Sci. STKE, 21 June 2005
CANCER Screening for Tumor Suppressors
Two separate groups, Westbrook et al. and Kolfschoten et al., used RNA interference (RNAi)-based screening in human cell lines to identify novel tumor suppressor genes. Westbrook et al. infected immortalized human mammary epithelial cells (TLM-HMECs) with a retroviral library of short-hairpin RNAs (shRNAs) in which each shRNA was tagged with a unique 60-nucleotide sequence (DNA "bar code") and looked for clones that showed anchorage-independent growth (indicative of malignant transformation). Combining sequencing of the proviral shRNA of individual clones with bar-code microarray analysis of pooled clones, the authors identified genes associated with formation of anchorage-independent colonies. Transforming growth factor-β receptor II (TGF-βRII), a known tumor suppressor, was identified in the screen, and shRNA knockdown of the tumor suppressor PTEN also elicited anchorage-independent cell growth. Array-based comparative genomic hybridization analysis indicated that two genes identified through RNAi screening, TGFBR2 and REST (for RE1-silencing transcription factor, also known as NRSF for neuron-restrictive silencing factor, a transcriptional repressor), were frequently deleted in colorectal cancers. Microdeletions involving REST were found in a primary colon cancer and a colon cancer cell line, and further analysis revealed a dominant-negative REST mutant in another colon cancer line. REST knockdown promoted signaling through the phosphoinositide 3-kinase (PI3K) pathway, whereas expression of a dominant-negative form of the PI3K regulatory subunit inhibited REST-shRNA-induced transformation. Thus, the authors conclude that REST is a good candidate for a tumor suppressor that may act by suppressing PI3K signaling.
Using a similar approach, Kolfschoten et al. infected immortalized human fibroblasts that can be transformed by oncogenic Ras with a retroviral RNAi library. Among the genes identified as targets of RNAi whose knockdown could substitute for oncogenic RAS signaling was the homeodomain pituitary transcription factor PITX1. PITX1 knockdown enhanced RAS signaling without affecting RAS abundance and produced a phenotype similar to that seen with overexpression of activated RAS. PITX1 expression was reduced compared with control tissues in human prostate and bladder cancers as well as in colon cancers that expressed wild-type RAS. When PITX1 was expressed in Caco-2 cells (a colon cancer line with reduced endogenous abundance of PITX1), it inhibited cell proliferation, growth in soft agar, and tumorigenicity in nude mice. Sequence analysis indicated that the promoter of the RAS-GAP (GTPase activating protein) RASAL1 contained a PITX1 binding site, and chromatin immunoprecipitation analysis indicated that PITX1 associated with the RASAL1 promoter. Transfection of human embryonic kidney (HEK) 293 cells with PITX1 enhanced RASAL1 mRNA abundance, whereas PITX1 knockdown reduced RASAL1 mRNA expression. Moreover, RASAL1 knockdown antagonized a reduction in ERK (extracellular signal-regulated kinase) phosphorylation produced by PITX1 overexpression and itself stimulated anchorage-independent cell growth. Thus, PITX1 appears to function as a tumor suppressor that acts through RASAL1 to repress RAS signaling. Downward discusses both articles in a Preview.
T. F. Westbrook, E. S. Martin, M. S. Schlabach, Y. Leng, A. C. Liang, B. Feng, J. J. Zhao, T. M. Roberts, G. Mandel, G. J. Hannon, R. A. DePinho, L. Chin, S. J. Elledge, A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837-848 (2005). [PubMed]
I. G. M. Kolfschoten, B. van Leeuwen, K. Berns, J. Mullenders, R. L. Beijersbergen, R. Bernards, P. M. Voorhoeve, R. Agami, A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849-858 (2005). [PubMed]
J. Downward, RNA interference libraries prove their worth in hunt for tumor suppressor genes. Cell 121, 813-815 (2005). [PubMed]
Citation: Screening for Tumor Suppressors. Sci. STKE 2005, tw226 (2005).
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