The ZEB1 Transcription Factor Acts in a Negative Feedback Loop with miR200 Downstream of Ras and Rb1 to Regulate Bmi1 Expression

Yongqing Liu,Ester Sánchez-Tilló,Xiaoqin Lu,Li Huang,Brian Clem,Sucheta Telang,Alfred B Jenson,Miriam Cuatrecasas,Jason Chesney,Antonio Postigo,Douglas C Dean

doi:10.1074/jbc.m113.533505

Abstract

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Highlights

Oncogenic Ras muations in cancer cells cause tumor-suppressing senescence in primary cells through repression of Bmi1
ZEB1 Is Repressed and miR-200 Is Induced by Mutant Ras in mouse embryo fibroblasts (MEFs)—Mutant Ras has been shown to induce expression of ZEB1 through activation of Raf and in turn Erk2 [6], and such induction of ZEB1 has been linked to epithelial-mesenchymal transcription factor (EMT) and tumor invasion in cancer cells [3,4,5]. miR-200 is tightly linked to ZEB1 in a mutual repression loop [4], and miR-200 is repressed by mutant Ras in cancer cells [3, 24, 25]
In contrast to these previously published findings in cancer cells, we found that mutant Ras caused repression of ZEB1 and induction of miR-200 in primary cultures of MEFs (Fig. 1B)

Summary

Background

Oncogenic Ras muations in cancer cells cause tumor-suppressing senescence in primary cells through repression of Bmi. Beyond its classic role in EMT, ZEB1 expression is closely linked to cell proliferation in vivo [26], and we found previously that heterozygous mutation of ZEB1 is sufficient for induction of cdki and premature senescence of MEFs in culture [26] As noted above, this is the same phenotype observed with Bmi mutation [9, 10], further suggesting that Bmi might be an important downstream target of ZEB1. In cancer cells where dominant repression by the Rb1 pathway is lost, Ras induces ZEB1 to cause repression of miR-200 and in turn induction of Bmi1 These results place Rb1 pathway status and ZEB1 in unanticipated roles as key downstream effectors that determine whether Bmi is repressed by mutant Ras, leading to oncogene-induced senescence, or induced, leading to tumor progression

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION