TGFβ1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of cell growth that targets gene-regulatory events, but also inhibits the function of CDC45-MCM-GINS helicases (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go-Ichi-Ni-San) through multiple mechanisms to achieve cell-cycle arrest. Early in G(1), TGF beta 1 blocks MCM subunit expression and suppresses Myc and Cyclin E/Cdk2 activity required for CMG assembly, should MCMs be expressed. Once CMGs are assembled in late-G(1), TGF beta 1 blocks CMG activation using a direct mechanism involving the retinoblastoma (Rb) tumor suppressor. Here, in cells lacking Rb, TGF beta 1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth arrest remains intact and Smad2/3/4-dependent. Such arrest occurs due to inhibition of MCM hexamer assembly by TGF beta 1, which is not seen when Rb is present and MCM subunit expression is normally blocked by TGF beta 1. Loss of Smad expression prevents TGF beta 1 suppression of MCM assembly. Mechanistically, TGF beta 1 blocks a Cyclin E-Mcm7 molecular interaction required for MCM hexamer assembly upstream of CDC10-dependent transcript-1 (CDT1) function. Accordingly, overexpression of CDT1 with an intact MCM-binding domain abrogates TGF beta 1 arrest and rescues MCM assembly. The ability of CDT1 to restore MCM assembly and allow S-phase entry indicates that, in the absence of Rb and other canonical mediators, TGF beta 1 relies on inhibition of Cyclin E-MCM7 and MCM assembly to achieve cell cycle arrest. Implications: These results demonstrate that the MCM assembly process is a pivotal target of TGF beta 1 in eliciting cell cycle arrest, and provide evidence for a novel oncogenic role for CDT1 in abrogating TGF beta 1 inhibition of MCM assembly.