Building a human kinase gene repository: Bioinformatics, molecular cloning, and functional validation

Self Cite

Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones, which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen.functional screen | estrogen receptor T he two thirds of women with estrogen receptor-(ER) or progesterone receptor-positive breast cancers are excellent candidates for antihormone therapy. Selective ER modulators (SERMs), like tamoxifen, block ER activation and have impacted both therapy and survival. However, the success of tamoxifen therapy is limited by intrinsic and acquired drug resistance. Several pathways have been implicated in antiestrogen resistance, including: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway, which is implicated in cell survival; the EGFR family; and the RAS/RAF/MEK1/2/ERK1/2 family, which regulate cell proliferation (1, 2). Loss of ER expression or function may also be an important mechanism of de novo resistance to tamoxifen, either through relatively rare ER mutations or changes in coactivators and corepressors (3).Several groups have used gene-expression analysis to identify genes regulated through ER (4) that are affected by SERMs in breast cancer cells (5, 6). Others have used tumor samples to develop gene signatures that can predict clinical responses to tamoxifen (7-10). Genetic strategies have also been used to identify genes that drive tamoxifen resistance. Receptor tyrosine kinases and MAPK signaling were detected using expression of pooled cDNA libraries in ZR-75-1, an approach often biased toward the most abundantly expressed genes and which requires recovery of hits by PCR (11). The analysis of antiestrogen-sensitive and -resistant MCF7 cells by SNP and comparative genomic hybridization pointed to changes in protein abundance rather than somatic genomic changes (12). An RNA interference screen of kinases identified CDK10, CRK7, and MAP2K7, whose knockdown cause tamoxif...

“…Kinase plasmids were fully sequence verified and validated (14). DNA preparation, transfections, and virus preparation have been described previously (15).…”

Section: Methodsmentioning

confidence: 99%

Section: Tamrmentioning

confidence: 99%

High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy

González-Malerva

Park

Zou

et al. 2011

Self Cite

“…The kinase cDNA library has been described elsewhere (36), and kinases were expressed from a retroviral vector based on the Moloney murine leukemia virus. The kinase shRNA library used for screens is a subset of the lentiviral shRNA library in the LKO.1 vector from the RNAi Consortium.…”

Section: Methodsmentioning

confidence: 99%

High-throughput screens in diploid cells identify factors that contribute to the acquisition of chromosomal instability

Conery

Harlow²

2010

Self Cite

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors, but the mechanisms by which a stable diploid cell loses the ability to maintain genomic integrity are not well characterized. We have approached this critical issue through the use of high-throughput screens in untransformed diploid epithelial cells. In a screen of a cDNA library, we identified 13 kinases whose overexpression leads to increased ploidy. In a series of shRNA screens, we identified 16 kinases whose loss leads to increased ploidy. In both cDNA and shRNA screens, the majority of hits have not been linked previously to genomic stability. We further show that sustained loss of the shRNA screening hits leads to multipolar spindles and heterogeneous chromosome content, two characteristics of chromosomal instability. Loss of several of the kinases leads to loss of contact inhibition and to anchorage-independent growth, vital traits acquired during tumor development. We anticipate that this work will serve as a template for the comprehensive identification of pathways whose dysregulation can drive tumorigenesis through impaired karyotypic maintenance.aneuploidy | kinases | RNAi screen | tumorigenesis G enomic instability promotes heterogeneity within a population of developing tumor cells, allowing for more rapid adaptation and acquisition of characteristics favorable for tumor development. Chromosomal instability is one type of genomic instability involving impaired maintenance of chromosome structure and number. Aneuploidy, defined here as an abnormal number of chromosomes, is a manifestation of chromosomal instability and was identified over 100 ago by von Hansemann (1). Aneuploidy is now known to characterize the majority of solid tumors, and chromosomal instability likely represents a common mechanism by which nascent tumor cells acquire beneficial mutations (2).If the acquisition of chromosomal instability and the resulting aneuploidy represent important steps in the development of many solid tumors, understanding how a chromosomally stable diploid cell becomes aneuploid is a critical issue. Evidence from in vitro and in vivo studies suggests that one route leading from diploidy to aneuploidy passes through tetraploidy (3-5). Little is known, however, about the events that result in tetraploidy.Here, we use cDNA and shRNA screens of human kinases in immortal but untransformed human diploid epithelial cells to look for proteins involved in the generation of tetraploidy and subsequent chromosomal instability. By approaching the question of chromosomal instability from the direction of a diploid cell, we hope to expand on screens that have been carried out in aneuploid tumor cells (6-11). This will allow us to follow in a controlled manner the progression from diploid to tetraploid to aneuploid. From these screens, we have identified a number of kinases whose overexpression or loss leads to increased ploidy. Further characterization reveals that certain o...

“…These vectors enable the rapid and efficient in-frame transfer of coding sequences from master vectors to virtually any protein expression vector in a single conservative biochemical step, allowing the transfer of entire libraries of genes and enabling the broadest possible range of experimentation (19). The FLEXGene effort has included large collections of human kinases and breast cancer-related genes (26,28), complete genome collections for Saccharomyces cerevisiae (22), Yersinia pestis, and Francisella tularensis (29), as well as a collection for Pseudomonas aeruginosa (30), which are available at http:// plasmid.hms.harvard.edu, as will be this collection.…”

mentioning

confidence: 99%

“…Assembling such clones requires automated and highly quality-controlled processes, followed by careful sequence analysis and evaluation to eliminate any inappropriate clones (e.g., truncation mutations). We and others have developed robust pipelines for the production of fully sequence-verified, genome-scale plasmid collections of protein coding sequences for a variety of organisms (19)(20)(21)(22)(23)(24)(25)(26)(27). In this context, we have initiated the full length expression-ready gene collection (FLEXGene), which comprises plasmid clones bearing complete ORFs situated in recombinational cloning vectors.…”

mentioning

confidence: 99%

Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae

Rolfs¹,

Montor²,

Yoon

et al. 2008

Self Cite

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae O1 biovar eltor str. N16961: a completegenome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, Ϸ15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications.cholera ͉ functional proteomics ͉ immunity ͉ ORF clones ͉ protein microarray