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.DOI: http://dx.doi.org/10.7554/eLife.13722.001

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 G while protein levels increased throughout the cell cycle, peaking in mitosis. Inhibition of CDK4/CDK6 revealed proteasome-mediated Ki-67 degradation in G After cell-cycle exit, low-level Ki-67 expression persisted but was undetectable in fully quiescent differentiated cells or senescent cells. CDK4/CDK6 inhibition and in tumors in mice caused G 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. .

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.

Senescence is an irreversible proliferation withdrawal that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 and RB 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 CDK inhibitor p21 and Chk1 controls D-type cyclin-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting Cyclin D1-CDK2/CDK4. The resulting G2 exit, which precedes appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and DNA damage foci reduction. In p53/RB-proficient cancer cells, 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 down-regulating CDK6, whereas Chk2 knockdown enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting DNA damage-induced senescence onset in G2.

22Ki-67 is a nuclear protein universally expressed in proliferating vertebrate cells (1), a 23 characteristic that underlies its widespread use as a cancer biomarker. It organises 24 heterochromatin (2,3) and the mitotic chromosome periphery (2,4-6), but it is dispensable for 25 cell proliferation in vivo2. It thus remains unclear whether Ki-67 confers any advantages to 26 cancer cells. Here, using mouse models of breast and intestinal cancer, we show that Ki-67 27 ensures gene expression programmes required for both carcinogenesis and tumour 28 immunogenicity. We find that germline disruption of Ki-67 protects mice against intestinal 29 tumourigenesis, while in mammary carcinoma cells, ablation of Ki-67 slows tumour growth 30 and inhibits metastasis, despite the failure of cells lacking Ki-67 to induce an effective anti-31 tumour immune response. Mechanistically, Ki-67 loss leads to widespread deregulation of 32 gene expression, including genes involved in the epithelial-mesenchymal transition (EMT), 33 immune responses and drug metabolism. This results in loss of mesenchymal and stem cell 34 characteristics, downregulation of MHC class I expression and sensitisation to various drug 35classes. Our results suggest that, by structuring chromatin to allow global transcriptome 36 changes, Ki-67 allows cancer cells to adapt to their environment. This is required for all stages 37 of tumourigenesis and drug resistance, but, by maintaining tumour immunogenicity, it also 38 confers a targetable vulnerability on cancer cells. 39 40 Results and discussion 41Although Ki-67 is universally expressed in dividing cancer cells, cell proliferation is not 42 affected by Ki-67 gene disruption in any cell type yet analysed in vitro, nor during mouse 43 development 2,5-7 . However, Ki-67 might still be important in carcinogenesis since it 44 contributes to organisation of chromatin 2 , which controls gene expression and thus cellular 45 plasticity, a characteristic of cancer cells 8,9 . We first tested the involvement of Ki-67 in tumour 46 initiation using an in vitro model. We transduced our previously generated Mki67 +/+ or TALEN-47

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<div>Abstract<p>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 <i>in vitro</i> 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. <i>Cancer Res; 77(10); 2722–34. ©2017 AACR</i>.</p></div>

<p>Dataset of high-confidence Ki-67 interactions</p>