A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.     

Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and?a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.     

An RNAi-based genetic screen for oxidative stress resistance reveals retinol saturase as a mediator of stress resistance

Oxidative stress has been implicated in the pathogenesis of numerous late-onset diseases as well as organismal longevity. Nevertheless, the genetic components that affect cellular sensitivity to oxidative stress have not been explored extensively at the genome-wide level in mammals. Here we report an RNA interference (RNAi) screen for genes that increase resistance to an organic oxidant, tert-butylhydroperoxide (tert-BHP), in cultured fibroblasts. The loss-of-function screen allowed us to identify several short hairpin RNAs (shRNAs) that elevated the cellular resistance to tert-BHP. One of these shRNAs strongly protected cells from tert-BHP and H(2)O(2) by specifically reducing the expression of retinol saturase, an enzyme that converts all-trans-retinol (vitamin A) to all-trans-13,14-dihydroretinol. The protective effect was well correlated with the reduction in mRNA level and was observed in both primary fibroblasts and NIH3T3 cells. The results suggest a novel role for retinol saturase in regulating sensitivity to oxidative stress and demonstrate the usefulness of large-scale RNAi screening for elucidating new molecular pathways involved in stress resistance.     

Identification of five human novel genes associated with cell proliferation by cell-based screening from an expressed cDNA ORF library

The development of functional profiling technologies provides opportunity for high-throughput functional genomics studies. We describe a cell-based screening system to identify novel human genes associated with cell proliferation. The method integrates luciferase reporter gene activity, fluorescence stain, automated microscopy and cellular phenotype assays. We successfully used the system to screen 409 novel human genes cloned by our lab and found that 27 genes significantly up-regulated promoter-Renilla luciferase reporter plasmid (pRL) activity. Among them, five genes, TRAF3IP3, ZNF306, ZNF250, SGOL1, and ZNF434, were determined through morphological observation, calcein AM fluorescence stain, MTT assay and cell cycle analysis to be associated with cell proliferation. Furthermore, we showed that the gene TRAF3IP3, which initially was identified to specifically interact with TRAF3, stimulated cell growth by modulating the c-Jun N-terminal kinase (JNK) pathway, and RNAi of TRAF3IP3 confirmed that the effect was physiological and necessary. In summary, we integrated a rapid and efficient system for screening novel growth regulatory genes. Using the new screening system we identified five genes associated with cell proliferation for the first time.     

RAB5A基因对肺腺癌细胞微丝的影响

为研究RAB5A基因对肺腺癌细胞中微丝的影响,通过FITC标记的鬼笔环肽对AGZY83-a细胞骨架中的微丝特异染色,利用共聚焦激光扫描显微镜发现RAB5A过表达后微丝束变密。经Superarray肿瘤转移相关基因微芯片分析RAB5A对肿瘤转移相关基因的表达影响．发现了3个与细胞骨架调节相关基因表达发生变化,NM23H1与Rac1的表达受到抑制．同时S100A4的表达增加。以前有研究认为S100A4基因可抑制NM23H1基因表达,为验证NM23H1基因的表达降低是否由于S100肖4表达增高所致,利用RNAi沉默AGZY83-a细胞中S100A4基因的表达,发现NM23H1基因表达增高,由此推断RAB5A基因可能通过上调S100A4基因表达来抑制NM23H1基因表达。     

Duplicated KOX zinc finger gene clusters flank the centromere of human chromosome 10: evidence for a pericentric inversion during primate evolution.

Two related zinc finger (ZNF) gene clusters from the pericentromeric region of human chromosome 10, defined by cDNAs of the KOX series, have been cloned in yeast artificial chromosomes (YACs). The two clusters evolved by duplication of an ancestral gene cluster before the divergence of the human and great ape lineages. Included in cluster A are the ZNF gene sequences ZNF11A, ZNF33A, and ZNF37A, while cluster B comprises the related sequences ZNF11B, ZNF33B and ZNF37B. Genes from both clusters are expressed: cDNAs KOX2, KOX31 and KOX21 derive from ZNF11B, ZNF33A and ZNF37A, respectively. Further YACs have been isolated which link ZNF11A and ZNF33A to another gene, ZNF25, defined by cDNA clone KOX19. Therefore ZNF25 also forms part of cluster A, but has no counterpart in cluster B. Surprisingly, the KOX ZNF gene clusters are located on opposite sides of the centromere: cluster A maps to 10p11.2, while cluster B is in 10q11.2. This suggests the occurrence during primate evolution of a previously undescribed pericentric inversion subsequent to the cluster duplication. The evolution of this subset of KOX ZNF genes has therefore involved three types of genetic event: local gene duplication, gene cluster duplication, and chromosome rearrangement.     

Glyceraldehyde-3-phosphate dehydrogenase mediates anoxia response and survival in Caenorhabditis elegans

Oxygen deprivation has a role in the pathology of many human diseases. Thus it is of interest in understanding the genetic and cellular responses to hypoxia or anoxia in oxygen-deprivation-tolerant organisms such as Caenorhabditis elegans. In C. elegans the DAF-2/DAF-16 pathway, an IGF-1/insulin-like signaling pathway, is involved with dauer formation, longevity, and stress resistance. In this report we compared the response of wild-type and daf-2(e1370) animals to anoxia. Unlike wild-type animals, the daf-2(e1370) animals have an enhanced anoxia-survival phenotype in that they survive long-term anoxia and high-temperature anoxia, do not accumulate significant tissue damage in either of these conditions, and are motile after 24 hr of anoxia. RNA interference was used to screen DAF-16-regulated genes that suppress the daf-2(e1370)-enhanced anoxia-survival phenotype. We identified gpd-2 and gpd-3, two nearly identical genes in an operon that encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. We found that not only is the daf-2(e1370)-enhanced anoxia phenotype dependent upon gpd-2 and gpd-3, but also the motility of animals exposed to brief periods of anoxia is prematurely arrested in gpd-2/3(RNAi) and daf-2(e1370);gpd-2/3(RNAi) animals. These data suggest that gpd-2 and gpd-3 may serve a protective role in tissue exposed to oxygen deprivation.     

Median absolute deviation to improve hit selection for genome-scale RNAi screens

High-throughput screening (HTS) of large-scale RNA interference (RNAi) libraries has become an increasingly popular method of functional genomics in recent years. Cell-based assays used for RNAi screening often produce small dynamic ranges and significant variability because of the combination of cellular heterogeneity, transfection efficiency, and the intrinsic nature of the genes being targeted. These properties make reliable hit selection in the RNAi screen a difficult task. The use of robust methods based on median and median absolute deviation (MAD) has been suggested to improve hit selection in such cases, but mean and standard deviation (SD)-based methods are still predominantly used in many RNAi HTS. In an experimental approach to compare these 2 methods, a genome-scale small interfering RNA (siRNA) screen was performed, in which the identification of novel targets increasing the therapeutic index of the chemotherapeutic agent mitomycin C (MMC) was sought. MAD values were resistant to the presence of outliers, and the hits selected by the MAD-based method included all the hits that would be selected by SD-based method as well as a significant number of additional hits. When retested in triplicate, a similar percentage of these siRNAs were shown to genuinely sensitize cells to MMC compared with the hits shared between SD- and MAD-based methods. Confirmed hits were enriched with the genes involved in the DNA damage response and cell cycle regulation, validating the overall hit selection strategy. Finally, computer simulations showed the superiority and generality of the MAD-based method in various RNAi HTS data models. In conclusion, the authors demonstrate that the MAD-based hit selection method rescued physiologically relevant false negatives that would have been missed in the SD-based method, and they believe it to be the desirable 1st-choice hit selection method for RNAi screen results.