Der pRb/APCCdh1-Komplex: Identifizierung neuer Substrate und Analyse der Regulation
Zusammenfassung der Projektergebnisse
The loss of pRB function is thought to occur during the development of most cancers and is a key event that allows tumor cells to undergo uncontrolled proliferation. pRB is a multifunctional protein, and different binding proteins mediate different aspects of pRB function. Exactly how pRB acts to mediate different phenotypes is unclear. pRB has been reported to associate with a very large number of cellular proteins and has several different cellular activities. Over 230 proteins are shown to physically interact with pRB and many of these have been proposed to be important for it to function. During the course of my project I aimed to identify players of the functional network surrounding pRB. I used shRNA screens for a systematic functional analysis of Kinases (representing signaling pathways) and pRB-associated proteins to find key effectors of pRBs tumor-suppressive action. Over the period of my fellowship I completed shRNA screens that identified kinases that influence the ability of the retinoblastoma tumor suppressor (pRB) to arrest cell proliferation and to establish a senescence-like state in the SaOS2 cells, an osteosarcoma tumor cell line. The results show that the ability of pRB to block tumor cell proliferation is sensitive to the levels of many different kinases. Changes in the expression of these proteins, resulting for example from alteration in gene copy number, may affect the ability of pRB to suppress tumor cell proliferation. Unexpectedly, one of the kinases found to be important for pRB's ability to induce senescence, a form of permanent cell cycle arrest, was LATS2. LATS2 is a component of the HIPPO tumor suppressor pathway and this functional interaction reveals that pRB and LATS2 act synergistically. Mechanistic studies show that pRB and LATS2 converge in the regulation of the E2F transcription factor. pRB-induced senescence in SaOS2 cells requires the DREAM complex, a repressor of E2F-dependent transcription. I found that LATS2 promotes the efficient repression of E2F targets by DREAM. Interestingly, the RB1 and LATS2 genes are physically linked on chromosome 13q and deletions that lead to loss of heterozygosity of both genes are very common in human tumors. These findings suggest that 13q deletions may undermine the synergistic action of LATS2 and pRB, making it difficult for cells to silence E2F-dependent transcription. During the course of my project I assembled a custom collection of shRNAs that targets 230 proteins that have either been reported to associate with pRB for high-throughput screening. I continued the project by looking for pRB interacting proteins that modify pRB's ability to block proliferation, alter S-phase entry and promote differentiation. Those results enable me to most likely link kinases that modified pRB function in the first round of screens directly to pRB. Furthermore, I investigate which interactors are essential for pRB-mediated suppression of proliferation in human pancreatic cancer cells treated with cdk4/6 inhibitors. This specific context of using cdk4/6 inhibitors provide a mechanism to re-activate pRB in an endogenous setting. Overall, my results using the screening approaches mentioned, show that pRB functions in a much larger regulatory network of interactions. Its ability to arrest tumor cells does not depend solely on its intrinsic activity, but is strongly influenced by co-operating pathways and interacting proteins that act in concert with pRB to block cell proliferation.
Projektbezogene Publikationen (Auswahl)
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A kinase shRNA screen links LATS2 and the pRb tumor suppressor pathway. Genes Dev. April 15, 2011 25: 814-830
Tschöp K., Conery A.R., Settleman J., Harlow E., and Dyson, N.
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Identifying players in the functional network around pRB. Cell Cycle. 2011 Nov15;10(22):3814-5
Tschöp K., Dyson N.
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Pumilio facilitates miRNA regulation of the E2F3 oncogene. Genes Dev. 2012 Feb 15;26(4):356-68
Miles WO, Tschöp K., Herr A., Ji, J.-Y.a nd Dyson, N.
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Loss of RBF1 changes glutamine catabolism. Genes Dev. 2013 Jan 15;27(2):182-96
Nicolay B.N., Gameiro P.A., Tschöp K., Korenjak M., Heilmann A.M., Asara J.M., Stephanopoulos G., Iliopoulos O., Dyson N.J.
