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1.  The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling 
Molecular and Cellular Biology  2013;33(16):3343-3353.
Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription. CTBP2 suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.
doi:10.1128/MCB.01213-12
PMCID: PMC3753894  PMID: 23775127
2.  MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling 
Cell  2012;151(5):937-950.
SUMMARY
Inhibitors of the ALK and EGF receptor tyrosine kinases provoke dramatic but short-lived responses in lung cancers harboring EML4-ALK translocations or activating mutations of EGFR, respectively. We used a large-scale RNAi screen to identify MED12, a component of the transcriptional MEDIATOR complex that is mutated in cancers, as a determinant of response to ALK and EGFR inhibitors. MED12 is in part cytoplasmic where it negatively regulates TGF-βR2 through physical interaction. MED12 suppression therefore results in activation of TGF-βR signaling, which is both necessary and sufficient for drug resistance. TGF-β signaling causes MEK/ERK activation, and consequently MED12 suppression also confers resistance to MEK and BRAF inhibitors in other cancers. MED12 loss induces an EMT-like phenotype, which is associated with chemotherapy resistance in colon cancer patients and to gefitinib in lung cancer. Inhibition of TGF-βR signaling restores drug responsiveness in MED12KD cells, suggesting a strategy to treat drug-resistant tumors that have lost MED12.
doi:10.1016/j.cell.2012.10.035
PMCID: PMC3672971  PMID: 23178117
3.  ZNF423 Is Critically Required for Retinoic Acid-Induced Differentiation and Is a Marker of Neuroblastoma Outcome 
Cancer cell  2009;15(4):328-340.
SUMMARY
Retinoids play key roles in differentiation, growth arrest and apoptosis and are increasingly used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Using a large-scale RNA interference-based genetic screen we identify ZNF423 (also known as Ebfaz, OAZ or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARα/RXRα nuclear receptor complex and is essential for transactivation in response to retinoids. Down-regulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome of neuroblastoma patients.
SIGNIFICANCE
Cancer biomarkers make it possible to foretell cancer outcome (prognosis) or responses to therapy (prediction). Human neuroblastoma is the most common childhood solid tumor with a broad range of clinical outcomes, ranging from spontaneous regression to extremely aggressive disease. We show here that ZNF423 is a prognostic biomarker for human neuroblastoma independent of MYCN amplification. We also establish here a causal role of ZNF423 in RA-induced differentiation and proliferation of neuroblastoma cells. Therefore, ZNF423 may also predict responses to RA-based therapies in the clinic. More generally, our results underscore that the identification of novel components of key signaling pathways using genetic screens can yield biomarkers having clinical utility.
doi:10.1016/j.ccr.2009.02.023
PMCID: PMC2693316  PMID: 19345331
Retinoic acid receptor; differentiation; neuroblastoma; RNAi
4.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
doi:10.1038/nmeth.1351
PMCID: PMC2789971  PMID: 19644458
5.  PI3K Hyperactivation Results in Lapatinib Resistance that is Reversed by the mTOR/PI3K Inhibitor NVP-BEZ235 
Cancer research  2008;68(22):9221-9230.
Small molecule inhibitors of HER2 are clinically active in women with advanced HER2 positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach we performed a genome wide loss-of-function shRNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumour suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we demonstrate that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that PI3K induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance which can be effectively reversed by NVP-BEZ235.
doi:10.1158/0008-5472.CAN-08-1740
PMCID: PMC2587064  PMID: 19010894
Breast cancer; lapatinib; barcode screen; PI3K pathway; PI3K inhibitors
6.  A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway 
PLoS ONE  2009;4(3):e4798.
Background
The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function.
Results
We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21CIP1.
Conclusions
We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer.
doi:10.1371/journal.pone.0004798
PMCID: PMC2653142  PMID: 19277210
7.  The Histone Demethylase Jarid1b (Kdm5b) Is a Novel Component of the Rb Pathway and Associates with E2f-Target Genes in MEFs during Senescence 
PLoS ONE  2011;6(9):e25235.
Senescence is a robust cell cycle arrest controlled by the p53 and Rb pathways that acts as an important barrier to tumorigenesis. Senescence is associated with profound alterations in gene expression, including stable suppression of E2f-target genes by heterochromatin formation. Some of these changes in chromatin composition are orchestrated by Rb. In complex with E2f, Rb recruits chromatin modifying enzymes to E2f target genes, leading to their transcriptional repression. To identify novel chromatin remodeling enzymes that specifically function in the Rb pathway, we used a functional genetic screening model for bypass of senescence in murine cells. We identified the H3K4-demethylase Jarid1b as novel component of the Rb pathway in this screening model. We find that depletion of Jarid1b phenocopies knockdown of Rb1 and that Jarid1b associates with E2f-target genes during cellular senescence. These results suggest a role for Jarid1b in Rb-mediated repression of cell cycle genes during senescence.
doi:10.1371/journal.pone.0025235
PMCID: PMC3181323  PMID: 21980403

Results 1-7 (7)