Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states

Significance Population heterogeneity can make the treatment of tumors more challenging. Whereas a therapeutic agent may be effective against one fraction of a population, it may be less effective against another fraction. Although heterogeneity can be genetic and attributed to mutations, there can also be nongenetic heterogeneity, where a clonal population can harbor distinct subpopulations. Here, we identified a single gene, p21, that was responsible for population heterogeneity in cell cycle activity and explain that this heterogeneity can arise from regulatory relationships of p21 with Cyclin-dependent kinase 2 (CDK2) and E3 ubiquitin ligases. We suggest that, instead of using CDK inhibitors (CKIs) in cancer therapy, CKIs themselves should be targeted. Given concurrently with chemotherapy agents, CKI inhibitors would reduce tumor heterogeneity and thus increase chemotherapy efficacy. Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states—quiescence and cell cycling—can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21–CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.