Abstract
Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.
Highlights
Introduction of mutant Ras into triple knock-out (TKO)mouse embryo fibroblasts (MEFs) leads to superinduction of ZEB1, to a level well beyond that seen in TKO MEFs
Induction of ZEB1 Is Required for Cell Immortalization and Loss of Contact Inhibition When the Rb1 Pathway Is Mutated— Rb1-E2F binds and represses the ZEB1 promoter, ZEB1 is induced in Rb1Ϫ/Ϫ MEFs, and it is further induced when all three Rb1 family members are mutated to generate TKO MEFs (Fig. 1A) [5, 6, 16]
We demonstrate two sequential inductions of ZEB1, the first resulting from mutation of Rb1 and the second from Ras mutation
Summary
Ras mutation drives tumor initiation as well as invasion. Results: The ZEB1 transcription factor is sequentially induced with mutation of Rb1 and Ras, and these inductions are required for Ras-mediated tumor initiation and invasion. ZEB1 acts in a negative feedback loop to repress expression of the miR-200 family, which has a key role in tumor invasion and metastasis [14, 18, 19]; ZEB1 is required to maintain repression of cell cycle inhibitory cdk inhibitors, and its down-regulation in ZEB1ϩ/Ϫ and ZEB1Ϫ/Ϫ MEFs leads to a ZEB1 concentration-dependent induction of these cdk inhibitors and premature senescence [11, 20]. The Rb1 pathway constitutively represses cdk inhibitors, and we show that induction of ZEB1 when this pathway is lost is required to maintain repression of these inhibitors, allowing for the classic immortalization and loss of cell contact inhibition seen with Rb1 pathway mutation in cultured MEFs. Importantly, we demonstrate that the block in senescence resulting from this induction of ZEB1 is required subsequently for Ras-initiated tumor formation in nude mice. When Ras is mutated, ZEB1 is further induced, and this induction by Ras is required to reach a threshold of ZEB1 sufficient to cause repression of miR-200, which we link to Ras-mediated tumor invasion
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
