Proteomic profiling of Palbociclib arrested cells ± APC/C inactivation.

ABSTRACT: Cellular proliferation is highly regulated to ensure proper tissue homeostasis and to prevent diseases such as cancer. In actively proliferating cells, entry into S phase of the cell cycle and initiation of DNA replication is promoted by inactivation of the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), though the critical S phase-promoting substrates have not been fully elucidated. The APC/C is also active in cells that have exited the cell cycle and entered an arrested state, but whether APC/C activity is essential to maintain arrest is unclear. We found that APC/C inactivation is sufficient to bypass cellular arrest induced by the CDK4/6 inhibitor Palbociclib. To identify potential APC/C substrates responsible for driving the escape from cell cycle arrest, we arrested cells using Palbociclib and then induced EMI1, inactivating the APC/C. Samples were collected at 0, 8, 16 and 20h after arrest and analyzed using proteomics. We found that inactivation of the APC/C promotes cell cycle re-entry by inducing RB phosphorylation and E2F-dependent gene transcription. Stabilization of both cyclin A and cyclin B contributes to arrest bypass, but only cyclin A accumulation is absolutely required. Direct expression of APC/C-resistant cyclin A, but not cyclin B, in arrested cells induces S phase entry analogous to APC/C inactivation. Cells bypassing arrest initiate DNA replication with severely reduced origin licensing, at least in part due to premature geminin accumulation. As a result, cells exhibit reduced rates of DNA synthesis, which leads to the accumulation of replication stress and long-term proliferation defects. Our findings suggest that CDK4/6 inhibition in cancers with reduced APC/C activity may be ineffective at promoting cytostatic cell cycle arrest but may nonetheless lead to elevated levels of replication stress that ultimately leads to a more durable cell cycle withdrawal.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christine Mills  

LAB HEAD: Jean Cook

PROVIDER: PXD046783 | Pride | 2024-07-18

REPOSITORIES: Pride