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We used whole-organ mapping to study loco-geographic molecular changes in evolution of human bladder cancer from mucosal field effects. The integrative multi-platform analyses based on genome-wide RNA sequencing, methylation, copy number variations, and whole exome sequencing identified over 100 dysregulated canonical pathways involving immunity, tissue differentiation and transformation as initiators of bladder carcinogenesis. Widespread dysregulation of interleukin signaling was the dominant change signifying the important role of inflammation and immunity in the incipient phases of urothelial carcinogenesis. The analyses of mutational patterns identified three types of mutations based on their geographic distribution and variant allele frequencies. The most common were low frequency subclonal mutations restricted to individual mucosal samples which were the progeny of their respective uroprogenitor cells. The two additional types of mutations were associated with clonal expansion and involved large areas of bladder mucosa. The first group referred to as α mutations, showed a low mutational frequency across the mucosa. The second group referred to as β mutations increased in their frequencies with disease progression and a large proportion of them represented mutated transcriptional regulators controlling proliferation. Time modeling revealed that bladder carcinogenesis is spanning 10-15 years and can be divided into dormant and progressive phases. The progressive phase lasted 1-2 years and was primarily driven by β mutations with high proliferative advantage. This is the first detailed molecular characterization of mucosal field effects initiating bladder carcinogenesis on the whole-organ scale. It provides novel insights into incipient phases of bladder carcinogenesis and biomarkers for early detection of bladder cancer as well as targets for preventive therapies.

Summary: Sarcomatoid urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs show a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations. They are related to the basal molecular subtype of conventional UCs and could be divided into epithelial-basal and more clinically aggressive mesenchymal subsets on the basis of TP63 and its target gene expression levels. Other analyses reveal that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of the EMT network, and nearly half exhibit a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer. : Guo et al. report that sarcomatoid carcinoma of the bladder evolves by the progression of the basal subtype of conventional urothelial carcinoma with the enrichment of mutagenesis signature 1 and mutations of TP53, RB1, and PIK3CA. It is driven by the dysregulation of the EMT network and shows increased immune infiltrate with overexpression of PD-L1. Keywords: bladder cancer, sarcomatoid carcinoma, urothelial carcinoma, epithelial-mesenchymal transformation, genomic expression, chromatin remodeling, microRNA expression, molecular classification, basal subtype, immune phenotype

Background Fusion genes form as a result of abnormal chromosomal rearrangements linking previously separate genes into one transcript. The FGFR3-TACC3 fusion gene (F3-T3) has been shown to drive gliomagenesis in glioblastoma (GBM), a cancer that is notoriously resistant to therapy. However, successful targeting of F3-T3 via small molecular inhibitors has not revealed robust therapeutic responses, and specific targeting of F3-T3 has not been achieved heretofore. Here, we demonstrate that depleting F3-T3 using custom siRNA to the fusion breakpoint junction results in successful inhibition of F3-T3+ GBMs, and that exosomes can successfully deliver these siRNAs. Methods We engineered 10 unique siRNAs (iF3T3) that specifically spanned the most common F3-T3 breakpoint with varying degrees of overlap, and assayed depletion by qPCR and immunoblotting. Cell viability assays were performed. Mesenchymal stem cell–derived exosomes (UC-MSC) were electroporated with iF3T3, added to cells, and F3-T3 depletion measured by qPCR. Results We verified that depleting F3-T3 using shRNA to FGFR3 resulted in decreased cell viability and improved survival in glioma-bearing mice. We then demonstrated that 7/10 iF3T3 depleted F3-T3, and importantly, did not affect levels of wild-type (WT) FGFR3 or TACC3. iF3T3 decreased cell viability in both F3T3+ GBM and bladder cancer cell lines. UC-MSC exosomes successfully delivered iF3T3 in vitro, resulting in F3-T3 depletion. Conclusion Targeting F3-T3 using siRNAs specific to the fusion breakpoint is capable of eradicating F3T3+ cancers without toxicity related to inhibition of WT FGFR3 or TACC3, and UC-MSC exosomes may be a plausible vehicle to deliver iF3T3.

Genomic profiling studies have demonstrated that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to frontline chemotherapy. We analyzed the mRNA expressions of signature luminal and basal genes in bladder cancer tumor samples from publicly available and MD Anderson Cancer Center cohorts. We developed a quantitative classifier referred to as basal to luminal transition (BLT) score which identified the molecular subtypes of bladder cancer with 80–94% sensitivity and 83–93% specificity. In order to facilitate molecular subtyping of bladder cancer in primary care centers, we analyzed the protein expressions of signature luminal (GATA3) and basal (KRT5/6) markers by immunohistochemistry, which identified molecular subtypes in over 80% of the cases. In conclusion, we provide a tool for assessment of molecular subtypes of bladder cancer in routine clinical practice.

Summary We report a comprehensive molecular analysis of 34 cases of small cell carcinoma (SCC) and 84 cases of conventional urothelial carcinoma (UC), with The Cancer Genome Atlas cohort of 408 conventional UC bladder cancers used as the reference. SCCs showed mutational landscapes characterized by nearly uniform inactivation of TP53 and were dominated by Sanger mutation signature 3 associated with loss of BRCA1/2 function. SCCs were characterized by downregulation of luminal and basal markers and were referred to as double-negative. Transcriptome analyses indicated that SCCs displayed lineage plasticity driven by a urothelial-to-neural phenotypic switch with a dysregulated epithelial-to-mesenchymal transition network. SCCs were depleted of immune cells, and expressed high levels of the immune checkpoint receptor, adenosine receptor A2A (ADORA2A), which is a potent inhibitor of immune infiltration. Our observations have important implications for the prognostication and development of more effective therapies for this lethal bladder cancer variant., Graphical Abstract, Highlights • SCCs show TP53/RB1 loss with mutational signature of BRCA1/2 loss of function • SCCs are driven by neural phenotypic switch with dysregulated EMT network • SCCs show depleted immune phenotype with upregulation of ADORA2A, Biological Sciences; Genomics; Cancer Systems Biology; Cancer; Transcriptomics

Background Fusion genes form as a result of abnormal chromosomal rearrangements linking previously separate genes into one transcript. The FGFR3-TACC3 fusion gene (F3-T3) has been shown to drive gliomagenesis in glioblastoma (GBM), a cancer that is notoriously resistant to therapy. However, successful targeting of F3-T3 via small molecular inhibitors has not revealed robust therapeutic responses, and specific targeting of F3-T3 has not been achieved heretofore. Here, we demonstrate that depleting F3-T3 using custom siRNA to the fusion breakpoint junction results in successful inhibition of F3-T3+ GBMs, and that exosomes can successfully deliver these siRNAs. Methods We engineered 10 unique siRNAs (iF3T3) that specifically spanned the most common F3-T3 breakpoint with varying degrees of overlap, and assayed depletion by qPCR and immunoblotting. Cell viability assays were performed. Mesenchymal stem cell–derived exosomes (UC-MSC) were electroporated with iF3T3, added to cells, and F3-T3 depletion measured by qPCR. Results We verified that depleting F3-T3 using shRNA to FGFR3 resulted in decreased cell viability and improved survival in glioma-bearing mice. We then demonstrated that 7/10 iF3T3 depleted F3-T3, and importantly, did not affect levels of wild-type (WT) FGFR3 or TACC3. iF3T3 decreased cell viability in both F3T3+ GBM and bladder cancer cell lines. UC-MSC exosomes successfully delivered iF3T3 in vitro, resulting in F3-T3 depletion. Conclusion Targeting F3-T3 using siRNAs specific to the fusion breakpoint is capable of eradicating F3T3+ cancers without toxicity related to inhibition of WT FGFR3 or TACC3, and UC-MSC exosomes may be a plausible vehicle to deliver iF3T3.

Background It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers. Methods We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n = 132), Lund (n = 308), and The Cancer Genome Atlas (TCGA) (n = 408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n = 89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer. Findings Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Interpretation The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification. Funding U.S. National Cancer Institute and National Institute of Health., Highlights • Bladder cancer can be assigned to two intrinsic molecular subtypes referred to as luminal and basal. • Luminal tumors are characterized by the expression signature of intermediate/superficial layers of normal urothelium and they show up-regulation of PARγ target genes with enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. • Basal tumors show the expression signature similar to the basal layer of normal urothelium and they show the up-regulation of p63 target genes with the enrichment of TP53 and RB1 mutations. • Muscle invasive tumors of basal type are more aggressive when compared to luminal cancers. • The immunohistochemical expression of only two markers, luminal (GATA3) and basal (KRT5/6) is sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Our analyses of several large cohorts comprising of a total of 937 bladder cancer tumors samples with annotated clinical data showed that bladder cancers can be classified into molecular subtypes referred to as luminal and basal. Luminal tumors are similar in their gene expression pattern to more differentiated intermediate and superficial layers of normal urothelium. Basal cancers recapitulate the gene expression signature of the less differentiated basal layer of the urothelium. These molecular subtypes have distinct up-regulation of transcription factors that regulate the expression pattern of their target genes and are enriched for specific mutations. Basal tumors which deeply invade the bladder wall are more aggressive when compared to luminal types and they show distinct sensitivities to frontline chemotherapy. The classification of bladder cancer into its intrinsic molecular subtypes can be accomplished with two immunohistochemical markers such as GATA3 for luminal tumors and KRT5/6 for basal tumors which can be performed in primary care centers.

Ovarian cancer is one of the most lethal malignant tumors in women. The prognosis of ovarian cancer patients depends, in part, on their response to platinum-based chemotherapy. Our recent analysis of genomics and clinical data from the Cancer Genome Atlas demonstrated that somatic mutations of ADAMTS 1, 6, 8, 9, 15, 16, 18 and L1 genes were associated with higher sensitivity to platinum and longer progression-free survival, overall survival, and platinum-free survival duration in 512 patients with high-grade serous ovarian carcinoma. Among the ADAMTS mutations, ADAMTS16 is the most commonly affected gene in ovarian cancer. However, the functional role of these mutations in ovarian cancer cells is largely unknown. We performed in vitro studies to compare the functional effects of the six identified ADAMTS missense mutations on the platinum sensitivity of ovarian cancer cells. We also used a well-characterized in vivo mouse model to evaluate the response of ovarian cancer cells with ADAMTS16 mutations to platinum-based therapy. Our results showed that exogenously expressed ADAMTS16 missense mutations inhibited cell growth or sensitized tumor cells to cisplatin and inhibited tumor growth in vivo. Orthotopic xenograft experiments showed that mice injected with ovarian cancer cells that exogenously expressed ADAMTS16 mutations had a better response to cisplatin treatment. Thus, these functional studies provide evidence that mutations of ADAMTS16 actively contribute to therapeutic response in ovarian cancer.

Insulin-like growth factor binding protein 2 (IGFBP2) overexpression is common in high-grade glioma and is both a strong biomarker of aggressive behaviour and a well-documented prognostic factor. IGFBP2 is a member of the secreted IGFBP family that functions by interacting with circulating IGFs to modulate IGF-mediated signalling. This traditional view of IGFBP2 activities has been challenged by the recognition of the diverse functions and cellular locations of members of the IGFBP family. IGFBP2 has been previously established as a driver of glioma progression to a higher grade. In this study, we sought to determine whether IGFBP2-overexpressing tumours are dependent on continued oncogene expression and whether IGFBP2 is a viable therapeutic target in glioma. We took advantage of the well-characterized RCAS/Ntv-a mouse model to create a doxycycline-inducible IGFBP2 model of glioma and demonstrated that the temporal expression of IGFBP2 has dramatic impacts on tumour progression and survival. Further, we demonstrated that IGFBP2-driven tumours are dependent on the continued expression of IGFBP2, as withdrawal of this oncogenic signal led to a significant decrease in tumour progression and prolonged survival. Inhibition of IGFBP2 also impaired tumour cell spread. To assess a therapeutically relevant inhibition strategy, we evaluated a neutralizing antibody against IGFBP2 and demonstrated that it impaired downstream IGFBP2-mediated oncogenic signalling pathways. The studies presented here indicate that IGFBP2 not only is a driver of glioma progression and a prognostic factor but is also required for tumour maintenance and thus represents a viable therapeutic target in the treatment of glioma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

// Yan Sun 1, 5 , Yuexin Liu 1 , David Cogdell 1 , George A. Calin 2, 4 , Baocun Sun 5 , Scott Kopetz 3 , Stanley R. Hamilton 1 , Wei Zhang 1, 4 1 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 4 The Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 5 Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China Correspondence to: Wei Zhang, e-mail: wzhang@mdanderson.org Yan Sun, e-mail: sunyan@tjmuch.com Keywords: microRNA, biomarker, plasma, colorectal cancer, stage Received: September 23, 2015 Accepted: January 23, 2016 Published: February 04, 2016 ABSTRACT Circulating microRNAs (miRNAs) have emerged as promising biomarkers; however, few miRNAs have been reproducible and can be used in clinical practice. In this study, we screened the levels of 754 miRNAs using TaqMan array in 50 individual plasma samples from 10 demographically matched healthy controls and 40 colorectal cancer (CRC) patients (10 each of stage I–IV) and identified 22 miRNAs associated with the presence of and stages of CRC. Then we performed the validation for 11 miRNAs in an independent cohort including 187 CRC cases and 47 healthy controls. Comprehensive analyses showed that plasma miR-96 distinguished stage I–IV CRC from healthy controls with an area under curve (AUC) of 0.740; miR-203 separated stage III–IV CRC patients from stage I–II with an AUC of 0.757; and miR-141 differentiated stage IV CRC from stage I–III patients with an AUC of 0.851. Survival analyses showed that plasma miR-96 and miR-200b were independent prognostic factors for overall survival. Thus, we propose four miRNAs (miR-96, miR-203, miR-141 and miR-200b) as clinically validated circulating biomarkers for CRC prognosis that warrant further evaluation for clinical utility.

SUMMARYThe sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment ofTP53, RB1, and PIK3CAmutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.

Background Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations. Objective We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine. Design, setting, and participants We performed genome-wide copy number variation analysis on cohorts from the University of Texas MD Anderson Cancer Center ( n =40) and The Cancer Genome Atlas ( n =129), and identified the most frequently amplified chromosomal regions. These data were used to select four of the amplified regions to design a multicolor FISH test, referred to as the quartet test. Assay validation was performed on urine samples from 98 patients with bladder cancer: 56 with low-grade papillary, 42 with high-grade invasive disease, and 48 benign controls. Intervention The quartet test can be used in clinical practice for noninvasive detection of bladder cancer. Outcome measurements and statistical analysis We initially analyzed samples using a fraction of abnormal cell scores and then by the quantitative score, which included not only the proportion of cells with abnormal copy numbers, but also the proportion of cells with numbers of altered copies and degree of amplification. We used receiver operator characteristic (ROC) curves to identify cutoff values for the scores at which performances of sensitivity and specificity were maximized. Results and limitations The copy number status assessed by probes detected in voided urine reflected the amplification status of the primary tumor. An ROC curve summarizing the proportion of assayed cells with any abnormal copy numbers gave specificity of 93.8% and sensitivity of 78.6% using the proportion of cells with abnormal copy numbers. The quantitative score giving extra weight to cells with multiple simultaneous amplifications provided 95.8% specificity and 76.8% sensitivity. Both percentage of abnormal cells and quantitative scores were highly effective for assessing the grade of the tumor. The full spectrum of potential clinical applications was not explored in the current study, and further validation studies are needed. Conclusions The quartet test shows promising specificity and sensitivity results, but it requires validation on a larger multi-institutional cohort of samples. Patient summary The quartet test can be used for noninvasive detection of bladder cancer in voided urine. It can also be used to assess the grade of the tumor and tumor recurrence as well as post-treatment effects.

We developed a whole organ mapping approach that combines microscopic histological assessment of the entire mucosal membrane of the bladder with comprehensive genomic profiling. We used the approach to study the locoregional molecular changes in a human bladder containing multi-focal cancer. Uniform and widespread DNA methylation changes were identified in almost the entire mucosa representing the initial field effect. The field effect was associated with sub-clonal low allele frequency mutations and a small numer of uniform DNA copy number alterations that were typically restricted to small areas. An inactivating founder mutation in the pro-apoptotic and RNA splicing gene, ACIN1, was identified in normal appearing mucosa and expanded clonally with an additional 21 mutations in the development of in situ precursor lesions, and progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and several foci of carcinoma were almost identical, confirming their clonal origins. Importantly, the pathways affected by the CNVs and DNA mutations, including the Kras pathway, appeared to be preceded by the field changes in DNA methylation, suggesting that the DNA-based changes reinforced mechanisms that had already been initiated by methylation. Overall, the results demonstrate that field changes in DNA methylation can serve as the initiators of carcinogenesis in the bladder.

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and lethal cancer. The role of autophagy in the pathobiology of PDAC is intricate, with opposing functions manifested in different cellular contexts. MIR506 functions as a tumor suppressor in many cancer types through the regulation of multiple pathways. In this study, we hypothesized that MIR506 exerted a tumor suppression function in PDAC by inducing autophagy-related cell death. Our results provided evidence that downregulation of MIR506 expression was associated with disease progression in human PDAC. MIR506 triggered autophagic flux in PDAC cells, which led to autophagy-related cell death through direct targeting of the STAT3 (signal transducer and activator of transcription 3)-BCL2-BECN1 axis. Silencing and inhibiting STAT3 recapitulated the effects of MIR506, whereas forced expression of STAT3 abrogated the effects of MIR506. We propose that the apoptosis-inhibitory protein BCL2, which also inhibits induction of autophagy by blocking BECN1, was inhibited by MIR506 through targeting STAT3, thus augmenting BECN1 and promoting autophagy-related cell death. Silencing BECN1 and overexpression of BCL2 abrogated the effects of MIR506. These findings expand the known mechanisms of MIR506-mediated tumor suppression to activation of autophagy-related cell death and suggest a strategy for using MIR506 as an anti-STAT3 approach to PDAC treatment.

Ovarian carcinoma is the most lethal gynaecological malignancy. Better understanding of the molecular pathogenesis of this disease and effective targeted therapies are needed to improve patient outcomes. MicroRNAs play important roles in cancer progression and have the potential for use as either therapeutic agents or targets. Studies in other cancers have suggested that miR-506 has anti-tumour activity, but its function has yet to be elucidated. We found that deregulation of miR-506 in ovarian carcinoma promotes an aggressive phenotype. Ectopic over-expression of miR-506 in ovarian cancer cells was sufficient to inhibit proliferation and to promote senescence. We also demonstrated that CDK4 and CDK6 are direct targets of miR-506, and that miR-506 can inhibit CDK4/6–FOXM1 signalling, which is activated in the majority of serous ovarian carcinomas. This newly recognized miR-506–CDK4/6–FOXM1 axis provides further insight into the pathogenesis of ovarian carcinoma and identifies a potential novel therapeutic agent. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Ovarian carcinoma is the most lethal gynecologic malignancy; the majority of patients succumb to the disease within 5 years of diagnosis. The poor survival rate is attributed to diagnosis at advanced stage, when the tumor has metastasized. The epithelial-to-mesenchymal transition (EMT) is a necessary step toward metastatic tumor progression. Through integrated computational analysis, we recently identified a master microRNA (miRNA) network that includes miR-101 and regulates EMT in ovarian carcinoma. In the present study, we characterized the functions of miR-101. Using reporter gene assays, we demonstrated that miR-101 suppressed the expression of the E-cadherin repressors ZEB1 and ZEB2 by directly targeting the 3'-untranslated region (3'UTR) of both ZEB1 and ZEB2. Introduction of miR-101 significantly inhibited EMT and cell migration and invasion. Introducing cDNAs of ZEB1 and ZEB2 without 3'UTR abrogated miR-101-induced EMT alteration, respectively. Our findings showed that miR-101 represents a redundant mechanism for the miR-200 family that regulates EMT through two major E-cadherin transcriptional repressors.

Summary: We used whole-organ mapping to study the locoregional molecular changes in a human bladder containing multifocal cancer. Widespread DNA methylation changes were identified in the entire mucosa, representing the initial field effect. The field effect was associated with subclonal low-allele frequency mutations and a small number of DNA copy alterations. A founder mutation in the RNA splicing gene, ACIN1, was identified in normal mucosa and expanded clonally with an additional 21 mutations in progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and foci of carcinoma were almost identical, confirming their clonal origins. The pathways affected by the DNA copy alterations and mutations, including the Kras pathway, were preceded by the field changes in DNA methylation, suggesting that they reinforced mechanisms that had already been initiated by methylation. The results demonstrate that DNA methylation can serve as the initiator of bladder carcinogenesis. : Majewski et al. report that methylation changes suppressing innate immunity and dysregulating Ras-related pathways initiate bladder carcinogenesis. Keywords: Whole-organ map, bladder cancer, DNA methylation, DNA copy alterations, founder mutation, field effect, clonal origins, clonal expansion, urothelial carcinoma, gene signature

Endometrial carcinoma (EC) is one of the most common malignancies of the female reproductive system. Migration and invasion inhibitory protein (MIIP) gene was recently discovered candidate tumor suppress gene which located at chromosome 1p36.22. 1p36 deletion was found in many types of tumor including EC. In the present study, we will determine the role and mechanism of MIIP in EC metastasis.Immunohistochemistry was used to measure MIIP expression in normal and EC tissue. Both gain-of-function (infection) and loss-of-function (siRNA) assays were used to alter MIIP expression levels. The effect of MIIP on cell migration and invasion was measured by transwell assay. F-actin immunofluorescence staining was used to observe the cell morphology. The activation of GTP-loaded Rac1 was evaluated by Rac activity assay kit. Immunoprecipitation/WB was used to measure the interaction between MIIP and PAK1.We demonstrate that MIIP expression was significantly decreased in EC patients comparing to the normal ones, and decreased MIIP expression in EC tissues is associated with deep myometrial invasion, advanced stage, and the presence of lymph node metastasis. Using both gain-of-function (infection) and loss-of-function (siRNA) assays, we show that MIIP markedly blocked EC cell migration, whereas loss of MIIP led to increase in EC cell migration. We demonstrate that elevated expression of MIIP resulted in cytoskeleton reorganization with decreased formation of lamellipodia. We also provide evidence that MIIP is a key molecule in directing Rac1 signaling cascades in EC. Ectopically expressed MIIP consistently competed with Rac1-GTP for binding with the PAK1 p21-binding domain. Our data show that MIIP and PAK1 bind each other and that a C-terminal polyproline domain of MIIP is required for PAK1 binding. Deletion of the PAK1-binding domain of MIIP reduced cell migration-inhibiting activity.MIIP may function as a tumor suppressor gene for endometrial carcinoma. MIIP attenuates Rac1 signaling through a protein interaction network, and loss of this regulator may contribute to EC metastasis.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.

Altered expression of oncogenic and tumour-suppressing microRNAs (miRNAs) is widely associated with tumourigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumours. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and examined expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression.

Introduction EasyChip HPV blot is a human papillomavirus (HPV) genotyping assay that can be potentially used for HPV assay validation or clinical HPV research. To evaluate its genotyping accuracy, we compared EasyChip HPV blot with quantitative real-time polymerase chain reaction (qRT-PCR)/type-specific PCR assays in the detection of 8 high-risk HPV genotypes. Materials and methods Archival SurePath Papanicolaou specimens with abnormal results and follow-up biopsy (n = 154) were selected retrospectively for HPV genotyping by EasyChip HPV blot. To determine the accuracy of the assay, qRT-PCR and type-specific PCR also were performed and results for 8 high-risk HPV genotypes were compared (HPV16, 18, 31, 33, 35, 45, 52, and 58). Results A total of 95 Papanicolaou specimens were qualified for data analysis. Concordance between EasyChip HPV blot and qRT-PCR/type-specific PCR assays was high, with a very good agreement for the 8 high-risk HPV genotypes (95%; kappa value: 0.894, 95% CI: 0.805-0.984) and for HPV16 and HPV18 (96%; kappa value: 0.899, 95% CI: 0.802-0.996). HPV16 was the most frequent HPV genotype by EasyChip HPV blot. The odds ratio of HPV16/18 for high-grade cervical intraepithelial neoplasia was 11.25 (95% CI: 3.93-32.31). Conclusions EasyChip HPV blot is a reliable HPV genotyping assay that can be used for HPV assay validation or clinical HPV studies.

BACKGROUND: Peripheral neuropathy is a major dose-limiting toxicity of chemotherapy, especially after multiple courses of paclitaxel. The development of paclitaxel-induced neuropathy is associated with the activation of microglia followed by the activation and proliferation of astrocytes, and the expression and release of proinflammatory cytokines in the spinal dorsal horn. Cannabinoid type 2 (CB2) receptors are expressed in the microglia in neurodegenerative disease models. METHODS: To explore the potential of CB2 agonists for preventing paclitaxel-induced neuropathy, we designed and synthesized a novel CB2-selective agonist, namely, MDA7. The effect of MDA7 in preventing paclitaxel-induced allodynia was assessed in rats and in CB2 / and CB 2 / mice. We hypothesized that the CB2 receptor functions in a negative-feedback loop and that early MDA7 administration can blunt the neuroinflammatory response to paclitaxel and prevent mechanical allodynia through interference with specific signaling pathways. RESULTS: We found that MDA7 prevents paclitaxel-induced mechanical allodynia in rats and mice in a dose- and time-dependent manner without compromising paclitaxel’s antineoplastic effect. MDA7’s neuroprotective effect was absent in CB2 / mice and was blocked by CB2 antagonists, suggesting that MDA7’s action directly involves CB 2 receptor activation. MDA7 treatment was found to interfere with early events in the paclitaxel-induced neuroinflammatory response as evidenced by relatively reduced toll-like receptor and CB2 expression in the lumbar spinal cord, reduced levels of extracellular signal-regulated kinase 1/2 activity, reduced numbers of activated microglia and astrocytes, and reduced secretion of proinflammatory mediators in vivo and in in vitro models. CONCLUSIONS: Our findings suggest an innovative therapeutic approach to prevent chemotherapyinduced neuropathy and may permit more aggressive use of active chemotherapeutic regimens with reduced long-term sequelae. (Anesth Analg 2012;114:1104–20)

The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells.

Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients.

Tribological thin-film coatings can enhance the performance of mechanical components such as bearings and gears. Although a lubricant is present in most applications, the interactions of the lubricant with the coated surfaces are not always well understood. In the present study, Stribeck curves (i.e., traction coefficient vs. dimensionless film thickness λ) were generated for lubricated rolling contact between coated and uncoated surfaces. Chromium nitride, tungsten carbide–reinforced amorphous hydrocarbon, and silicon-incorporated diamond-like carbon coatings were evaluated. Compositions, hydrogen concentrations, Raman spectra, and surface energies are reported for the films. A ball-on-flat test configuration was used in 5%, 50%, and 100% slide-to-roll conditions. The test lubricant was a polyalphaolefin containing rust and oxidation inhibitor additives only. Differences in traction performance were observed for different coating types. Traction coefficients decreased at high λ with increased hydrocarbon...

To evaluate a commercialized in situ hybridization (ISH) assay for detecting human papillomavirus (HPV) DNA, we compared the ability of a new ISH probe, Inform HPV III (Ventana Medical Systems, Tucson, AZ), to that of PCR assays to detect HPV DNA in cervical tissue specimens with normal cervix (20 cases), cervical intraepithelial neoplasia (CIN; CIN 1, 27 cases; CIN 2, 28 cases; and CIN 3, 33 cases), and cervical carcinoma (29 cases). General HPV DNA was detected using consensus primer-mediated PCR assays. HPV genotyping was performed by using EasyChip HPV blot (King Car Yuan Shan Institute, I-Lan, Taiwan). HPV16 integration status (E2/E6 ratio) was determined by using quantitative real-time PCR. Our findings showed that the ISH and PCR had fair to good agreements in detecting HPV DNA across all CIN categories without significant differences (Kappa coefficient, 0.34 to 0.63; P = 0.13 to 1.0). However, ISH detected significantly fewer HPV-positive cases in carcinoma than PCR did (Kappa coefficient, 0.2; P = 0.03). Eleven cases with ISH − PCR + results had HPV types that can be detected by Inform HPV III. Five carcinoma cases with ISH − PCR + results showed a significantly higher level of integrated HPV16 ( P = 0.008) than did the ISH + cases. As a consequence, lower copy numbers of episomal HPV16 in carcinoma might be the cause for the false-negative ISH results. Although the punctate signal pattern of HPV significantly increased with the severity of disease ( P trend = 0.01), no significant difference in the HPV16 integration status was observed between the cases with a punctate signal only and the cases with mixed punctate and diffuse signals ( P = 0.4). In conclusion, ISH using the Inform HPV III probe seems comparable to PCR for detecting HPV DNA in cervical tissue with CINs. False-negative ISH results appear to be associated with the lower copy numbers of the episomal HPV16 but not with the ability of the Inform HPV III probe to detect specific HPV types. In addition, signal patterns, especially a mixed punctate and diffuse pattern of HPV, cannot be reliably used to predict viral integration status.

This note describes a method for measuring probe alignment errors on precision cylindrical coordinate measuring machines. Specifically, this method is used to determine the minimum distance between the line of travel of the center of a spherical probe tip and the axis of rotation of a rotary axis. Within the Timken facility, we refer to this error as intersection error [Bryan JB. Private communication; 7 June 1992]. In addition to intersection error, this method determines the position along the probe line of travel at which the center of the probe tip passes nearest to the axis of rotation. This position is commonly referred to as probe offset error. It is used to adjust the probe location such that its radial position is zero where it intersects (or nearly intersects) the axis of rotation. In this way, the probe tip location is datumed to the axis of rotation.

Multiple signal transduction pathways play a crucial role in cancer development, progression, and response to different therapies. An important issue is whether common signal transduction pathways are ubiquitously altered in all cancer types and some unique pathways are involved in different cancer types. Another important issue is whether and how transduction signaling molecules are heterogeneously expressed and activated in different cancer cells within and between cancer cell types.To gain insight into these issues, we assembled a protein lysate array with 90 different cell lines of 12 different cell types. Each sample is diluted 2-fold six times, and samples from the dilution series were printed three times on the array. We then measured the expression levels and phosphorylation status of 52 different signaling proteins with specific antibodies and carried out statistical hierarchical clustering analysis.The most significant finding based on the cluster analysis was that the cell lines did not group based on tumor types, suggesting that the signaling pathways studied were commonly activated in most of the tumor types cultured in vitro. As expected, related proteins associated with specific signaling pathways clustered together, and analysis of the 30 most differentially expressed proteins revealed the PI3-K signaling pathway was upregulated in several different tumor types and the VEGF-angiogenesis pathway was downregulated in hematopoetic cancers. Another important observation, with clinical implications was that EGFR was the most heterogeneous among all the cell lines. We also observed signaling pathways unique to specific types of cancers such as the inverse relationship between p16ink and Rb, and the EGFR mediated pathway activation characteristic of pancreatic cancers.Using reverse phase lysate array analysis in this study, we were able to determine potential relationships and signaling pathways, both common and unique, to different types of cancer using cell lines in vitro. This data could be utilized for mining information related to an individual cancer of interest and combined with morphological and genomic profiles would help in creating a combination of expression markers and/or functional signaling maps for specific cancer diagnosis and therapy.

Typically, the contact of two rough bodies is transformed into an equivalent rough surface contacting a flat. Due to the mathematics involved, this was usually conducted for surfaces assumed to be Gaussian, where distribution of peaks and valleys are equal. These assumptions were acceptable when dealing with ground surfaces, which are typically Gaussian; however, other finishing techniques often result in skewed surface distributions. As such, an equation was developed numerically within, based on the Weibull distribution, to estimate the composite skewness of contacting rough bodies. The assumption of using the Weibull distribution to describe the surface variation was validated with measured data from different finishing techniques, as was the equation for composite skewness developed within. Presented at the STLE Annual Meeting in Las Vegas, Nevada, May 15-19, 2005 Review led by Alan Lebeck

Gastrointestinal stromal tumor (GIST) has emerged as a clinically distinct type of sarcoma with frequent overexpression and mutation of the c-Kit oncogene and a favorable response to imatinib mesylate [also known as STI571 (Gleevec)] therapy. However, a significant diagnostic challenge remains in the differentiation of GIST from leiomyosarcomas (LMSs). To improve on the diagnostic evaluation and to complement the immunohistochemical evaluation of these tumors, we performed a whole-genome gene expression study on 68 well characterized tumor samples. Using bioinformatic approaches, we devised a two-gene relative expression classifier that distinguishes between GIST and LMS with an accuracy of 99.3% on the microarray samples and an estimated accuracy of 97.8% on future cases. We validated this classifier by using RT-PCR on 20 samples in the microarray study and on an additional 19 independent samples, with 100% accuracy. Thus, our two-gene relative expression classifier is a highly accurate diagnostic method to distinguish between GIST and LMS and has the potential to be rapidly implemented in a clinical setting. The success of this classifier is likely due to two general traits, namely that the classifier is independent of data normalization and that it uses as simple an approach as possible to achieve this independence to avoid overfitting. We expect that the use of simple marker pairs that exhibit these traits will be of significant clinical use in a variety of contexts.

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P

Epithelial ovarian cancer remains the most lethal gynecological malignancy (1). The current standard of care consists of radical surgery and platinum-based chemotherapy. The five-year survival rate for patients with advanced ovarian cancer is only 30% to 40%, and acquired resistance to platinum is considered a major factor in disease relapse. Platinum-based drugs form intra- and interstrand adducts with DNA, which causes DNA double-strand breaks and triggers DNA damage and repair pathways. Homologous recombination is a critical pathway for DNA double-strand break repair (2) and is responsible for the resistance of high-grade serous ovarian cancer to frontline platinum-based chemotherapy (3). Cells with compromised homologous recombination machinery are highly sensitive to apoptosis triggered by platinum-induced DNA damage through a mechanism termed synthetic lethality (4). Thus, the ability to block homologous recombination-mediated repair is a focus of intense investigation as an approach to improve treatment outcomes in high-grade serous ovarian cancers. Recent studies demonstrated that BRCA2 mutations, and to a lesser extent BRCA1 mutations/methylation, are associated with improved survival and response to therapy in serous ovarian cancer (5,6). Whereas BRCA1 plays diverse roles in DNA damage pathways, the primary role of BRCA2 is to mediate homologous recombination by directly loading the RAD51 protein onto damage sites or stalled replication forks (7,8). RAD51 is a critical component of the homologous recombination-mediated double-strand DNA break repair machinery and assembles onto single-stranded DNA as a nucleoprotein filament and catalyzes the exchange of homologous DNA sequences (9). RAD51 suppression can sensitize cancer cells to DNA-damaging drugs (10–14), and RAD51 overexpression contributes to chemotherapy resistance in human soft tissue sarcoma cells (15). MicroRNAs (miRNAs) are a class of small noncoding RNAs (~22 nt) that regulate gene expression. MiRNAs bind to the 3′-untranslated region (3′-UTR) of target genes, which either leads to mRNA degradation or inhibits protein translation (16). Nearly 2578 miRNAs have been identified in the human genome and are thought to regulate 30% of the transcriptome (17). Increasing evidence has demonstrated that miRNA are highly deregulated in cancer, suggesting they may function as therapeutic tools (17–20). In a recent high-throughput miRNA signature screen, decreased expression of the chrXq27.3-miRNA cluster that included miR-506 was associated with early relapse in patients with advanced-stage epithelial ovarian cancer (21). Our studies established that miR-506 is a potent inhibitor of the epithelial-to-mesenchymal transition (EMT) (22,23), which is also associated with chemoresistance. In addition, we found that miR-506 could suppress proliferation and induce senescence by directly targeting the CDK4/6-FOXM1 axis in ovarian cancer (24). However, it is unknown whether miR-506 is involved in the chemotherapy response.

Signal transduction pathways play a crucial role in breast cancer development, progression, and response to different therapies. A major problem in breast cancer therapy is the heterogeneity among different tumor types and cell lines commonly used in preclinical studies. To characterize the signaling pathways of some of the commonly used breast cancer cell lines and dissect the relationship among a number of pathways and some key genetic and molecular events in breast cancer development, such as p53 mutation, ErbB2 expression, and estrogen receptor (ER)/progesterone receptor (PR) status, we performed pathway profiling of 14 breast cancer cell lines by measuring the expression and phosphorylation status of 40 different cell signaling proteins with 53 specific antibodies using a protein lysate array. Cluster analysis of the expression data showed that there was close clustering of phosphatidylinositol 3-kinase, Akt, mammalian target of rapamycin (mTOR), Src, and platelet-derived growth factor receptor β (PDGFRβ) in all of the cell lines. The most differentially expressed proteins between ER- and PR-positive and ER- and PR-negative breast cells were mTOR, Akt (pThr308), PDGFRβ, PDGFRβ (pTyr751), panSrc, Akt (pSer473), insulin-like growth factor-binding protein 5 (IGFBP5), Src (pTyr418), mTOR (pSer2448), and IGFBP2. Many apoptotic proteins, such as apoptosis-inducing factor, IGFBP3, bad, bax, and cleaved caspase 9, were overexpressed in mutant p53-carrying breast cancer cells. Hexokinase isoenzyme 1, ND2, and c-kit were the most differentially expressed proteins in high and low ErbB2-expressing breast cancer cells. This study demonstrated that ER/PR status, ErbB2 expression, and p53 status are major molecules that impact downstream signaling pathways.

The progression of gliomas has been extensively studied at the genomic level using cDNA microarrays. However, systematic examinations at the protein translational and post-translational levels are far more limited. We constructed a glioma protein lysate array from 82 different primary glioma tissues, and surveyed the expression and phosphorylation of 46 different proteins involved in signaling pathways of cell proliferation, cell survival, apoptosis, angiogenesis, and cell invasion. An analysis algorithm was employed to robustly estimate the protein expressions in these samples. When ranked by their discriminating power to separate 37 glioblastomas (high-grade gliomas) from 45 lower-grade gliomas, the following 12 proteins were identified as the most powerful discriminators: IBalpha, EGFRpTyr845, AKTpThr308, phosphatidylinositol 3-kinase (PI3K), BadpSer136, insulin-like growth factor binding protein (IGFBP) 2, IGFBP5, matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), phosphorylated retinoblastoma protein (pRB), Bcl-2, and c-Abl. Clustering analysis showed a close link between PI3K and AKTpThr308, IGFBP5 and IGFBP2, and IBalpha and EGFRpTyr845. Another cluster includes MMP9, Bcl-2, VEGF, and pRB. These clustering patterns may suggest functional relationships, which warrant further investigation. The marked association of phosphorylation of AKT at Thr308, but not Ser473, with glioblastoma suggests a specific event of PI3K pathway activation in glioma progression.

Laboratories use different laser-based scanners to scan microarray images. To assess whether results from different scanners are comparable, and thus whether data from different laboratories can be compared, we scanned the same microarray slide with three commercial scanners that use different imaging techniques. After the acquisition of the microarray images produced by the three scanners, the images were quantified using a single imaging software package and protocol. The results were compared, and we found that the data obtained from the three scanners were comparable and that the variations caused by the use of different instruments were negligible, in spite of the fact that the scanners were based on different optical imaging techniques.

A finite element model was developed to investigate the effects of a spherical debris on elastohydrodynamically lubricated rolling/sliding contacts. Three dimensional dent profiles were obtained using finite element method showing horseshoe shape material pile-up along the rolling direction. The dent profiles obtained from the finite element analysis (FEA) were compared with the experimental results. There is good qualitative agreement between FEA and experimental dent profiles. The FEA dent profiles were then used in a time dependent thermal elastohydrodynamic lubrication (EHL) point contact model to analyze the dent effects on the pressure, film thickness and temperature profiles. The presence of a dent in lubricated contacts generates high pressure spikes and increases the peak temperature. The internal stresses were calculated based on the pressure and traction data obtained from the EHL analysis. The results indicate that a dent created by a debris will cause the internal maximum Von Mises stress to occur near the surface, which contributes to surface initiated failures.

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Fusions can lead to production of oncogenic fusion proteins or to enhanced expression of oncogenes. Several recent studies have reported that some fusion genes can escape microRNA regulation via 3'-untranslated region (3'-UTR) deletion. We performed whole transcriptome sequencing to identify fusion genes in glioma and discovered FGFR3-TACC3 fusions in 4 of 48 glioblastoma samples from patients both of mixed European and of Asian descent, but not in any of 43 low-grade glioma samples tested. The fusion, caused by tandem duplication on 4p16.3, led to the loss of the 3'-UTR of FGFR3, blocking gene regulation of miR-99a and enhancing expression of the fusion gene. The fusion gene was mutually exclusive with EGFR, PDGFR, or MET amplification. Using cultured glioblastoma cells and a mouse xenograft model, we found that fusion protein expression promoted cell proliferation and tumor progression, while WT FGFR3 protein was not tumorigenic, even under forced overexpression. These results demonstrated that the FGFR3-TACC3 gene fusion is expressed in human cancer and generates an oncogenic protein that promotes tumorigenesis in glioblastoma.

Our previous study shows that PURNE2 mRNA plays an important role in the differential diagnosis of leiomyosarcoma and gastrointestinal stromal tumor (GIST). Non-coding RNA PCA3 locates in the intron of PRUNE2 and may play a role in PRUNE2 expression. The aim of this study was to explore the expression of PCA3 mRNA and PRUNE2 in leiomyosarcoma and their correlation.The expression of PRUNE2 mRNA was analyzed by agilent gene expression microarray CHIP in 31 leiomyosarcomas and 37 GISTs, and the correlation of the PRUNE2 expression and prognosis of leiomyosarcoma was predicted. Real-Time PCR assay was used to detect the mRNA levels of PCA3 and PRUNE2 in 13 leiomyosarcomas and to investigate their correlation. Seven prostate cancer tissues were used as control of PCA3.The level of PRUNE2 mRNA expression was significantly higher in the 31 leiomyosarcomas than that in the 37 GISTs, and the level of PRUNE2 mRNA expression was correlated with survival of the leiomyosarcoma patients. Compared with prostate cancer, the non-coding RNA PCA3 expression level was significantly lower in leiomyosarcoma, and it had no correlation with the prognosis of leiomyosarcoma. Most importantly, the PRUNE2 and PCA3 mRNA expressions were both upregulated in leiomyosarcoma and showed a significant positive correlation.Our findings demonstrate for the first time that PRUNE2 expression is correlated with the survival of leiomyosarcoma patients. Furthermore, non-coding RNA PCA3, which locates in the intron of PRUNE2, has a significant positive correlation with PRUNE2 and may play an important role in the pathogenesis of leiomyosarcoma.

Altered expression of oncogenic and tumor-suppressing microRNAs (miRNAs) is widely associated with tumorigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumors. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and interrogated expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression.

The recently described transcriptomic switch to a mitosis program in castration-resistant prostate cancer (CRPC) suggests that mitotic proteins may be rationally targeted at this lethal stage of the disease. In this study, we showed upregulation of the mitosis-phase at the protein level in our cohort of 51 clinical CRPC cases and found centrosomal aberrations to also occur preferentially in CRPC compared with untreated, high Gleason-grade hormone-sensitive prostate cancer (P