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Find video protocols related to scientific articles indexed in Pubmed.
Clinically Relevant microRNAs in Ovarian Cancer.
Mol. Cancer Res.
PUBLISHED: 10-12-2014
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MicroRNAs (miRNAs/miRs) belong to a class of small non-coding RNAs that can negatively regulate messenger RNA (mRNA) expression of target genes. miRNAs are involved in multiple aspects of ovarian cancer cell dysfunction and the phenotype of ovarian cancer cells can be modified by targeting miRNA expression. miRNA profiling has detected a number of candidate miRNAs with the potential to regulate many important biological functions in ovarian cancer, but their role still needs to be clarified, given the remarkable heterogeneity among ovarian cancers and the context dependent role of miRNAs. This review summarizes the data collected from The Cancer Genome Atlas (TCGA) and several other genome-wide projects to identify dysregulated miRNAs in ovarian cancers. Copy number variations (CNVs), epigenetic alterations, and oncogenic mutations are also discussed that impact miRNA levels in ovarian disease. Emphasis is given to the role of particular miRNAs in altering expression of genes in human ovarian cancers with the potential to provide diagnostic, prognostic and therapeutic targets. Particular attention has been given to TP53, BRCA1/2, CA125 (MUC16), HE4 (WFDC2), and imprinted genes such as ARHI (DIRAS3). Better understanding of the abnormalities in miRNA expression and downstream transcriptional and biological consequences will provide leads for more effective biomarkers and translational approaches in the management of ovarian cancer.
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Reverse-phase protein array for prediction of patients at low risk of developing bone metastasis from breast cancer.
Oncologist
PUBLISHED: 08-12-2014
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A biomarker that predicts bone metastasis based on a protein laboratory assay has not been demonstrated. Reverse-phase protein array (RPPA) enables quantification of total and phosphorylated proteins, providing information about their functional status. The aim of this study was to identify bone-metastasis-related markers in patients with primary breast cancer using RPPA analysis.
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Focal adhesion kinase: an alternative focus for anti-angiogenesis therapy in ovarian cancer.
Cancer Biol. Ther.
PUBLISHED: 04-23-2014
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This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAK(Y397) were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P<0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P<0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P<0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors.
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Molecular biomarkers of residual disease after surgical debulking of high-grade serous ovarian cancer.
Clin. Cancer Res.
PUBLISHED: 04-22-2014
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Residual disease following primary cytoreduction is associated with adverse overall survival in patients with epithelial ovarian cancer. Accurate identification of patients at high risk of residual disease has been elusive, lacking external validity and prompting many to undergo unnecessary surgical exploration. Our goal was to identify and validate molecular markers associated with high rates of residual disease.
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BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis.
Mol Oncol
PUBLISHED: 04-21-2014
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Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent.
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Notch3 pathway alterations in ovarian cancer.
Cancer Res.
PUBLISHED: 04-17-2014
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The Notch pathway plays an important role in the growth of high-grade serous ovarian (HGS-OvCa) and other cancers, but its clinical and biologic mechanisms are not well understood. Here, we found that the Notch pathway alterations are prevalent and significantly related to poor clinical outcome in patients with ovarian cancer. Particularly, Notch3 alterations, including amplification and upregulation, were highly associated with poor patient survival. Targeting Notch3 inhibited ovarian cancer growth and induced apoptosis. Importantly, we found that dynamin-mediated endocytosis was required for selectively activating Jagged-1-mediated Notch3 signaling. Cleaved Notch3 expression was the critical determinant of response to Notch-targeted therapy. Collectively, these data identify previously unknown mechanisms underlying Notch3 signaling and identify new, biomarker-driven approaches for therapy.
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Preclinical and early clinical evaluation of the oral AKT inhibitor, MK-2206, for the treatment of acute myelogenous leukemia.
Clin. Cancer Res.
PUBLISHED: 02-28-2014
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Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206. Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA).
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2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.
Nat Commun
PUBLISHED: 02-17-2014
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Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.
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Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy.
Cancer Cell
PUBLISHED: 01-13-2014
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Muscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered three molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPAR? and estrogen receptor transcription and were enriched with activating FGFR3 mutations and potential FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.
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Autocrine effects of tumor-derived complement.
Cell Rep
PUBLISHED: 01-11-2014
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We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.
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Metabolic shifts induced by fatty acid synthase inhibitor orlistat in non-small cell lung carcinoma cells provide novel pharmacodynamic biomarkers for positron emission tomography and magnetic resonance spectroscopy.
Mol Imaging Biol
PUBLISHED: 11-20-2013
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Abnormal fatty acid (FA) synthesis is one of the common features of cancer. Fatty acid synthase (FASN), a multifunctional enzyme playing a key role in biosynthesis of FA, is up-regulated in prostate, breast, and lung carcinomas. Orlistat is a FDA-approved anti-obesity drug that inhibits the thioesterase domain of FASN, interferes with cellular FA synthesis, can arrest tumor cell proliferation, and induces tumor cell apoptosis. The current study was aimed to investigate the metabolic changes associated with FASN inhibition by orlistat and to understand the molecular mechanisms behind the observed metabolic changes in non-small cell lung carcinoma (NSCLC) cell lines.
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Independent validation of a model using cell line chemosensitivity to predict response to therapy.
J. Natl. Cancer Inst.
PUBLISHED: 08-20-2013
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Methods using cell line microarray and drug sensitivity data to predict patients chemotherapy response are appealing, but groups may be reluctant to release details to preserve intellectual property. Here we describe a case study to validate predictions while treating the methods as a "black box."
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Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes.
Clin. Cancer Res.
PUBLISHED: 08-15-2013
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The clinical relevancy of the 7-subtype classification of triple-negative breast cancer (TNBC) reported by Lehmann and colleagues is unknown. We investigated the clinical relevancy of TNBC heterogeneity by determining pathologic complete response (pCR) rates after neoadjuvant chemotherapy, based on TNBC subtypes.
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The emerging role of HE4 in the evaluation of epithelial ovarian and endometrial carcinomas.
Oncology (Williston Park, N.Y.)
PUBLISHED: 08-06-2013
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HE4 (human epididymis protein 4) is overexpressed in both ovarian and endometrial cancers. Levels of the shed HE4 protein are elevated in sera from ovarian and endometrial cancer patients. HE4 is less frequently elevated than cancer antigen 125 (CA 125) in benign gynecologic conditions and is found in a fraction of endometrial and ovarian cancers that lack CA 125 expression. Consequently, HE4 has emerged as an important biomarker that complements CA 125 in discriminating between benign and malignant pelvic masses, monitoring response to treatment, and detecting recurrences of both ovarian and endometrial cancer. The "risk of ovarian malignancy algorithm" (ROMA) incorporates CA 125, HE4, and menopausal status to distinguish benign from malignant adnexal masses, and has been approved by the US Food and Drug Administration to aid in referring patients who are likely to have ovarian cancer to specially trained gynecologic oncologists for surgery. HE4 also promises to augment the sensitivity of CA 125 for detecting early-stage ovarian cancer. In this review, we discuss the discovery and biologic significance of HE4 and evaluate available evidence regarding the utility of HE4 as a biomarker for ovarian and endometrial cancer.
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Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies.
Neuromuscul. Disord.
PUBLISHED: 07-18-2013
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The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.
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Bisphosphorylated PEA-15 sensitizes ovarian cancer cells to paclitaxel by impairing the microtubule-destabilizing effect of SCLIP.
Mol. Cancer Ther.
PUBLISHED: 03-29-2013
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Paclitaxel is a standard chemotherapeutic agent for ovarian cancer. PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) regulates cell proliferation, autophagy, apoptosis, and glucose metabolism and also mediates AKT-dependent chemoresistance in breast cancer. The functions of PEA-15 are tightly regulated by its phosphorylation status at Ser104 and Ser116. However, the effect of PEA-15 phosphorylation status on chemosensitivity of cancer cells remains unknown. Here, we tested the hypothesis that PEA-15 phosphorylated at both Ser104 and Ser116 (pPEA-15) sensitizes ovarian cancer cells to paclitaxel. We first found that knockdown of PEA-15 in PEA-15-high expressing HEY and OVTOKO ovarian cancer cells resulted in paclitaxel resistance, whereas re-expression of PEA-15 in these cells led to paclitaxel sensitization. We next found that SKOV3.ip1-DD cells (expressing phosphomimetic PEA-15) were more sensitive to paclitaxel than SKOV3.ip1-AA cells (expressing nonphosphorylatable PEA-15). Compared with SKOV3.ip1-vector and SKOV3.ip1-AA cells, SKOV3.ip1-DD cells displayed reduced cell viability, inhibited anchorage-independent growth, and augmented apoptosis when treated with paclitaxel. Furthermore, HEY and OVTOKO cells displayed enhanced paclitaxel sensitivity when transiently overexpressing phosphomimetic PEA-15 and reduced paclitaxel sensitivity when transiently overexpressing nonphosphorylatable PEA-15. These results indicate that pPEA-15 sensitizes ovarian cancer cells to paclitaxel. cDNA microarray analysis suggested that SCLIP (SCG10-like protein), a microtubule-destabilizing protein, is involved in pPEA-15-mediated chemosensitization. We found that reduced expression and possibly posttranslational modification of SCLIP following paclitaxel treatment impaired the microtubule-destabilizing effect of SCLIP, thereby promoting induction of mitotic arrest and apoptosis by paclitaxel. Our findings highlight the importance of pPEA-15 as a promising target for improving the efficacy of paclitaxel-based therapy in ovarian cancer.
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Tumour angiogenesis regulation by the miR-200 family.
Nat Commun
PUBLISHED: 03-02-2013
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The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200s role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent.
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Comparison of molecular subtype distribution in triple-negative inflammatory and non-inflammatory breast cancers.
Breast Cancer Res.
PUBLISHED: 01-29-2013
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Because of its high rate of metastasis, inflammatory breast cancer (IBC) has a poor prognosis compared with non-inflammatory types of breast cancer (non-IBC). In a recent study, Lehmann and colleagues identified seven subtypes of triple-negative breast cancer (TNBC). We hypothesized that the distribution of TNBC subtypes differs between TN-IBC and TN-non-IBC. We determined the subtypes and compared clinical outcomes by subtype in TN-IBC and TN-non-IBC patients.
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Reproducibility of SELDI Spectra Across Time and Laboratories.
Cancer Inform
PUBLISHED: 03-14-2011
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This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited.The reproducibility of mass spectrometry (MS) data collected using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) has been questioned. This investigation was designed to test the reproducibility of SELDI data collected over time by multiple users and instruments. Five laboratories prepared arrays once every week for six weeks. Spectra were collected on separate instruments in the individual laboratories. Additionally, all of the arrays produced each week were rescanned on a single instrument in one laboratory. Lab-to-lab and array-to-array variability in alignment parameters were larger than the variability attributable to running samples during different weeks. The coefficient of variance (CV) in spectrum intensity ranged from 25% at baseline, to 80% in the matrix noise region, to about 50% during the exponential drop from the maximum matrix noise. Before normalization, the median CV of the peak heights was 72% and reduced to about 20% after normalization. Additionally, for the spectra from a common instrument, the CV ranged from 5% at baseline, to 50% in the matrix noise region, to 20% during the drop from the maximum matrix noise. Normalization reduced the variability in peak heights to about 18%. With proper processing methods, SELDI instruments produce spectra containing large numbers of reproducibly located peaks, with consistent heights.
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Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.
Eur. J. Hum. Genet.
PUBLISHED: 03-02-2011
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Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy with an estimated prevalence of 1/8000. There are two genetically distinct types, DM1 and DM2. DM2 is generally milder with more phenotypic variability than the classic DM1. Our previous data on co-segregation of heterozygous recessive CLCN1 mutations in DM2 patients indicated a higher than expected DM2 prevalence. The aim of this study was to determine the DM2 and DM1 frequency in the general population, and to explore whether the DM2 mutation functions as a modifier in other neuromuscular diseases (NMD) to account for unexplained phenotypic variability. We genotyped 5535 Finnish individuals: 4532 normal blood donors, 606 patients with various non-myotonic NMD, 221 tibial muscular dystrophy patients and their 176 healthy relatives for the DM2 and DM1 mutations. We also genotyped an Italian idiopathic non-myotonic proximal myopathy cohort (n = 93) for the DM2 mutation. In 5496 samples analyzed for DM2, we found three DM2 mutations and two premutations. In 5511 samples analyzed for DM1, we found two DM1 mutations and two premutations. In the Italian cohort, we identified one patient with a DM2 mutation. We conclude that the DM2 mutation frequency is significantly higher in the general population (1/1830; P-value = 0.0326) than previously estimated. The identification of DM2 mutations in NMD patients with clinical phenotypes not previously associated with DM2 is of particular interest and is in accord with the high overall prevalence. On the basis of our results, DM2 appears more frequent than DM1, with most DM2 patients currently undiagnosed with symptoms frequently occurring in the elderly population.
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Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ?EGFR.
J. Proteome Res.
PUBLISHED: 02-14-2011
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An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ?EGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ?EGFR play a significant role in malignancy. To understand the ?EGFR signal, we compared it to that of a kinase-inactivated mutant of ?EGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ?EGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ?EGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ?EGFR expressing tumors.
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Altered MEF2 isoforms in myotonic dystrophy and other neuromuscular disorders.
Muscle Nerve
PUBLISHED: 11-25-2010
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Because of their central role in muscle development and maintenance, MEF2 family members represent excellent candidate effectors of the muscle pathology in myotonic dystrophy (DM). We investigated the expression and alternative splicing of all four MEF2 genes in muscle from neuromuscular disorder (NMD) patients, including DM1 and DM2. We observed MEF2A and MEF2C overexpression in all NMD muscle, including 12 MEF2-interacting genes. Exon 4 and 5 usage in MEF2A and MEF2C was different between DM and normal muscle, with DM showing the embryonic isoform. Similar splicing differences were observed in other NMD muscle. For MEF2C, missplicing was more pronounced in DM than in other dystrophies. Our data confirm dysregulation of MEF2A and MEF2C expression and splicing in several NMD, including DM. Our findings demonstrate that aberrant splicing in NMD is independent from expression of mutant repeats, and suggests that some aberrant splicing, even in DM, may be compensatory rather than primary.
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Blasted cell line names.
Cancer Inform
PUBLISHED: 10-14-2010
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While trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. Most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name.
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Tackling the widespread and critical impact of batch effects in high-throughput data.
Nat. Rev. Genet.
PUBLISHED: 09-14-2010
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High-throughput technologies are widely used, for example to assay genetic variants, gene and protein expression, and epigenetic modifications. One often overlooked complication with such studies is batch effects, which occur because measurements are affected by laboratory conditions, reagent lots and personnel differences. This becomes a major problem when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. Using both published studies and our own analyses, we argue that batch effects (as well as other technical and biological artefacts) are widespread and critical to address. We review experimental and computational approaches for doing so.
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Evaluation of changes in serum protein profiles during neoadjuvant chemotherapy in HER2-positive breast cancer using an LC-MALDI-TOF/MS procedure.
Proteomics
PUBLISHED: 09-10-2010
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Comparison of protein profiles of sera acquired before and after preoperative chemotherapy for breast cancer may reveal tumor markers that could be used to monitor tumor response. In this study, we analyzed pre- and post-chemotherapy protein profiles of sera from 39 HER2-postive breast cancer patients (n=78 samples) who received 6 months of preoperative chemotherapy using LC-MALDI-TOF/MS technology. We detected qualitative and quantitative differences in pair-wise comparison of pre- and post chemotherapy samples that were different in patients who achieved pathological complete response (pCR, n=21) compared with those with residual disease (n=18). We identified 2329 and 3152 peaks as differentially expressed in the pre-chemotherapy samples of the responders and non-responders. Comparison of matching pre- and post-chemotherapy samples identified 34 (32 decreased, two increased) and 304 peaks (157 decreased, 147 increased) that significantly changed (p<0.01, false discovery rate ? 20%) after treatment in responders and non-responders, respectively. The top 11 most significantly altered peptide peaks with the greatest change in intensity were positively identified. These corresponded to eight proteins including ?-2-macroglobulin, complement 3, hemopexin, and serum amyloid P in the responder group and chains C and A of apolipoprotein A-I, hemopexin precursor, complement C, and amyloid P component in the non-responding groups. All proteins decreased after therapy, except chain C apolipoprotein A and hemopexin precursor that increased. These results suggest that changes in serum protein levels occur in response to chemotherapy and these changes partly appear different in patients who are highly sensitive to chemotherapy compared with those with lesser response.
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Statistical contributions to proteomic research.
Methods Mol. Biol.
PUBLISHED: 04-22-2010
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Proteomic profiling has the potential to impact the diagnosis, prognosis, and treatment of various diseases. A number of different proteomic technologies are available that allow us to look at many proteins at once, and all of them yield complex data that raise significant quantitative challenges. Inadequate attention to these quantitative issues can prevent these studies from achieving their desired goals, and can even lead to invalid results. In this chapter, we describe various ways the involvement of statisticians or other quantitative scientists in the study team can contribute to the success of proteomic research, and we outline some of the key statistical principles that should guide the experimental design and analysis of such studies.
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Methylation of the candidate biomarker TCF21 is very frequent across a spectrum of early-stage nonsmall cell lung cancers.
Cancer
PUBLISHED: 04-07-2010
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The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, further characterization of the stages and types of nonsmall cell lung cancer (NSCLC) that are hypermethylated and the frequency of hypermethylation and associated "second hits" were assessed.
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Prospective comparison of clinical and genomic multivariate predictors of response to neoadjuvant chemotherapy in breast cancer.
Clin. Cancer Res.
PUBLISHED: 01-12-2010
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Several different multivariate prediction models using routine clinical variables or multigene signatures have been proposed to predict pathologic complete response to combination chemotherapy in breast cancer. Our goal was to compare the performance of four conceptually different predictors in an independent cohort of patients.
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Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.
Acta Neuropathol.
PUBLISHED: 01-01-2010
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Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.
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Dual targeting of EphA2 and FAK in ovarian carcinoma.
Cancer Biol. Ther.
PUBLISHED: 06-24-2009
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EphA2 gene silencing has been shown to result in antitumor efficacy. Here we considered whether silencing additional targets downstream of EphA2 would further enhance the therapeutic effect. EphA2 targeted siRNA was tested in combination with either FAK or Src targeted siRNA using DOPC nanoliposomes in orthotopic models of ovarian carcinoma. The effects of therapy were determined by changes in tumor weight, proliferation (Ki-67), and microvessel density (CD31). In our initial in vivo study, EphA2 plus FAK silencing resulted in the greatest reduction in tumor growth (by 73%, p < 0.005) as compared to control siRNA alone. In the SKOV3ip1 and HeyA8 ovarian cancer models, EphA2 siRNA-DOPC treatment resulted in a 50-67% decrease in tumor growth (p < 0.02, for both), and FAK siRNA-DOPC resulted in a 61-62% decrease in tumor growth (p < 0.009, p < 0.05, respectively). EphA2 plus FAK siRNA-DOPC treatment resulted in a significant reduction (SKOV3ip1: 76%, p < 0.007, HeyA8: 90%, p < 0.003) in tumor growth compared to control siRNA-DOPC. Combination treatment with EphA2 + FAK siRNA-DOPC resulted in significant decreases in tumor cell proliferation (p < 0.001) and microvessel density compared to control siRNA-DOPC (80%; p < 0.001), or the monotherapy groups (p values <0.001). These data suggest that the antitumor efficacy of in vivo EphA2 targeting is enhanced in combination with FAK silencing. Dual targeting of EphA2 and FAK may have therapeutic implications for ovarian cancer management.
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Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines.
Brain
PUBLISHED: 06-08-2009
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Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4.
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Clinical evaluation of chemotherapy response predictors developed from breast cancer cell lines.
Breast Cancer Res. Treat.
PUBLISHED: 06-06-2009
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The goal of this study was to develop pharmacogenomic predictors in response to standard chemotherapy drugs in breast cancer cell lines and test their predictive value in patients who received treatment with the same drugs. Nineteen human breast cancer cell lines were tested for sensitivity to paclitaxel (T), 5-fluorouracil (F), doxorubicin (A) and cyclophosphamide (C) in vitro. Baseline gene expression data were obtained for each cell line with Affymetrix U133A gene chips, and multigene predictors of sensitivity were derived for each drug separately. These predictors were applied individually and in combination to human gene expression data generated with the same Affymetrix platform from fine needle aspiration specimens of 133 stage I-III breast cancers. Tumor samples were obtained at baseline, and each patient received 6 months of preoperative TFAC chemotherapy followed by surgery. Cell line-derived prediction results were correlated with the observed pathologic response to chemotherapy. Statistically robust differentially expressed genes between sensitive and resistant cells could only be found for paclitaxel. False discovery rates associated with the informative genes were high for all other drugs. For each drug, the top 100 differentially expressed genes were combined into a drug-specific response predictor. When these cell line-based predictors were applied to patient data, there was no significant correlation between observed response and predicted response either for individual drug predictors or combined predictions. Cell line-derived predictors of response to four commonly used chemotherapy drugs did not predict response accurately in patients.
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Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics.
Cancer Res.
PUBLISHED: 05-12-2009
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Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a "tumorigenic" signature defined using CD44(+)/CD24(-) breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.
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Variable slope normalization of reverse phase protein arrays.
Bioinformatics
PUBLISHED: 03-31-2009
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Reverse phase protein arrays (RPPA) measure the relative expression levels of a protein in many samples simultaneously. A set of identically spotted arrays can be used to measure the levels of more than one protein. Protein expression within each sample on an array is estimated by borrowing strength across all the samples, but using only within array information. When comparing across slides, it is essential to account for sample loading, the total amount of protein printed per sample. Currently, total protein is estimated using either a housekeeping protein or the sample median across all slides. When the variability in sample loading is large, these methods are suboptimal because they do not account for the fact that the protein expression for each slide is estimated separately.
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Genome-wide hypomethylation in head and neck cancer is more pronounced in HPV-negative tumors and is associated with genomic instability.
PLoS ONE
PUBLISHED: 02-13-2009
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Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.
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Epigenetic analysis of the Notch superfamily in high-grade serous ovarian cancer.
Gynecol. Oncol.
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Gene methylation and other epigenetic modifications of gene regulation have been implicated in the growth of ovarian cancer, but the clinical significance of such modifications in the Notch pathway in high-grade serous ovarian cancer (HGS-OvCa) is not well understood. We used The Cancer Genome Atlas (TCGA) data to study the clinical relevance of epigenetic modifications of Notch superfamily genes.
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Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment.
Blood
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The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.
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Surface Adjustment of Reverse Phase Protein Arrays using Positive Control Spots.
Cancer Inform
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Reverse phase protein arrays (RPPA) measure the relative expression levels of a protein in many samples simultaneously. Observed signal from these arrays is a combination of true signal, additive background, and multiplicative spatial effects. Background subtraction alone is not sufficient to remove all nonbiological trends from the data. We developed a surface adjustment that uses information from positive control spots to correct for spatial trends on the array beyond additive background. This method uses a generalized additive model to estimate a smoothed surface from positive controls. When positive controls are printed in a dilution series, a nested surface adjustment performs an intensity-based correction. When applicable, surface adjustment is able to remove spatial trends and increase within slide replicate agreement better than background subtraction alone as demonstrated on two sets of arrays. This work demonstrates the importance of including positive control spots on the array.
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Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures.
Proteome Sci
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Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.