A Systematic Screen for CDK4/6 Substrates Links FOXM1 Phosphorylation to Senescence Suppression in Cancer Cells

Abstract

SUMMARY Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry, and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition.

Modification of the DNA Damage Response by Therapeutic CDK4/6 Inhibition

“…Considering the well-documented mechanism of action of CDK4/6 inhibitors through the RB pathway, it is unclear whether a cytostatic agent such as PD0332991 would ultimately function cooperatively or antagonistically with cytotoxic chemotherapies. Recently, studies have demonstrated an importance for CDK4/6 activity in cellular senescence mediated by FOXM1 (31). While this systematic screen identified the first potentially relevant targets for CDK4/6 inhibition outside of the RB protein, FOXM1 protein levels were found to be regulated in an RB-dependent manner within our model systems (supplemental Fig.…”

Tumor suppressor p16 INK4A is necessary for survival of cervical carcinoma cell lines

“…An unbiased screen for CDK4 and CDK6 substrates yielded 68 potential phosphorylation targets, in addition to pRB, p107, and p130 (34). Several of these putative substrates, including FOXM1 and SMAD3, have been implicated in senescence signaling (34)(35)(36). Thus, the tumor-suppressive activities of CDK4/6 in pRB/p107/p130-defective cells may be mediated through one or several of these, or involve as yet unidentified pathways (Fig.…”

“…Our results, however, suggest that there are other relevant CDK4/CDK6 substrates that need to be retained in an unphosphorylated state when pRB is inactivated, and that continued p16 INK4A expression is required to quench their aberrant phosphorylation in such cells. An unbiased screen for CDK4 and CDK6 substrates yielded 68 potential phosphorylation targets, in addition to pRB, p107, and p130 (34). Several of these putative substrates, including FOXM1 and SMAD3, have been implicated in senescence signaling (34)(35)(36).…”

Targeting CDK6 in cancer: State of the art and new insights

“…Multisite phosphorylation by CDK4/6-cyclin D complexes has been shown to both stabilize and activate FOXM1. 17 Similarly, phosphorylation of SMAD2 and SMAD3 results in the elimination of the activation function of a SMAD2/3/4 trimer such as the expression of p15 and p21. 18 CDK6, but not CDK4, binds to EYA2 protein to reduce its half-life.…”