Genome scale molecular analysis has revolutionized the experimental process by which the genes and pathways that influence the initiation, progression, and treatment of cancer can be identified. This has opened up many exciting avenues of biomedical research, but it has also served to confirm the genetic and cellular complexity that underlies tumorigenesis. The identification of the gene silencing mechanism, RNA interference (RNAi), initially in invertebrates and later in mammalian cells, has had enormous implications for our understanding of the regulation of gene expression and our ability to modulate it experimentally. This project is focused on the hypothesis that molecular and phenotypic perturbations induced by RNAi will give insight into the biology of cancer and identify novel anti-cancer molecular targets. The intention of most studies exploiting the RNAi mechanism for loss of function (LOF) analysis is the gene-specific cleavage of protein encoding mRNAs. Technologies that exploit the endogenous RNA-based gene silencing mechanism, RNAi, have developed rapidly for the dissection of gene-function relationships and as a means of furthering molecular target analysis. The overall goal of this project is the enhanced application of RNAi-based technologies for the study of cancer biology. To do this we are focusing on establishing protocols and assays for the reproducible assessment of the effects of RNAi at a molecular and functional level that can be used in mammalian cell line model systems appropriate for the study of tumorigenesis. We are applying optimized and robust protocols for inducing RNAi in cells using synthetic siRNAs and other RNAi effectors, such as short hairpin RNAs (shRNAs) and the use of quantitative assays for analyzing the efficacy of RNA. To date we have examined the silencing mediated by RNAi effectors corresponding to several hundred human genes. Following the induction of RNAi we are applying methods that can assay multiple molecular and phenotypic end-points to assess the function of a protein within specific cancer related pathways and at a broader systems level. Methods we employ include multiplex mRNA assays that allow us to study the downstream effects of LOF on specific pathways and whole transcriptome expression profiling (microarray based analysis) that enable us to identify new roles for genes in an unbiased manner. To support this work we have begun to develop and assess bioinformatic tools that can help us to identify non-targeted, but sequence-specific, affects on gene expression following introduction an RNAi effector into a cell. Our molecular based assays are coupled with functional studies chosen on the basis of the putative or defined role of the specific protein under study. During FY13 have been involved in studies that are using RNAi based technologies to (1) study genes required for the survival of ovarian cancer cells, (2) investigate genes associated with susceptibility to prostate cancer, and (3) identify genes required for the survival of pediatric sarcoma. Finally, this project continues to be critical for assisting and training CCR Investigators in the application of RNAi analysis, and for the establishment of protocols for RNAi screens targeting thousands of human genes (see Z01 BC 010615).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010613-10
Application #
8763150
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2013
Total Cost
$311,174
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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Camps, Jordi; Pitt, Jason J; Emons, Georg et al. (2013) Genetic amplification of the NOTCH modulator LNX2 upregulates the WNT/β-catenin pathway in colorectal cancer. Cancer Res 73:2003-13
Tandle, Anita T; Kramp, Tamalee; Kil, Whoon J et al. (2013) Inhibition of polo-like kinase 1 in glioblastoma multiforme induces mitotic catastrophe and enhances radiosensitisation. Eur J Cancer 49:3020-8
Hummon, Amanda B; Pitt, Jason J; Camps, Jordi et al. (2012) Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells. Mol Cancer 11:1
Grade, Marian; Hummon, Amanda B; Camps, Jordi et al. (2011) A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets. Int J Cancer 128:1069-79
Martin, S E; Wu, Z-H; Gehlhaus, K et al. (2011) RNAi screening identifies TAK1 as a potential target for the enhanced efficacy of topoisomerase inhibitors. Curr Cancer Drug Targets 11:976-86
Mackiewicz, Mark; Huppi, Konrad; Pitt, Jason J et al. (2011) Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA. Breast Cancer Res Treat 130:663-79
Murrow, Lyndsay M; Garimella, Sireesha V; Jones, Tamara L et al. (2010) Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome. Breast Cancer Res Treat 122:347-57
Zhang, Yong-Wei; Jones, Tamara L; Martin, Scott E et al. (2009) Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response. J Biol Chem 284:18085-95
Ryan, Michael C; Zeeberg, Barry R; Caplen, Natasha J et al. (2008) SpliceCenter: a suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi, microarray, and peptide-based studies. BMC Bioinformatics 9:313