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

Abstract

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.

Keywords: Ki-67; cancer; genetically modified mice; transcription.

Fig. 1.

Ki-67 is dispensable for human cancer cells but is rarely mutated. (A) Expression levels of Ki-67 (MKI67), PCNA, cyclin D1 (CCND1), and c-Myc (MYC) in cell lines from the Cancer Cell Line Encyclopedia. Values are log₂(transcripts per kilobase million [TPM] + 1) of TPM-normalized RNA-Seq data. (B) Relative copy number values for each gene from Sanger and Broad Institute whole-exome sequencing data of cancer cell lines. (C) DepMap analysis of dependency of cancer cell lines on each gene, from genome-wide CRISPR (blue) and RNAi (violet) screens. Lower values of gene effect indicate higher likelihood of dependency in a given cell line. Zero corresponds to a nonessential gene; −1 is the median of all common essential genes. (D) cBioPortal mutation analysis of each gene in pancancer TCGA data.

Fig. 2.

Germline disruption of Ki-67 protects mice against intestinal tumorigenesis. (A and C) immunohistochemistry staining of β-catenin in whole intestines from 6- to 7-mo-old mice. (A) WT, Mki67^+/2ntΔ and Mki67^2ntΔ/2ntΔ mice were treated with AOM-DSS and analyzed 16 wk later. Zoomed Insets show accumulation of β-catenin in nuclei. (Scale bars: Left, 2.5 mm; Inset, 500 µm.) (C) Apc^+/Δ14Mki67^+/2ntΔ and Apc^+/Δ14Mki67^2ntΔ/2ntΔ mice were analyzed after 6 mo (scale bars: 3 mm). (B and D) Quantification of the number (Upper) and total area (Lower) of neoplastic lesions. Error bars, SEM (n = 7 Apc^+/Δ14Mki67^+/2ntΔ mice; n = 6 Apc^+/Δ14Mki67^2ntΔ/2ntΔ mice). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Ki-67 ablation deregulates global gene expression programs in 4T1 cells. Dot plot analysis of differentially expressed genes (DEGs) in NIH/3T3 Mki67^−/− cells (A), 4T1 Mki67^−/− cells (B), and MDA-MB-231 MKI67^−/− cells (D). Red dots: DEGs with P value < 0.05; purple dots: log₂ fold change (LFC) >1 or <−1, P value < 0.05; gray dots: not significant (NS). (C) Venn diagrams of DEGs in NIH/3T3 and 4T1 Mki67^−/− cells under condition of P value < 0.05 (Upper) and P value (LFC > 1 or < −1) < 0.05 (Lower). Gene set enrichment analysis of highly deregulated genes in 4T1 Mki67^−/− cells (E) and MDA-MB-231 MKI67^−/− cells (H). (F) qRT-PCR analysis of DEGs in 4T1 Mki67^−/− cells; fold change in expression ± SD is shown. Gene set enrichment analysis of ENCODE and ChEA Consensus transcription factors from ChIP-X gene sets in Mki67^−/− 4T1 cells (G) and MDA-MB-231 MKI67^−/− cells (I). False discovery rate-adjusted P values. (J, Left) Heat maps of ChIP-Seq analysis of H3K27me3 in most down- and up-regulated genes in WT (CTRL) and Mki67^−/− cells (TSS, transcription start site; TES, transcription end site). (J, Right) Gene set enrichment analysis associated with these genes (ER, estrogen receptor; HSV1, Herpex simplex virus 1; mTOR, mechanistic target of rapamycin; MAPK, mitogen-activated protein kinase; CML, chronic myeloid leukemia; HTLV1, human T cell lymphotropic virus type 1). (K) The average values of the H3K27me3 (in green) and H3K4me3 (in apricot) ChIP-Seq reads over the 10-kb region surrounding the gene in 4T1 Mki67^−/− cells (AvgKO) were subtracted from the values of WT cells (AvgCTL) and then plotted against the log of the fold change for each gene in RNA-Seq. (L) ChIP-Seq profiles of histone marks at the Twist1 locus.

Fig. 4.

Ki-67 promotes stem cell characteristics and controls the EMT in mammary carcinoma. (A) Immunoblot analysis of Hes1 expression in CTRL and two different clones (#1 and #2) of Mki67^−/− 4T1 cells. Immunoblot (B) and immunofluorescence (C) analysis of vimentin and E-cadherin (E-Cad) expression in CTRL, Mki67^−/− 4T1, and MDA-MB-231 cells. DNA was stained with DAPI. (Scale bars: 30 µm.) (D) ALDH activity measured using a flow cytometry assay in 4T1 CTRL and Mki67^−/− cells. DAEB (N,N-diethylaminobenzaldehyde), an inhibitor of ALDH, was used as a negative control. (Upper) Flow cytometry profiles (FSC-A, forward scatter area). (Lower) Quantification of ALDH+ cells. Error bars, SEM (n = 2 independent analyses). (E) Mammosphere formation assay of 4T1 CTRL or Mki67^−/− cells. Representative images (Left) and quantification (Right; error bars, SEM, n = 10) after 7 d. (Scale bars: 400 µm.)*P < 0.05.

Fig. 5.

Ki-67 promotes tumor growth. (A) Dot plot analysis of differentially expressed genes in 4T1 Mki67^−/− cells vs. tumors derived from grafting 4T1 Mki67^−/− cells into nude mice, showing a highly significant correlation. LFC, log₂ fold change. (B) Fluorescent immunohistochemistry analysis of vimentin in 4T1 CTRL (WT) and Mki67^−/− tumors in NOD/SCID mice. (Scale bars: 30 µm.) (C) Fluorescent IHC analysis of H3S10ph in 4T1 CTRL and Mki67^−/− tumors in NOD/SCID mice (red; pan cytokeratin [PCK] in green, and DNA stained with DAPI in blue; Upper) and quantification (Lower). (Scale bars: 30 µm.) (D) IHC staining for Ki-67 and PCNA in 4T1 CTRL or Mki67^−/− tumors. (Scale bars: 100 µm.) The 4T1 CTRL or Mki67^−/− orthotopic xenografts in NOD/SCID (E) and nude (F) mice. Tumor growth was monitored for 3 wk. Error bars, SEM (n = 6 mice). CD-31 staining of endothelial cells in CTRL and Mki67^−/− tumors in NOD-SCID mice: quantification of mean vessel density (MVD; G) and representative IHC images (H). (Scale bar: 500 µm.) (I) Sirius red staining of collagen in CTRL and Mki67^−/− tumors in NOD-SCID mice. Insets indicate tumor areas presented at higher magnification. (Scale bar: 250 µm.) (J) Quantification of MVD in CTRL and Mki67^−/− tumors in nude mice. **P < 0.001; ****P < 0.0001; ns, nonsignificant.

Fig. 6.

Ki-67 promotes metastasis and antitumor immune responses. (A) Quantification of lung metastases in nude mice injected orthotopically with 4T1 CTRL (WT) or Mki67^−/− cells. Error bars, SEM. (A, Upper) Representative images of lungs stained to visualize metastases (white nodules). (background scale is mm; ***P < 0.0001). (B) Lung tissue from nude mice injected via tail vein with 4T1 CTRL or Mki67^−/− cells (two per condition) was dissociated after 2 wk, and resulting cells were maintained in the presence of 6-thioguanine to select for 4T1 cells. (B, Left) Crystal violet staining of resulting colonies. (B, Right) Quantification. (C) Tumor growth over 6 wk of 4T1 CTRL or Mki67^−/− orthotopic xenografts in immunocompetent BALB/c mice. Error bars, SEM (n = 8 mice), and quantification of lung metastases in each group (D). Error bars, SEM; ns, non significant. (E) The 4T1 CTRL or Mki67^−/− #2 cells were injected via tail vein into immune-competent BALB/c mice. Representative images of stained lungs (day 21 postinjection); metastases are white. (background scale is mm) (F) Immunochistochemistry analysis of 4T1 CTRL or Mki67^−/− tumors (week 4 posttransplantation) stained for Gr-1, an MDSC marker. (Scale bars: 100 µm.) (G) The 4T1 CTRL and Mki67^−/− cells were stained with anti–H2D^d fluorescein isothiocyanate (FITC)-conjugated antibody and anti–H2K^d Phycoerythrin (PE)-conjugated antibody, or control isotypes. (G, Left) Flow cytometry profiles. (G, Right) Quantification (n = 1 of each clone).

Fig. 7.

Ki-67 promotes cancer cell drug resistance. (A) Xenobiotic metabolism is a hallmark of genes down-regulated in Mki67^−/− cells. LFC, log2 fold change. (B) IC₅₀ (concentration of drug needed for 50% growth inhibition) of 4T1 WT (CTRL) or Mki67^−/− cells to indicated compounds, derived from dose–response experiments.