually significantly associated with survival in neuroblastoma. The subsequent initial functional characterisation of these miRNAs revealed that only miR-497 significantly impacted cell proliferation and apoptosis levels in the MYCN amplified cell lines examined. MiR-497 had previously been identified as a member of the 15 miRNA expression profile associated with survival and in the present study it is demonstrated that miR-497 is individually significantly associated with both event free and overall survival in neuroblastoma. Low miR-497 expression was also significantly associated with MYCN amplification and the over-expression of miR- 497 resulted in the inhibition of cell proliferation and caspase mediated apoptosis in MYCN amplified high-risk neuroblastoma cells. A novel target for miR-497, WEE1, was also discovered in neuroblastoma and it was determined that miR-497 exerts a tumour suppressive function through the efficient down-regulation of WEE1 protein. WEE1 is a tyrosine kinase regulator of the cell cycle that functions by negatively regulating CDC2 mediated apoptosis. The over-expression of WEE1 has been demonstrated in a variety of cancer types and has been associated with a poor clinical prognosis in these cancers. This study demonstrated that high WEE1 expression was associated with poor clinical outcome in neuroblastoma and that high WEE1 expression was significantly associated with MYCN amplification. Treatment of high-risk neuroblastoma cells with MK-1775, a small molecule inhibitor of WEE1 currently undergoing clinical trials, resulted in the same phenotypic effect of increased apoptosis. A comparative study between the downstream effects of MK-1775 treatment and miR-497 over-expression revealed that both methods of WEE1 inhibition resulted in decreased CDC2 phosphorylation, increased cleaved PARP and increased levels of gamma-H2AX, demonstrating the similarities between with small molecule inhibitor and the natural antisense inhibitor of WEE1. Additionally this study identified a second novel target for miR-497 in neuroblastoma, CHK1. The over-expression of both WEE1 and CHK1 has been previously demonstrated in neuroblastoma. CHK1 functions as a key cell cycle checkpoint regulator by positively modulating WEE1 activity and inhibiting CDC25a. CDC25a functions to dephosphorylate and subsequently reactivate CDC2, therefore promoting apoptosis induction. This study revealed that high expression of CHK1 is significantly associated with both poor event free and overall survival in neuroblastoma and that CHK1 expression is significantly associated with MYCN amplification. The identification of both WEE1 and CHK1 as novel targets of miR-497 in neuroblastoma has further emphasised the tumour suppressive role of miR-497 and highlights the therapeutic potential for miR-497 mediated therapy in high-risk neuroblastoma

Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer.

INTRODUCTION: HOX genes play vital roles in growth and development, however, atypical redeployment of these genes is often associated with steroidal adaptability in endocrine cancers. We previously identified HOXC11 to be an indicator of poor response to hormonal therapy in breast cancer. In this study we aimed to elucidate genes regulated by HOXC11 in the endocrine resistant setting. METHODS: RNA-sequencing paired with transcription factor motif-mapping was utilised to identify putative HOXC11 target genes in endocrine resistant breast cancer. Validation and functional evaluation of the target gene, prosaposin (PSAP), was performed in a panel of endocrine sensitive and resistant breast cancer cell lines. The clinical significance of this finding was explored in clinical cohorts at both mRNA and protein level. RESULTS: PSAP was shown to be regulated by HOXC11 in both tamoxifen and aromatase inhibitor (AI) resistant cell lines. Transcript levels of HOXC11 and PSAP correlated strongly in samples of primary breast tumours (r = 0.7692, n = 51). PSAP has previously been reported to activate androgen receptor (AR) in prostate cancer cells. In a panel of breast cancer cell lines it was shown that endocrine resistant cells exhibit innately elevated levels of AR compared to their endocrine sensitive counterparts. Here, we demonstrate that stimulation with PSAP can drive AR recruitment to a hormone response element (HRE) in AI resistant breast cancer cells. Functionally, PSAP promotes cell migration and invasion only in AI resistant cells and not in their endocrine sensitive counterparts. In a cohort of breast cancer patients (n = 34), elevated serum levels of PSAP were found to associate significantly with poor response to endocrine treatment (p = 0.04). Meta-analysis of combined PSAP and AR mRNA are indicative of poor disease-free survival in endocrine treated breast cancer patients (hazard ratio (HR): 2.2, P = 0.0003, n = 661). CONCLUSION: The HOXC11 target gene, PSAP, is an AR activator which facilitates adaptation to a more invasive phenotype in vitro. These findings have particular relevance to the development of resistance to AI therapy which is an emerging clinical issue. PSAP is a secreted biomarker which has potential in identifying patients failing to exhibit sustained response to hormonal treatment

MicroRNA-497 increases apoptosis in MYCN amplified neuroblastoma cells by targeting the key cell cycle regulator WEE1.

BACKGROUND: Neuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted target of miR-497, has emerged as an oncogene in several cancer types and therefore represents an attractive potential target for novel therapy approaches in high-risk neuroblastoma. Our aim was to investigate the potential tumor suppressive role of miR-497 in high-risk neuroblastoma. METHODS: Expression levels of miR-497 and WEE1 in tissues and cells were determined using RT-PCR. The effect of miR-497 and siWEE1 on cell viability was evaluated using MTS assays, apoptosis levels were determined using FACS analysis of Annexin V/PI stained cells, and target protein expression was determined using western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean±S.E.M and differences were tested for significance using 2-tailed Students t-test. RESULTS: We determined that miR-497 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and that low miR-497 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-497 reduced cell viability and increased apoptosis in MNA cells. We identified WEE1 as a novel target for miR-497 in neuroblastoma. Furthermore, our analysis showed that high WEE1 levels are significantly associated with poor EFS and OS in neuroblastoma and that siRNA knockdown of WEE1 in MNA cell lines results in significant levels of apoptosis, supporting an oncogenic role of WEE1 in neuroblastoma. Cisplatin (CDDP) treatment of both miR-497 over-expressing cells and WEE1 inhibited cells, resulted in a significant increase in apoptosis in MNA cells, describing a synergistic effect and therefore a potential therapeutic for high-risk neuroblastoma. CONCLUSION: Our study's results are consistent with miR-497 being a candidate tumor suppressor in neuroblastoma, through the direct targeting of WEE1. These findings re-enforce the proposal of WEE1 as a therapeutic target in neuroblastoma.</p

Rabbinic Elements in the Verbal System of Maskilic Hebrew Fiction 1857-1881

a Hazard ratio (HR) curve was generated for prosaposin (PSAP) using the TCGA dataset. PSAP expressions between 25 and 75 % quantiles have a HR consistently >1. b Androgen receptor (AR) mRNA does not associate with poor disease-free survival (DFS) in endocrine-treated breast cancer. Kaplan-Meier survival curves were generated to assess the impact of high androgen receptor transcript levels on survival of endocrine-treated patients with breast cancer (n = 661). (PDF 204 kb

Fracture and dislocation classification compendium - 2007: Orthopaedic Trauma Association classification, database and outcomes committee.

The purpose of this new classification compendium is to republish the Orthopaedic Trauma Association\u27s (OTA) classification. The OTA classification was originally published in a compendium of the Journal of Orthopaedic Trauma in 1996. It adopted The Comprehensive Classification of the Long Bones developed by Müller and colleagues and classified the remaining bones. In this compendium, the introductory chapter reviews new scientific information about classifying fractures that has been published in the last 11 years. The classification is presented in a revised format that is easier to follow. The OTA and AO classification will now have a unified alpha-numeric code eliminating the differences that have existed between the 2 codes. The code was significantly revised for the clavicle and scapula, foot and hand, and patella. Dislocations have been expanded on an anatomic basis and for most joints will be coded separately. This publication should stimulate new developments and interest in a unified language to code and classify fractures. Further improvements in classification will result in better patient care and clinical research

Additional file 6: of Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer

Table: Estrogen receptor (ER) motif results ( p <0.001). A 400-bp-sized window surrounding starting sites of HOXC11 target genes was selected for ER motif searching. The searching process was performed using the FIMO program available in MEME-suite with the p value significant cutoff set at 0.001. (PDF 198 kb