Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial-mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels.

The cell proliferation antigen Ki-67 is widely used in cancer histopathology, but estimations of Ki-67 expression levels are inconsistent and understanding of its regulation is limited. Here we show that cell-cycle regulation underlies variable Ki-67 expression in all situations analyzed, including nontransformed human cells, normal mouse intestinal epithelia and adenomas, human cancer cell lines with or without drug treatments, and human breast and colon cancers. In normal cells, Ki-67 was a late marker of cell-cycle entry; Ki-67 mRNA oscillated with highest levels in G2 while protein levels increased throughout the cell cycle, peaking in mitosis. Inhibition of CDK4/CDK6 revealed proteasome-mediated Ki-67 degradation in G1 After cell-cycle exit, low-level Ki-67 expression persisted but was undetectable in fully quiescent differentiated cells or senescent cells. CDK4/CDK6 inhibition in vitro and in tumors in mice caused G1 cell-cycle arrest and eliminated Ki-67 mRNA in RB1-positive cells but had no effect in RB1-negative cells, which continued to proliferate and express Ki-67. Thus, Ki-67 expression varies due to cell-cycle regulation, but it remains a reliable readout for effects of CDK4/CDK6 inhibitors on cell proliferation. Cancer Res; 77(10); 2722-34. ©2017 AACR.

The cell proliferation antigen Ki-67 organises heterochromatin.

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.