Additional file 2 of Identification and targeting of selective vulnerability rendered by tamoxifen resistance
Additional file 2:Table S1. Patient's information for survival, Immunofluorescence, and qPCR.... more Additional file 2:Table S1. Patient's information for survival, Immunofluorescence, and qPCR. Table S2. Patient's information for microarray analysis.
Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, ... more Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annota-tion. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA se-quences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annota-tion changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, compari-son and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-...
Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a dire... more Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with imp...
Background The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast ca... more Background The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. Experimental design To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. Results Here, for the first time, we provide an ample ...
Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial ste... more Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis, and transcriptomics to identify a common MoA for the anticancer compounds RITA, aminoflavone (AF), and oncrasin-1 (Onc-1). Global thermal proteome profiling revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability, transcriptional addiction. This led to the preferential loss of expression of oncogenes involved in PDGF, EGFR, VEGF, insulin/IGF/MAPKK, FGF, Hedgehog, TGFβ, and PI3K signaling pathways. Increased reactive oxygen species level in cancer cells was a prerequisite for targeting the mRNA transcription machinery, thus conferring cancer selectivity to these compounds. Furthermore, DNA repair factors involved in homologous recombination were amon...
For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. The... more For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs. To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner. This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced. The resulting method, called double-mismatch allelespecific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test. Various methods are available for single nucleotide polymorphism (SNP) genotyping, each with their benefits and limitations. While SNP genotyping on a genome-scale is currently dominated by microarray analysis or massively parallel sequencing, assessment of a targeted selection of SNPs (or single nucleotide variants) is mainly done through PCR. Restriction fragment length polymorphism (RFLP) 1 is possibly the oldest and simplest method of this type relying on restriction digest followed by size separation of the differential fragments. PCR based methods, including high-resolution melting (HRM) 2,3 and real-time PCR using hydrolysis probes 4 are faster and less laborious. Although these PCR based methods can be performed in high-throughput, are amenable to automation, and generate data that is relatively easy to analyze, a major drawback is the dependency on probes-either labeled or not-increasing design complexity and cost, especially when the number of samples to be genotyped is low. As an alternative, we present a cost-effective quantitative PCR (qPCR) based genotyping assay consisting of two parallel qPCR reactions, each including a modified allele-specific (AS) forward and common wild-type (WT) reverse primer (or vice versa). The combined information of quantification cycle values (Cq) from the two allele-specific reactions enables robust and straightforward genotyping. The simple setup avoids the necessity for probes, dedicated software and specialized instrumentation (and accompanying software) such as a high-resolution melting or sequencing instrument. Our method gives accurate genotyping results with all types of SNPs across a wide range of DNA input concentrations. Allele-specific qPCR assay design is available through our in-house developed primerXL web tool (www.primerxl.org) to increase adoption throughout the community.
Neuroblastoma is one of the most common and most deadly childhood tumors. There is an unmet need ... more Neuroblastoma is one of the most common and most deadly childhood tumors. There is an unmet need to develop new therapeutic modalities for this malignancy that preferentially should be guided by our increasing knowledge of the biology of neuroblastoma. Proliferation and survival of neuroblastoma cells is critically dependent on suppression of the activity of the tumor suppressor protein p53, which is often mediated by increased activity of the MDM2 oncoprotein. Accordingly, small-molecule inhibitors of the interaction between MDM2 and p53 may provide a useful therapeutic option for the treatment of neuroblastoma by restoring the potent antitumor activity of wild-type p53. One of the most promising classes of selective inhibitors of the p53-MDM2 interaction are the nutlins, which have been extensively studied over the last years in several tumor types, including neuroblastoma. We discuss here preclinical data that support the notion that nutlin drugs may offer therapeutic benefit for children with neuroblastoma, on condition that wild-type p53 is present.
The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 pub... more The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression ... more Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data. Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for quantifying gene transcription. In order to accurately and reproducibly compare mRNA concentrations across different samples, it is essential to normalize the raw data 1. The preferred normalization strategy is to use expression levels of reference genes that are stably expressed in the conditions used in the study 2. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines highlight the importance of properly selecting and validating reference genes for qPCR normalization and suggest testing multiple candidate reference genes for a specific experimental design, followed by using the geometric mean of an experimentally determined optimal number of genes (2 or more) with the most stable expression 1,3. Failure to validate the applied reference genes is likely to result in misinterpretation of the data 3. To date, commercial "endogenous control gene panels" for validation of RT-qPCR mouse reference genes are available. These panels comprise the most commonly used reference genes in mouse research and these genes are considered to be generally stable under a wide variety of circumstances (e.g. across different tissues, mouse strains, experimental conditions, etc.). Various reports, however, have shown that the most popular reference
Inhibition of CDK4/6 as a novel therapeutic option for neuroblastoma
Cancer Cell International, 2015
Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than... more Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than 1 year of age. We hypothesized that aberrations in genes that control the cell cycle could play an important role in the pathogenesis of neuroblastoma and could provide a tractable therapeutic target. In this study, we screened 131 genes involved in cell cycle regulation at different levels by analyzing the effect of siRNA-mediated gene silencing on the proliferation of neuroblastoma cells. Marked reductions in neuroblastoma cellular proliferation were recorded after knockdown of CCND1 or PLK1. We next showed that pharmacological inhibition of cyclin D1 dependent kinases 4/6 (CDK4/6) with PD 0332991 (palbociclib) reduced the growth of neuroblastoma cell lines, induced G1 cell cycle arrest, and inhibited the cyclin D1-Rb pathway. Selective inhibition of CDK4/6 using palbociclib may provide a new therapeutic option for treating neuroblastoma.
Restoration of the antitumor activity of p53 could offer a promising approach for the treatment o... more Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells. Genome-wide miRNA expression analysis showed that miR-34a-5p, miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p are upregulated following nutlin-3 treatment in a p53 dependent manner. The function of miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p was analyzed by ectopic overexpression of miRNA mimics. We observed that these p53-regulated miRNAs inhibit the proliferation of neuroblastoma cells to varying degrees, with the most profound growth inhibition recorded for miR-182-5p. Overexpression of miR-182-5p promoted apoptosis in some neuroblastoma cell lines and induced neuronal differentiati...
The selection and validation of stably expressed reference genes is a critical issue for proper R... more The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.
Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging fr... more Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis. Patients and Methods: We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays. Results and Conclusion: We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8
Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative po... more Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T &am... more While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma.
Additional file 2 of Identification and targeting of selective vulnerability rendered by tamoxifen resistance
Additional file 2:Table S1. Patient's information for survival, Immunofluorescence, and qPCR.... more Additional file 2:Table S1. Patient's information for survival, Immunofluorescence, and qPCR. Table S2. Patient's information for microarray analysis.
Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, ... more Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annota-tion. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA se-quences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annota-tion changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, compari-son and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-...
Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a dire... more Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with imp...
Background The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast ca... more Background The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. Experimental design To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. Results Here, for the first time, we provide an ample ...
Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial ste... more Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis, and transcriptomics to identify a common MoA for the anticancer compounds RITA, aminoflavone (AF), and oncrasin-1 (Onc-1). Global thermal proteome profiling revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability, transcriptional addiction. This led to the preferential loss of expression of oncogenes involved in PDGF, EGFR, VEGF, insulin/IGF/MAPKK, FGF, Hedgehog, TGFβ, and PI3K signaling pathways. Increased reactive oxygen species level in cancer cells was a prerequisite for targeting the mRNA transcription machinery, thus conferring cancer selectivity to these compounds. Furthermore, DNA repair factors involved in homologous recombination were amon...
For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. The... more For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs. To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner. This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced. The resulting method, called double-mismatch allelespecific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test. Various methods are available for single nucleotide polymorphism (SNP) genotyping, each with their benefits and limitations. While SNP genotyping on a genome-scale is currently dominated by microarray analysis or massively parallel sequencing, assessment of a targeted selection of SNPs (or single nucleotide variants) is mainly done through PCR. Restriction fragment length polymorphism (RFLP) 1 is possibly the oldest and simplest method of this type relying on restriction digest followed by size separation of the differential fragments. PCR based methods, including high-resolution melting (HRM) 2,3 and real-time PCR using hydrolysis probes 4 are faster and less laborious. Although these PCR based methods can be performed in high-throughput, are amenable to automation, and generate data that is relatively easy to analyze, a major drawback is the dependency on probes-either labeled or not-increasing design complexity and cost, especially when the number of samples to be genotyped is low. As an alternative, we present a cost-effective quantitative PCR (qPCR) based genotyping assay consisting of two parallel qPCR reactions, each including a modified allele-specific (AS) forward and common wild-type (WT) reverse primer (or vice versa). The combined information of quantification cycle values (Cq) from the two allele-specific reactions enables robust and straightforward genotyping. The simple setup avoids the necessity for probes, dedicated software and specialized instrumentation (and accompanying software) such as a high-resolution melting or sequencing instrument. Our method gives accurate genotyping results with all types of SNPs across a wide range of DNA input concentrations. Allele-specific qPCR assay design is available through our in-house developed primerXL web tool (www.primerxl.org) to increase adoption throughout the community.
Neuroblastoma is one of the most common and most deadly childhood tumors. There is an unmet need ... more Neuroblastoma is one of the most common and most deadly childhood tumors. There is an unmet need to develop new therapeutic modalities for this malignancy that preferentially should be guided by our increasing knowledge of the biology of neuroblastoma. Proliferation and survival of neuroblastoma cells is critically dependent on suppression of the activity of the tumor suppressor protein p53, which is often mediated by increased activity of the MDM2 oncoprotein. Accordingly, small-molecule inhibitors of the interaction between MDM2 and p53 may provide a useful therapeutic option for the treatment of neuroblastoma by restoring the potent antitumor activity of wild-type p53. One of the most promising classes of selective inhibitors of the p53-MDM2 interaction are the nutlins, which have been extensively studied over the last years in several tumor types, including neuroblastoma. We discuss here preclinical data that support the notion that nutlin drugs may offer therapeutic benefit for children with neuroblastoma, on condition that wild-type p53 is present.
The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 pub... more The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression ... more Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data. Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for quantifying gene transcription. In order to accurately and reproducibly compare mRNA concentrations across different samples, it is essential to normalize the raw data 1. The preferred normalization strategy is to use expression levels of reference genes that are stably expressed in the conditions used in the study 2. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines highlight the importance of properly selecting and validating reference genes for qPCR normalization and suggest testing multiple candidate reference genes for a specific experimental design, followed by using the geometric mean of an experimentally determined optimal number of genes (2 or more) with the most stable expression 1,3. Failure to validate the applied reference genes is likely to result in misinterpretation of the data 3. To date, commercial "endogenous control gene panels" for validation of RT-qPCR mouse reference genes are available. These panels comprise the most commonly used reference genes in mouse research and these genes are considered to be generally stable under a wide variety of circumstances (e.g. across different tissues, mouse strains, experimental conditions, etc.). Various reports, however, have shown that the most popular reference
Inhibition of CDK4/6 as a novel therapeutic option for neuroblastoma
Cancer Cell International, 2015
Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than... more Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than 1 year of age. We hypothesized that aberrations in genes that control the cell cycle could play an important role in the pathogenesis of neuroblastoma and could provide a tractable therapeutic target. In this study, we screened 131 genes involved in cell cycle regulation at different levels by analyzing the effect of siRNA-mediated gene silencing on the proliferation of neuroblastoma cells. Marked reductions in neuroblastoma cellular proliferation were recorded after knockdown of CCND1 or PLK1. We next showed that pharmacological inhibition of cyclin D1 dependent kinases 4/6 (CDK4/6) with PD 0332991 (palbociclib) reduced the growth of neuroblastoma cell lines, induced G1 cell cycle arrest, and inhibited the cyclin D1-Rb pathway. Selective inhibition of CDK4/6 using palbociclib may provide a new therapeutic option for treating neuroblastoma.
Restoration of the antitumor activity of p53 could offer a promising approach for the treatment o... more Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells. Genome-wide miRNA expression analysis showed that miR-34a-5p, miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p are upregulated following nutlin-3 treatment in a p53 dependent manner. The function of miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p was analyzed by ectopic overexpression of miRNA mimics. We observed that these p53-regulated miRNAs inhibit the proliferation of neuroblastoma cells to varying degrees, with the most profound growth inhibition recorded for miR-182-5p. Overexpression of miR-182-5p promoted apoptosis in some neuroblastoma cell lines and induced neuronal differentiati...
The selection and validation of stably expressed reference genes is a critical issue for proper R... more The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.
Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging fr... more Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis. Patients and Methods: We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays. Results and Conclusion: We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8
Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative po... more Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T &am... more While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma.
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