The focus of my research group is on the application of innovative functional genomics tools to identify novel genes that have a role in the biology of cancer. We use both high-throughput loss-of-function RNA interference genetic screens and gain-of-function genetic screens with retroviral cDNA expression libraries to identify novel components of cancer-relevant pathways and genes that modulate cellular responses to cancer drugs.
Loss-of-function genetic screens
We have generated tens of thousands short hairpin RNA vectors to silence the majority of human and murine genes through RNA interference. Moreover, we have generated a number of “gene family” knockdown libraries, in which all members of a gene family are individually targeted for suppression by shRNA vectors. One particular focus has been the family of de-ubiquitinating enzymes (DUBs), which act as antagonists of ubiquitin ligases to remove ubiquitin moieties from proteins. Using this DUB knockdown library, we have identified the cylindromatosis tumor suppressor gene (CYLD) as a regulator of the anti apoptotic transcription factor NF-kB and USP1 as a regulator of the Fanconi anemia D2 protein (FANCD2).
Identification of drug response biomarkers through functional genetic screens
A). Gain-of-function genetic screens. Collections of cDNAs are ectopically expressed in drug-sensitive cells, either in polyclonal format or in an arrayed format in multi-well plates, often through use of a viral vector system (retroviral, lentiviral, or adenoviral). Cells are exposed to a cancer drug and resistant cells will continue to proliferate. From drug-resistant colonies, cDNAs can be recovered, identified by sequence analysis, and re-tested for their ability to confer drug resistance.
B). Short hairpin RNA (shRNA) barcode loss-of-function genetic screens. Collections of shRNA vectors are expressed polyclonally in drug-sensitive cells and subjected to drug selection. Cells harboring an shRNA vector that confers drug resistance will become enriched in the population, shRNAs that enhance the sensitivity to a cancer drug will become depleted under drug selection compared to a reference population that is not exposed to drug.
Each shRNA vector contains a unique identifier sequence (the barcode), which can be recovered by polymerase chain reaction (PCR) and its abundance quantified on a dedicated DNA microarray containing the barcode sequences.
shRNAs that cause drug resistance are enriched and appear red on the microarray, depleted shRNAs appear green.
Gain-of-function genetic screens
We use retroviral cDNA expression libraries to identify novel genes that act in pathways, which are frequently deregulated in human cancer. In short, these genetic screens involve the infection of a cell population with a high-complexity retroviral cDNA expression library, selection of cells with altered phenotype, followed by identification of the cDNA responsible for the phenotype. In the past years, we have successfully used this approach to identify novel genes that act in several cancer-relevant pathways and to identify biomarkers of drug resistance.
Prahallad, A., Sun, C., Huang, S., Di Nicolantonio, F., Salazar, R., Zecchin, D., Beijersbergen, R.L., Bardelli, A., and Bernards, R. (2012). Unresponsiveness to BRAF(V600E) inhibition of colon cancer through feedback activation of EGFR. Nature 483, 100-103.
Epping, M.T., Meijer, L.A.T., Krijgsman, O., Bos, J.L., Pandolfi, P.P., and Bernards, R. (2011). TSPYL5 suppresses p53 levels and function by physical interaction with USP7. Nature Cell Biol. 13, 102-108.
Hölzel, M., Huang, S., Koster, J., Øra, I., Lakeman, A., Caron, H., Nijkamp, W., Xie, J., Callens, T., Asgharzadeh, S., Seeger, R.C., Messiaen, L., Versteeg, R., and Bernards, R. (2010). NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell 142, 218-229.
Huang, S., Laoukili, J., Epping, M.T., Koster, J., Holzel, M., Westerman, B.A., Nijkamp, W., Hata, A., Asgharzadeh, S., Seeger, R.C., Versteeg, R., Beijersbergen, R.L., and Bernards, R. (2009). ZNF423 Is Critically Required for Retinoic Acid-Induced Differentiation and Is a Marker of Neuroblastoma Outcome. Cancer Cell. 15, 328-340.
Van de Vijver, M.J., He, Y.D., van ’t Veer, L.J., Dai, H., A M Hart, A.A.M., Voskuil, D., Schreiber, G.J., Peterse, J.L., Roberts, C., Marton, M.J., Parrish, M., Atsma, D., Witteveen, A., Glas, A., Delahaye, L., van der Velde, T., Bartelink, H., Rodenhuis, S., Rutgers,E. Th., Friend, S.H. and Bernards, R.. (2002). A gene expression signature as a predictor of survival in breast cancer. New England J. Med. 347, 1999-2009.
Berns, K., Horlings, H., Hennessy, B.T., Madiredjo, M., Hijmans, E.M., Beelen, K., Linn, S.C., Gonzalez-Angulo, A.M., Stemke-Hale, K., Hauptmann, M., Beijersbergen, R.L., Mills, G.B., van de Vijver, M.J., and Bernards, R. (2007). A functional genetic approach identifies the PI3K pathway as a major determinant of Trastuzumab resistance in breast cancer. Cancer Cell 12, 395-402.
Brummelkamp, T.R., Fabius, A., Mullenders, J., Madiredjo, M., Velds, A., Kerkhoven, R.M., Bernards, R., and Beijersbergen, R.L. (2006). An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nature Chem. Biol. 2, 202-206.
Epping, M.T., Wang, L, Edel, M.J., Hernandez, M, and Bernards, R. (2005). The human tumor antigen PRAME is a dominant repressor of retinoic acid receptor signaling. Cell, 122, 835-847.
Nijman, S.M.B., Huang, T.T., Dirac, A.M.G., Brummelkamp, T.R., Kerkhoven, R.M., D’Andrea, A.D and Bernards, R. (2005). The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol. Cell, 17, 331-339.
Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, Kerkhoven, R. M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beijersbergen, R.L. and Bernards, R. (2004). A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature, 428, 431-437.
Brummelkamp, T.R., Nijman, S.M.B., Dirac, A.M.G., and Bernards, R. (2003). Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kB. Nature, 424, 797-801.
Agami, R., and Bernards, R. (2000). Distinct initiation and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage. Cell 102, 55-66.
Majewski, I., and Bernards, R. (2011). Taming the dragon: Biomarkers to individualize the treatment of cancer. Nature Med. 17, 304-312.
Bernards, R. (2010). It’s diagnostics, stupid. Cell, 141, 13-17
Mullenders, J and Bernards, R. (2009). Loss of function genetic screens as a tool to improve the diagnosis and treatment of cancer, Oncogene, 28, 4409-4420.
Eichhorn, P.J.A., Creyghton, M.P., and Bernards, R. (2009). Protein phosphatase 2A regulatory subunits and cancer. Biochem. Biophys. Acta, 1795, 1-15.
Van ‘t Veer, L.J., and Bernards, R (2008). Enabling personalized cancer medicine through analysis of gene expression patterns. Nature, 452, 564-570.
Bernards, R., Brummelkamp, T.R., and Beijersbergen, R.L. (2006). shRNA libraries and their use in cancer genetics. Nature Methods 3, 701-706.
Rowland, B.D., and Bernards, R. (2006). Re-evaluating cell cycle regulation by E2Fs. Cell 127, 871-874.
Nijman, S.M.B., Luna-Vargas, M.P.A., Velds, A., Brummelkamp, T.R., Dirac, A.M.G., Sixma, T.K., and Bernards, R. (2005). A Genomic and Functional Inventory of Deubiquitinating Enzymes. Cell, 123, 773-786.
Bernards, R. and Weinberg, R.A. (2002). Metastasis genes: A progression puzzle. Nature, 418, 823.
More publications by René Bernards on PubMed
List of relevant websites
Centre for Biomedical Genetics:
René Bernards received his Ph.D. in 1984 from the University of Leiden. He joined the laboratory of Robert Weinberg at the Whitehead Institute in Cambridge, USA for his postdoctoral training. Here, he studied the function of both oncogenes and tumor suppressor genes. He continued this work when he joined the Massachusetts General Hospital Cancer Center as an assistant professor in 1988. In 1992 he was appointed senior staff scientist at the Netherlands Cancer Institute. In 1994 he was appointed part time professor of molecular carcinogenesis at Utrecht University, The Netherlands.
In the last decade, his laboratory has focused on the development of new tools to carry out genome-wide loss-of-function genetic screens to identify novel genes that act in cancer-relevant pathways. In July of 2003 he co-founded “Agendia”, a genomics-based diagnostic company that started offering the first microarray-based diagnostic test for the clinical management of breast cancer (MammaPrint) in 2004. He received several awards for his research, including the Pezcoller Foundation-FECS Recognition for Contribution to Oncology, the Ernst W. Bertner Award for Cancer Research from the M.D. Anderson Cancer Center, the ESMO Lifetime Achievement Award in Translational Research in Breast Cancer and the Spinoza award from the Netherlands Organization for Scientific Research. He is a member of the Royal Netherlands Academy of Arts and Sciences.
Katrien Berns PhD Academic staff
Valentina Gambino Post-doc
Sidong Huang PhD Post-doc
Prasanth Kumar PhD Post-doc
Ian Majewski PhD Post-doc
Lorenza Mittempergher PhD Post-doc
Chong Sun PhD Post-doc
Zheng Xue PhD Post-doc
Floris Groenendijk Graduate Student
Guus Heynen MSc Graduate Student
Anirudh Prahallad Graduate Student
Annemiek Bes-Gennissen Technical staff
Astrid Bosma Technical staff
Winny Grernrum Technical staff
Marielle Hijmans MSc Technical staff