Ki-67 Regulates Global Gene Expression and Promotes Sequential Stages of Carcinogenesis

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

Karim Mrouja,b,1, Nuria Andrés-Sáncheza,b, Geronimo Dubraa,b, Priyanka Singha,b,2, Michal Sobeckia,b,3, Dhanvantri Chahara,b, Emile Al Ghoula,b, Ana Bella Aznara,b, Susana Prietoa,b, Nelly Pirotc,d, Florence Bernexc,d, Benoit Bordignone, Cedric Hassen-Khodjae, Martin Villalbaf, Liliana Krasinskaa,b, and Daniel Fishera,b,4

aInstitut de Génétique Moléculaire de Montpellier, CNRS, INSERM, University of Montpellier, 34293 Montpellier, France; bEquipe Labellisée Ligue 2018, Ligue Nationale Contre le Cancer, 75013 Paris, France; cInstitut de Recherche en Cancérologie de Montpellier, Institut du Cancer de Montpellier, INSERM, University of Montpellier, 34090 Montpellier, France; dBioCampus, Le Réseau d’Histologie Expérimentale de Montpellier, CNRS, INSERM, University of Montpellier, 34090 Montpellier, France; eBioCampus, Montpellier Ressources Imagerie, CNRS, INSERM, University of Montpellier, 34090 Montpellier, France; and fInstitute of Regenerative Medicine and Biotherapies, INSERM, University of Montpellier, 34295 Montpellier, France

Edited by Anton Berns, Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved January 28, 2021 (received for review December 29, 2020) Ki-67 is a nuclear protein that is expressedinallproliferatingverte- mediated biallelic disruption of the Ki-67 gene or human cancer cells brate cells. Here, we demonstrate that, although Ki-67 is not required with stable knockdown of Ki-67, repressive histone marks histone for cell proliferation, its genetic ablation inhibits each step of tumor H3 lysine-9 tri-methylation (H3K9me3) and histone H4 lysine-20 tri- initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to methylation (H4K20me3) are dispersed, the heterochromatin is less chemical or genetic induction of intestinal tumorigenesis. In estab- compact, and association between centromeres and nucleoli is dis- lished cancer cells, Ki-67 knockout causes global transcriptome rupted. Conversely, overexpression of Ki-67 caused ectopic hetero- remodeling that alters the epithelial–mesenchymal balance and sup- chromatin formation (11). Involvement of Ki-67 in chromatin presses stem cell characteristics. When grafted into mice, tumor organization was corroborated by a study showing that Ki-67 is re- growth is slowed, and metastasis is abrogated, despite normal cell quired to maintain heterochromatin marks at inactive X chromo- proliferation rates. Yet, Ki-67 loss also down-regulates major histo- somes in nontransformed cells (16). Heterochromatin is a compatibility complex class I antigen presentation and, in the 4T1 phenotypic marker of multiple cancers (17), suggesting that it might syngeneic model of mammary carcinoma, leads to an immune- be involved in carcinogenesis. We thus tested whether and how Ki-67 suppressive environment that prevents the early phase of tumor re- CELL BIOLOGY is required for different steps of carcinogenesis. gression. Finally, genes involved in xenobiotic metabolism are down- regulated, and cells are sensitized to various drug classes. Our results Results suggest that Ki-67 enables transcriptional programs required for cel- Ki-67 Is Dispensable in Cancer Cell Lines yet Is Rarely Mutated in lular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more Human Cancers. Ki-67 expression is widely used as a marker for susceptible to antitumor immune responses. cell proliferation in cancer, but whether it is important for

Ki-67 | cancer | genetically modified mice | transcription Significance

i-67 is a nuclear protein expressed only in proliferating Ki-67 is a nuclear protein present in all proliferating vertebrate Kvertebrate cells, a property underlying its widespread use in cells and is widely used as a marker in clinical cancer histopa- oncology as a biomarker (1). Its expression is routinely assessed thology. However, its cellular functions have remained largely in histopathology to grade tumors; there are also indications for mysterious, and whether it plays any roles in cancer was un- its use as a prognostic marker (2), although uncertainty over the known. In this work, we show genetically that Ki-67 is not relationship between Ki-67 index and prognosis remains. The required for cell proliferation in tumors, but it is required for all cellular functions of Ki-67 are not well understood, and whether stages of carcinogenesis. The effects on cell transformation, tumor growth, metastasis, and drug sensitivity correlate with it is involved in tumorigenesis is unclear. genome-scale changes in gene expression that modify cellular For a long time, Ki-67 was thought to be required for cell programs implicated in cancer. Thus, Ki-67 expression is ad- proliferation (3–8), and early work suggested that it promotes vantageous for cancer cells, but it comes with an Achilles’ heel: ribosomal RNA transcription (4, 9). However, recent genetic it makes them more visible to the immune system. studies have shown that despite promoting formation of the – perichromosomal layer of mitotic chromosomes (9 12), it is not Author contributions: K.M., L.K., and D.F. designed research; K.M., N.A.-S., P.S., M.S., D.C., required for cell proliferation (10–13). It is also dispensable for E.A.G., A.B.A., S.P., N.P., F.B., B.B., C.H.-K., and M.V. performed research; K.M., N.A.-S., ribosomal RNA synthesis and processing, and mice lacking Ki-67 G.D., P.S., M.S., D.C., E.A.G., N.P., F.B., B.B., C.H.-K., M.V., L.K., and D.F. analyzed data; and develop and age normally (11). In addition, Ki-67 is not over- L.K. and D.F. wrote the paper. expressed in cancers; rather, Ki-67 expression is controlled by The authors declare no competing interest. cell cycle regulators, including cyclin-dependent kinases (CDKs), This article is a PNAS Direct Submission. the activating subunit of the ubiquitin ligase APC/C-CDH1, Published under the PNAS license. which is required for destruction of mitotic cyclins, and the cell 1Present address: Institute for Stem Cell Biology and Regenerative Medicine, Stanford cycle transcription factor B-Myb (11, 14, 15). These recent University School of Medicine, Stanford, CA 94305. 2 studies raise the question of whether Ki-67 plays any role in Present address: Section on Nutrient Control of Gene Expression, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, tumorigenesis, which has not been addressed genetically. MD 20892. Despite not being essential for cell proliferation, Ki-67 might be 3Present address: Institute of Anatomy, University of Zurich, CH - 8057 Zurich, important in carcinogenesis for other reasons. We previously iden- Switzerland. tified over 50 Ki-67–interacting proteins that are involved in tran- 4To whom correspondence may be addressed. Email: [email protected]. scription and chromatin regulation. We also found that Ki-67 This article contains supporting information online at https://www.pnas.org/lookup/suppl/ organizes heterochromatin: in NIH/3T3 mouse embyonic fibroblast doi:10.1073/pnas.2026507118/-/DCSupplemental. cells with transcription activator-like effector nucleases (TALEN)- Published March 3, 2021.

PNAS 2021 Vol. 118 No. 10 e2026507118 https://doi.org/10.1073/pnas.2026507118 | 1of12 Downloaded by guest on September 26, 2021 carcinogenesis is unclear. We previously found no adverse effects of 739 cell lines (Fig. 1B), confirming that it is generally dispensable on cell proliferation of either shRNA-mediated knockdown of for cell proliferation in human cancer cells. Nevertheless, like Ki-67 in U2OS or HeLa cells or TALEN-mediated disruption of PCNA, MKI67 showed almost no copy number variations among the gene encoding Ki-67 in NIH/3T3 cells (11). To see whether the different cell lines, in contrasttothetwocancerdriveronco- this is true across different cancer types, we interrogated the genes MYC and CCND1, which were frequently amplified Cancer Dependency Map project Dependency Mapper (Dep- (Fig. 1C).Thesameistrueindatafromclinicalsamplesofcancer Map) (https://depmap.org/portal/) (18) using the latest CRISPR patients in the cBioPortal database (https://www.cbioportal.org/), (Avana 20Q1) and RNA interference (RNAi; Broad Institute) which further showed that only 5% of cancers presented mutations datasets. As controls, we compared Ki-67 with two proto- in MKI67 (Fig. 1D). Almost all of these were missense mutations, oncogenes that have roles in cell proliferation, MYC and CCND1 which might be passenger mutations. encoding c-Myc and cyclin D1, respectively, and with PCNA, Thus, even though Ki-67 is dispensable for cell proliferation in encoding Proliferating Cell Nuclear Antigen, whose expression virtually all cancer cells, it is ubiquitously expressed at similar levels profile is similar to that of Ki-67. in all cancers, corroborating our previous findings that variability in Ki-67, PCNA, and c-Myc were universally expressed, while Ki-67 expression is accounted for by its regulation through the cell cyclin D1 was expressed in most cell lines, although not all cycle (14). Together, these observations suggest that Ki-67 may (Fig. 1A), possibly due to compensation by a different D-type provide benefit to cancer cells. In contrast, its overexpression may cyclin. In CRISPR-Cas9 screens, PCNA was essential for pro- be counterselected in cancers, which would fit with our finding that liferation of 775 of 777 cell lines tested, while 719 of 726 lines increased levels of Ki-67 arrest cell proliferation (11). required c-Myc, and 579 of 726 required cyclin D1 (Fig. 1B). In The lack of overexpression or deletion of Ki-67 in cancers contrast, Ki-67 knockout did not affect cell proliferation in 725 implies that correlating Ki-67 expression with patient survival is

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.

2of12 | PNAS Mrouj et al. https://doi.org/10.1073/pnas.2026507118 Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis Downloaded by guest on September 26, 2021 likely to simply reflect the impact of the fraction of proliferating finding that Ki-67 interacts with many general chromatin regu- cells. If so, then PCNA expression should give similar results. To lators in the U2OS cancer cell line (11). We therefore expected address this question, we queried survival correlations in differ- that Ki-67 knockout would also extensively affect the tran- ent cancer types with MKI67 and PCNA messenger RNA levels scriptome of established cancer cells, with possible consequences in The Cancer Genome Atlas expression data using OncoLnc for tumorigenicity. To investigate this, we used the syngeneic 4T1 (19). For both genes, there was either no correlation (breast and mouse mammary carcinoma model, which is derived from colorectal cancer), a modest positive correlation (lung cancer), BALB/c mice. This cell line mimics human triple-negative breast or a strong negative correlation (liver cancer and renal cancer) of cancer, is highly invasive, and spontaneously metastasizes to expression levels with survival (SI Appendix, Fig. S1). Thus, in distant organs (22, 23). As expected, 4T1 cell proliferation rates several different cancers, despite the contrasting requirements were unaffected by CRISPR-Cas9–mediated Mki67 gene for PCNA and Ki-67 for cell proliferation, the correlation of knockout (SI Appendix, Fig. S4). In 4T1 cells, Mki67 knockout expression of each gene with survival is very similar. caused even more extensive gene expression alterations than in NIH/3T3 cells: 4,979 genes were deregulated, of which 1,239 and Mice Lacking Ki-67 Are Resistant to Intestinal Tumorigenesis. The 585 genes were more than twofold down-regulated and up- fact that Ki-67 is ubiquitously expressed in cancers but is not regulated, respectively (Fig. 3 B and C and Dataset S2). There −/− required for cell proliferation raises the question of whether it was little overlap in the deregulated genes between Mki67 4T1 has functional roles in carcinogenesis. To see whether Ki-67 (epithelial) and NIH/3T3 (mesenchymal) cells (Fig. 3C and SI knockout affects initiation of tumorigenesis in vivo, we used a Appendix, Tables S1 and S2) in accordance with our hypothesis 2ntΔ/2ntΔ germline TALEN disruption of Ki-67 (Mki67 )thatwegen- that, by organizing chromatin, Ki-67 enables global gene regu- erated (11). We first employed chemical induction of colon carci- lation in different cell types rather than directly controlling nogenesis by azoxymethane/dextran sodium sulfate (AOM-DSS) specific genes. 2ntΔ/2ntΔ treatment (20) in wild-type (WT) and Mki67 mice. We ob- To see whether the global effect of Ki-67 knockout on gene served that dextran sodium sulfate (DSS) alone induced a similar expression is conserved across cancer cell types and species, we decrease in body weight in control and mutated mice compared with next disrupted the MKI67 gene by CRISPR-Cas9 in human the controls (SI Appendix,Fig.S2A). Histopathological examination MDA-MB-231 triple-negative breast cancer cells (SI Appendix, of colonic sections revealed a typical DSS-induced colitis in both Fig. S5 A and B), which is a highly mesenchymal-like cell line due genotypes (SI Appendix,Fig.S2B), with increased numbers of im- to an extensive epithelial–mesenchymal transition (EMT). As −/− mune cells in the lamina propria, moderate crypt cells damage, and expected, MKI67 MDA-MB-231 cells proliferated normally hyperplasia. As expected, AOM-DSS efficiently induced colon tu- in vitro (SI Appendix, Fig. S5 C and D). Transcriptome analysis CELL BIOLOGY 2ntΔ/+ mors within 16 wk in both WT and Mki67 mice (indeed, het- by RNA-Seq showed that Ki-67 knockout in this cell line also erozygous mice had bigger lesions; future studies will address effects caused genome-scale alterations in gene expression (Fig. 3D), of Ki-67 gene dosage on tumor growth). However, no macroscopic with 9,127 genes deregulated, 914 of which were up- or down- 2ntΔ/2ntΔ lesions were observed in Mki67 mice (Fig. 2 A and B). This regulated by a factor of more than two (Dataset S3). suggests that Ki-67 is specifically required for initiation of tumori- We investigated whether the extensive transcriptome changes genesis. To confirm these findings, we used a genetic model of in- seen in cancer cells upon Ki-67 knockout affect pathways in- 2ntΔ/2ntΔ Δ14/+ testinal tumorigenesis. We crossed Mki67 mice with Apc volved in tumorigenesis. In 4T1 cells, bioinformatic analysis of mice, which rapidly develop tumors in the intestine due to loss of the the most up- and down-regulated genes revealed deregulation of second allele of the Apc tumor suppressor gene (21). While as various components of inflammation, apoptosis, p53, the EMT, Δ14/+ 2ntΔ/+ expected, Apc Mki67 mice formed multiple colon tumors, estrogen response, K-Ras signaling, and hypoxia (Fig. 3E). We Δ14/+ 2ntΔ/2ntΔ tumor burden was strongly reduced in Apc Mki67 mice also manually analyzed transcriptome data and noticed up- (Fig. 2 C and D). regulation of the Notch pathway, down-regulation of the EMT, Thus, Ki-67 is required for efficient initiation of tumorigenesis the Wnt pathway, antigen presentation, and aldehyde metabo- induced by chemical mutagenesis or loss of a tumor suppressor lism, which we validated by qRT-PCR (Fig. 3F). Down-regulated in vivo, suggesting that it might be required for cell transfor- genes were enriched in targets of nuclear factor erythroid mation. To test this, we transduced our previously generated 2-related factor 2, one of the major orchestrators of responses to +/+ −/− Mki67 or TALEN-mutated Mki67 NIH/3T3 fibroblasts oxidative stress; polycomb-repression complex 2 (PRC2), which (11) with oncogenic mutant H-Ras (G12V), and evaluated col- mediates Histone H3 lysine-27 trimethylation (H3K27me3) and ony formation as an indicator of transformation. While is a well-characterized regulator of the EMT (24, 25); the plu- H-RasG12V-transduced control 3T3 cells efficiently formed col- ripotency factors Nanog and Sox2; and interferon regulatory −/− onies, Mki67 3T3 cells did not, despite having similar rates of factor 8 (Fig. 3G). All of these pathways have previously been cell proliferation (SI Appendix, Fig. S3). This indicates that Ki-67 implicated in tumorigenesis. In MDA-MB-231 cells, like 4T1 expression facilitates oncogene-induced transformation in cells, pathway analysis also revealed genes involved in the EMT, these cells. inflammatory response, early estrogen response, K-RAS signaling, and hypoxia, while a significant portion of the deregulated Loss of Ki-67 Causes Global Transcriptome Changes and Deregulates genes was under the control of PRC2 and estrogen receptor 1 Pathways Involved in Cancer. Since we previously found that (Fig. 3 H and I and Dataset S3). In summary, similar pathways knockdown of Ki-67 in cancer cells altered their chromatin or- involved in cancer are affected upon Ki-67 knockout in different ganization and affected gene expression (11), we hypothesized cancer cell lines. that the resistance of Ki-67 knockout cells to transformation The prevalence of down-regulation of gene expression in Ki- might also result from gene expression changes. We first per- 67 knockout cells, the enrichment in PRC2 targets among these formed RNA-sequencing (RNA-seq) analysis of Ki-67 WT and genes, and our previous observations that Ki-67 associates with knockout NIH/3T3 cells. This revealed surprisingly wide-ranging the essential PRC2 component SUZ12 (11) prompted us to ask effects of Ki-67 loss on the transcriptome, with 2,558 genes sig- whether loss of Ki-67 affects genome-wide distribution of −/− nificantly deregulated in independent clones of Mki67 cells H3K27me3. To answer this question, we performed chromatin (q < 0.05) (Fig. 3A and Dataset S1). This level of transcriptome immunoprecipitation with high-throughput sequencing alteration suggested a global effect on chromatin rather than a (ChIP-Seq) on WT and Ki-67 knockout 4T1 cells. While there direct involvement of Ki-67 in controlling specific pathways or were no genome-wide changes in H3K27me3 distribution, a transcription factors, which is consistent with our previous substantial subfraction of genes showed an increase in this mark

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Fig. 2. Germline disruption of Ki-67 protects mice against intestinal tumorigenesis. (A and C) immunohistochemistry staining of β-catenin in whole intestines Δ Δ Δ from 6- to 7-mo-old mice. (A) WT, Mki67+/2nt and Mki672nt /2nt mice were treated with AOM-DSS and analyzed 16 wk later. Zoomed Insets show accu- mulation of β-catenin in nuclei. (Scale bars: Left, 2.5 mm; Inset,500μm.) (C) Apc+/Δ14Mki67+/2ntΔ and Apc+/Δ14Mki672ntΔ/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; Δ Δ Δ n = 6 Apc+/ 14Mki672nt /2nt mice). *P < 0.05; **P < 0.01; ***P < 0.001.

(SI Appendix, Fig. S6A). This was particularly evident for genes Ki-67 Promotes Stem Cell Characteristics and Controls the EMT in strongly repressed in Ki-67 knockout cells (Fig. 3 J, Left). To Mammary Carcinoma. We next investigated the biological conse- investigate correlations between changes in the levels of histone quences resulting from these global transcriptome alterations in modifications and of gene expression, we assigned an average cancer cells lacking Ki-67. Importantly, although the Notch value of the repressive H3K9me3, as well as activatory H3K4me3 pathway is oncogenic in T cell acute lymphoid leukemia, it can and histone H3 lysine-27 acetylation (H3K27ac) reads across 10 act as a tumor suppressor in specific cellular contexts (26), can kb surrounding the transcription start site, and plotted the dif- block Wnt signaling (27, 28) (a driver of tumorigenesis, cell ferences in these values between WT and Ki-67 knockout cells stemness, and the EMT), and induce drug resistance (29). We with gene expression changes. This showed a strong correlation confirmed Notch pathway up-regulation at the protein level between the change in gene expression, H3K27me3, and (Fig. 4A). While the EMT is closely associated with a stem-like H3K4me3 (Fig. 3K), while H3K27ac levels only correlated well state (30–32), the most stem-like states appear to show a hybrid with the most highly down-regulated genes (SI Appendix, Fig. expression of both epithelial and mesenchymal characteristics, S6B). Of nine genes whose down-regulated expression in Ki-67 with the most epithelial and mesenchymal cells losing stemness knockout cells we confirmed by qRT-PCR, we only found an (33–35). We found that 4T1 cells express both E-cadherin and obvious increase of H3K27me3 on the EMT-promoting tran- vimentin, suggesting a highly stem-like state (Fig. 4B), but Ki-67 scription factor Twist1. This correlated with down-regulation of knockout 4T1 cells had reduced expression of the mesenchymal active promoter-associated H3K4me3 and H3K27ac (Fig. 3L). marker vimentin and up-regulated E-cadherin (Fig. 4 B and C). There was also a slight increase of H3K27me3 and reduction in To see whether this translates to a loss of stem-like character, we H3K27ac at the Vimentin promoter (SI Appendix, Fig. S6C). analyzed aldehyde dehydrogenase (ALDH) activity, which is a Taken together, these results suggest that Ki-67 loss generally bona fide marker of stem and progenitor cells (36, 37). ALDH increases PRC2-mediated repressive histone marks at down- activity was strongly reduced in 4T1 Ki-67 knockout cells regulated genes, but most of the expression alterations result- (Fig. 4D). Furthermore, the ability to form spheroids in the ab- ing from Ki-67 knockout may not be directly due to modulation sence of adhesion to a surface, another characteristic of stem by PRC2. Instead, they are likely knock-on effects of altered cells (30, 38), was also largely decreased (Fig. 4E). expression of other transcriptional regulators that result in To test whether repression of the EMT in Ki-67 knockouts changes in active promoter-associated histone marks. depends on PRC2, we additionally disrupted PRC2 components

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CD F

E H CELL BIOLOGY

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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) >1or<−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 > 1or< −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.

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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.

−/− Suz12 or Ezh2 in WT and Mki67 4T1 cells using CRISPR- cells, we first engrafted WT and Ki-67 knockout 4T1 cells Cas9 (SI Appendix,Fig.S7A). This partially rescued vimentin expression, orthotopically into mouse mammary fat pads. Since Ki-67 indicative of an increased ability to undergo an EMT (SI Appendix,Fig. knockout caused alteration of inflammatory response genes S7B), but did not restore it to levels observed in WT cells. This cor- (Fig. 3E), we initially used athymic nude and NOD/SCID mice, roborates the results of the H3K27me3 ChIP-Seq analysis described allowing us to assess roles of Ki-67 in tumor growth and me- above, indicating that the influence of PRC2 activity on gene expression tastasis while minimizing possible confounding effects of an al- changes observed in Ki-67 knockout cells is limited. tered immune response. RNA-Seq of early-stage tumors from Analysis of EMT markers in MDA-MB-231 cells showed that WT and Ki-67 mutant 4T1 cells grafted into nude mice showed Ki-67 knockout cells retained a mesenchymal character, with that Ki-67–dependent transcriptome changes were well pre- vimentin but no E-cadherin staining (Fig. 4 B and C), suggesting served in vivo (Fig. 5A and Dataset S4), including down- that they are far from a hybrid-like EMT state. Importantly, Ki- regulation of mesenchymal genes and up-regulation of epithe- 67 knockout cells had even higher vimentin expression, implying lial genes and of the Notch pathway, which was validated by a further distance from the putative stem-like hybrid state. In increased HES1 staining in tumors (SI Appendix, Fig. S9A). agreement with this hypothesis, neither WT nor Ki-67 knockout Reduced vimentin staining of Ki-67 mutant tumors confirmed MDA-MB-231 cells showed significant ALDH activity (SI Ap- the loss of EMT in vivo (Fig. 5B). As assessed by PCNA and pendix, Fig. S8A). Importantly, Ki-67 knockout cells were unable phosphohistone H3S10 staining, cell proliferation in vivo was to form mammospheres, indicating a conserved requirement for unaffected by loss of Ki-67 (Fig. 5 C and D). However, tumors −/− Ki-67 in maintaining ability to seed formation of new cell colo- from Mki67 4T1 cells grew significantly slower than from nies (SI Appendix, Fig. S8B). As such, loss of Ki-67 reverses cells control cells in both types of immunodeficient mice (Fig. 5 E and to a more epithelial or a more mesenchymal state, depending on F). There were no apparent differences in apoptosis upon Ki-67 the cell type of origin. This perturbation of the EMT correlates knockout in 4T1 cells in vitro (SI Appendix, Fig. S9B), and while with a reduction of stem-like character in different cell lines. there was significantly increased apoptosis in one of the two 4T1 Ki-67 knockout clones in vivo (SI Appendix, Fig. S9 C and Ki-67 Expression Promotes Tumor Growth. To determine whether D), both clones had similarly slowed tumor progression. Analysis these phenotypic alterations affect the tumorigenicity of cancer of necrosis revealed variability between tumors and clones (SI

6of12 | PNAS Mrouj et al. https://doi.org/10.1073/pnas.2026507118 Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis Downloaded by guest on September 26, 2021 -/- -/- 10 4T1 CTRL Mki67 #1 Mki67 #2 A R = 0.36, p < 2.2e−16 D

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(mm p < 0.0001

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6110 316 days Vimentin Athymic nude F 2500 4T1 CTRL C 4T1 CTRL Mki67-/- #1 Mki67-/- #2 Mki67-/- #1 DAPI ) -/- 3 Mki67 #2

+ 1500 p=0.0056** (mm PCK 500 + Tumour volume 123 weeks

H3S10P NOD/SCID G H 4T1 CTRL Mki67-/- #1 Mki67-/- #2 p<0.0001 10 **** **** p<0.0001 ns ns CD31 5 MVD CELL BIOLOGY NOD/SCID % H3S10ph+ cells 0 CTRL Mki67-/- Mki67-/- CTRL Mki67-/- Mki67-/- Athymic nude #1 #2 #1 #2 J ns I 4T1 CTRL Mki67-/- #1 Mki67-/- #2 ** p=0.0037 MVD Sirious red CTRL Mki67-/- Mki67-/- NOD/SCID #1 #2

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.

Appendix, Fig. S9 E and F). As assessed by γ-H2A.X (histone both in cell culture and in tumors (Datasets S2 and S4), we an- H2A.X phosphorylated on serine-139) staining, we did not find evi- alyzed angiogenesis in WT and Ki-67 knockout tumors by CD31 dence for increased DNA damage in Ki-67 knockout cells nor tumors staining of endothelial cells. Indeed, we found that the mean (SI Appendix,Fig.S9G and H). Thus, in vivo differences in tumor blood vessel density was significantly reduced in tumors from growth between WT and Ki-67 knockout tumors cannot be explained both Ki-67 knockout clones in NOD/SCID mice (Fig. 5 G and either by reduced cell proliferation or by increased DNA damage or H). Tumors lacking Ki-67 also appeared more fibrotic than cell death. It is theoretically possible that the slower tumor growth of controls, as indicated by Sirius red staining of collagen (Fig. 5I). Ki-67 knockouts might be explained by a marginally lower intrinsic cell However, blood vessel density was comparable between WT and proliferation rate that we did not detect when culturing WT and Ki-67 knockout tumors in nude mice (Fig. 5J). Moreover, the knockout cell lines individually but might become visible over longer distribution of vessels of different sizes was similar between all timescalesandwhencoculturedwithWTcells.Totestthis,weper- genotypes and mouse backgrounds (SI Appendix, Fig. S9I). formed competition experiments in vitro between WT and knockout Therefore, while we cannot rule out a contribution of either 4T1 cells over 12 d. We found that the initial 50:50 ratio of WT and reduced or altered angiogenesis to the slow growth of knockout Ki-67 knockout 4T1 cells was maintained after 2 wk of coculture (SI tumors, this cannot explain the differences seen in all situations. Appendix,Fig.S10), ruling out this possible explanation. We also analyzed tumors from xenografts of WT and Ki-67 Next, since Ki-67 4T1 knockout cells had down-regulated ex- mutant MDA-MB-231 cells in nude mice. We similarly observed pression of several angiogenic factors, including angiopoietin-1, reduced tumor growth rate despite normal cell proliferation (SI

Mrouj et al. PNAS | 7of12 Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis https://doi.org/10.1073/pnas.2026507118 Downloaded by guest on September 26, 2021 Appendix,Fig.S11A–C). As with 4T1 cells, there was no detectable initial regression has been attributed to a strong antitumor im- increase in apoptosis in MDA-MB-231 cells lacking Ki-67 in vitro mune response (40). Surprisingly, no such initial regression oc- −/− (SI Appendix,Fig.S9B). However, apoptosis, but not DNA damage curred when Mki67 4T1 cells were engrafted (Fig. 6C), (SI Appendix,Fig.S11D), was significantly increased in Ki-67 suggesting that Ki-67 knockout cells fail to trigger an efficient knockout MDA-MB-231 tumors (SI Appendix,Fig.S11E). Necro- immune response. Nevertheless, despite the consequently higher sis was reduced (SI Appendix,Fig.S11F), and fibrosis was more tumor burden, there were either similar or less metastases than apparent (SI Appendix,Fig.S11G), while mean blood vessel density from control 4T1 cells (Fig. 6D), although differences failed to and distribution of vessels of different sizes were comparable with reach statistical significance (P = 0.13). We surmised that in- control tumors (SI Appendix,Fig.S11H and I). jection of 4T1 cells directly into the circulation via the tail vein To assess the generality of these observations in a different should circumvent the immune targeting of tumor cells at the tumor type, we stably knocked down Ki-67 by short hairpin RNA primary site, allowing direct assessment of the capacity of cells to (shRNA) in a commonly used aggressive human cervical cancer establish a metastatic niche. Importantly, WT 4T1 cells again −/− cell line, HeLa S3, and grafted the resulting cells and an shRNA more efficiently colonized lungs in this setting than Mki67 4T1 control line into opposing flanks of athymic nude mice. Ki-67 cells (Fig. 6E), despite the higher overall tumor burden in the knockdown was maintained in vivo (SI Appendix, Fig. S12 A and latter. Together, the results of these experiments underline the B). Again, tumor growth was severely impaired (SI Appendix, requirement for Ki-67 in seeding metastasis. They also suggest a Fig. S12C), despite unaffected cell proliferation (as shown by defective immune response to Ki-67 knockout tumors. In unchanged PCNA and mitotic indices) (SI Appendix, Fig. S12 D agreement, immunohistological analysis revealed increased in- and E), while there was increased necrosis and apoptosis (SI filtration of myeloid-derived suppressor cells (MDSCs) (41) in Appendix, Fig. S12 F and G). It is interesting to note that shRNA knockout tumors (using the MDSC marker, GR1) (Fig. 6F), of Ki-67 was sufficient to induce strong phenotypes. possibly indicating an immunosuppressive environment that In conclusion, Ki-67 knockout or knockdown in several different protects the tumors against immune-mediated cytotoxicity. To established cancer cell lines consistently results in slower tumor investigate why Ki-67 knockout cells fail to induce an efficient growth upon grafts in mice despite unchanged cell proliferation and antitumor immune response, we analyzed the expression of the often leads to noncell-autonomous increases in apoptosis or fibro- mouse major histocompatibility complex (MHC) class I, re- sis, with necrosis and angiogenesis more variably affected. However, sponsible for antigen presentation. Transcriptome analysis none of these plausible explanations for the reduced tumor growth showed down-regulated expression of factors implicated in an- are true in all experimental situations. These results show that ef- tigen processing and loading on MHC I: Tapasin, Tap1, Tap2, −/− fects of Ki-67 loss are wide ranging and multifactorial. Psmb8, and Psmb9 in Mki67 4T1 cells; tumors showed a Lastly, since many of the genes repressed in the absence of Ki- further down-regulation of Erap1 and B2M (Fig. 3 B and F and 67 are under the control of PRC2 and concurrent knockout of Datasets S2 and S4). Flow cytometry revealed lower expression PRC2 genes partly restores the EMT to Ki-67 KO cells (SI of MHC class I molecules H2D and H2K (Fig. 6G). MHC class I −/− Appendix, Fig. S7), we tested whether the inactivation of PRC2 in expression was also down-regulated in MKI67 MDA-MB-231 cells lacking Ki-67 could restore tumor growth. We injected cells (SI Appendix, Fig. S14), suggesting that roles of Ki-67 in −/− −/− −/− −/− Suz12 , Mki67 , and Suz12 Mki67 4T1 cells orthotopi- maintaining MHC expression are conserved across species. cally into nude mice and found that Suz12 knockout did not −/− restore Mki67 4T1 tumor growth rates, nor affect tumorige- Cells Lacking Ki-67 Are Sensitized to Drugs. The above results show nicity of control cells (SI Appendix, Fig. S13). As such, PRC2 that Ki-67 enables cell transformation, tumor growth, and me- contributes to suppressing the EMT in the absence of Ki-67, but tastasis, yet also confers efficient targeting of tumors by the it does not in itself have either pro- or antitumor activity in this immune system. Finally, we asked whether Ki-67 would also context, in contrast to other systems (25, 39). This result also influence drug responses, as, in addition to stem-like character- suggests that Ki-67 roles in tumorigenesis depend on several istics, the EMT has also been associated with resistance to che- chromatin regulatory pathways, consistent with it being a hub for motherapeutic drugs (42). We noticed that 26 genes involved in interactions with multiple chromatin regulators. drug metabolism were down-regulated in Ki-67 knockout 4T1 cells, while only 1 was up-regulated, suggesting that Ki-67 ex- Ki-67 Promotes Metastasis but Enables Efficient Antitumor Immune pression might affect sensitivity to chemotherapeutic drugs Responses. We observed that in orthotopic grafts, control 4T1 cells (Fig. 7A and SI Appendix, Fig. S15A). To test this, we performed metastasized to the lungs in 4 wk in nude mice, but metastases were an automated gene–drug screen using the Prestwick chemical −/− largely absent at this point in mice bearing Mki67 tumors library, composed of 1,283 Food and Drug Administration– (Fig. 6A). We tested whether this was due to differences in de- approved small molecules. We also included salinomycin, a tachment from the primary tumor or ability to seed metastases. To positive control found to target cancer stem cells (CSCs) (43), do this, we quantified rates of metastasis formation of control and and 6-thioguanine, which was originally used to isolate 4T1 cells Ki-67 knockout 4T1 cells by injecting cells directly into the tail vein, (22). Control 4T1 cells were sensitive to 102 drugs at 10 μM −/− then dissociating lung tissue after 3 wk, and growing cells in the concentration, while the two Mki67 clones were sensitive to 99 presence of 6-thioguanine, to which 4T1 cells are resistant. The and 98, with 82 hits common to the three cell lines (SI Appendix, number of metastatic cells that formed colonies was reduced nearly Fig. S15B and Dataset S5). This suggests that Ki-67 loss does not 100-fold in Ki-67 knockouts (Fig. 6B). This points to an essential qualitatively alter the drug sensitivity profiles. We next deter- requirement for Ki-67 in seeding metastasis, in accordance with its mined the concentration of drug needed for 50% growth inhi- apparent role in conferring stem-like characteristics. We could not bition (IC50) of 10 hits commonly used in cancer therapy. −/− test whether this reduced metastatic capacity was conserved in Importantly, Mki67 cells were markedly more sensitive to all MDA-MB-231 cells since, in our experiments, neither Ki-67 the molecules tested (Fig. 7B). As such, by supporting expression knockout nor control MDA-MB-231 xenografts generated any visi- of xenobiotic metabolism genes, Ki-67 provides cancer cells with ble lung metastases, consistent with their lack of stem-like character. a degree of protection against therapeutic drugs. We next investigated how Ki-67 expression affects tumorigenesis and metastasis in the context of an intact immune system Discussion −/− by engrafting WT or Mki67 4T1 cells into immune-proficient The above results show that Ki-67 is not required for cancer cell BALB/c mice. As expected (40), control 4T1 tumors established proliferation in vitro or in vivo in any cell type tested, but its quickly and initially regressed before regrowing (Fig. 6C). This expression critically influences all steps of tumorigenesis, including

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CDE CELL BIOLOGY

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–H2Dd fluorescein isothiocyanate (FITC)-conjugated antibody and anti–H2Kd Phycoerythrin (PE)-conjugated antibody, or control isotypes. (G, Left) Flow cytometry profiles. (G, Right) Quantification (n = 1 of each clone).

initiation, progression, and metastasis, as well as immune responses environment. This is indicated by the failure to generate intestinal anddrugsensitivity.Inbothhumanandmousemammarycarci- tumors in Ki-67 mutant mice; the reduced ability of some Ki-67 noma cells, we present evidence that this is because Ki-67 sustains knockout cancer cells to induce angiogenesis; the presence of both transcriptional programs needed for tumor cells to adapt to their epithelial and mesenchymal characteristics in Ki-67–positive

Mrouj et al. PNAS | 9of12 Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis https://doi.org/10.1073/pnas.2026507118 Downloaded by guest on September 26, 2021 AB

− − 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.

epithelial cancer cells, which are altered in cells lacking Ki-67; the consistent increase in necrosis. Additionally, it is possible that inability of the latter to colonize other tissues and give rise to tumors might arise from a smaller number of initiating cells, metastases; their increased sensitivity to drugs; and their reduced due to a loss of stem-like character. In support of this, interactions with the immune system. These phenotypes correlate spheroid formation is decreased in both highly epithelial (4T1) well with alterations of expression of genes involved in the EMT, and highly mesenchymal (MDA-MB-231) cancer cells. The antigen presentation, drug metabolism, and other cancer-associated fact that Ki-67 knockout reduces ability to seed metastases is hallmarks. consistent with this idea. Our data suggest that Ki-67 does not work by directly con- Whether or not Ki-67 expression maintains CSCs as such is trolling expression of specific genes, since in Ki-67 knockout debatable. A recent study in the human colorectal cancer cell mouse fibroblast or epithelial cancer cells, there are relatively line DLD-1 showed that Ki-67 knockout reduces the number of few common deregulated genes. Instead, we find evidence that cells expressing the antigen CD133, commonly assumed to be a Ki-67 regulates general chromatin states that affect expression marker of CSCs (13). However, in intestinal crypts, CD133 ex- of genes in fundamental biological processes. This is sup- pression is not specific to stem cells, and CD133-negative cancer ported by the fact that gene expression alterations are global cells were equally capable of sustaining tumorigenesis in a long- (i.e., well within an order of magnitude of the number of genes term serial transplantation model (44). Furthermore, the con- in the genome), with around 2,500 genes altered in non- cept that CSCs are rare preexisting populations of cells with transformed fibroblasts, nearly 5,000 genes deregulated in Ki- hard-wired CSC properties, a model that emerged from xeno- 67 knockout mouse mammary carcinoma cells, and over 9,000 graft experiments using sorted populations of hematopoietic (i.e., the majority of expressed genes) in human mammary stem cells, no longer appears valid, at least for solid tumors (45). cancer cells. We compared changes in expression of genes Lineage-tracing experiments in the intestine showed that tumors involved in biological processes that are significantly enriched arise from stem cells, which divide rapidly, make up around 10% in Ki-67 knockout 3T3, 4T1, and 4T1 tumor grafts and human of the cell in intestinal crypts, and can be regenerated from MDA-MB-231 cells (SI Appendix,Fig.S16). This clearly shows nonstem cells (45). A more recent concept is that epithelial cells the similarity of affected pathways, and even genes, among are phenotypically plastic, in a manner dependent on EMT- these different knockouts, and highlights the fact that within inducing transcription factors (46), with no such fixed entity as common pathways different individual genes may be affected CSC or non-CSC. Emerging evidence suggests that cells can among different cell types. The similarity between the changes reside in various phenotypic stages along the EMT spectrum with in different mammary carcinoma cells is apparent both in vitro concurrent expression of epithelial and mesenchymal traits and andintumors. that such hybrid states are important for carcinogenesis (33–35). Our interpretation is also consistent with the extensive alter- Our data showing that ALDH activity, which is a bona fide ations in chromatin histone marks. With such wide-ranging marker for intestinal stem cells and breast and colon CSCs (36, transcriptome changes, it is almost impossible to attribute the 37), is expressed by a significant fraction (∼10%) of WT epi- loss of tumorigenicity to changes in expression of candidate thelial cancer cells but far fewer (<3%) Ki-67 knockout cells, are genes, or even single regulatory mechanisms. Indeed, inactiva- consistent with this model. tion of the PRC2 complex did not restore tumorigenicity to Ki-67 The EMT and stemness have previously been correlated with knockout 4T1 cells. However, similar cancer-associated pathways drug metabolism (42, 47). We find that, in cancer cells with a are deregulated in 4T1 and MDA-MB-231 cells, suggesting reversion of the EMT, Ki-67 loss leads to a reduced expression of general conservation of regulatory mechanisms. genes encoding xenobiotic metabolism factors, which translates A role in cancer-promoting transcriptional programs pro- into increased sensitivity to all drugs tested in 4T1 cells. This is vides a plausible explanation for the ubiquitous expression of likely a conserved phenotype of Ki-67 loss since Ki-67 knockout Ki-67 across essentially all cancer types. Indeed, we show that HeLa, DLD1, and MCF10A cells were also found to be more Ki-67 is required for efficient tumor growth, independently of sensitive to all drugs tested (10, 13). cell proliferation, in different human and mouse cancer cell Previous studies of links between stem cell characteristics types. Although to some observers it might appear paradoxical and carcinogenesis have focused on transcriptional states, that tumor progression is reduced in Ki-67 knockouts without converging on Myc-regulated targets as the strongest link changes in cell proliferation rates, our data suggest several between stem cell and cancer cell transcriptional signatures possible mechanisms. We observed noncell-autonomous ef- (48). However, while in Ki-67 knockout NIH/3T3 cells, Myc fects in vivo, including increases in apoptosis or fibrosis, to was slightly down-regulated (Dataset S1), it was significantly which altered angiogenesis could be a contributor. Other up-regulated in 4T1 cells (Dataset S2), ruling out the possi- forms of cell death cannot be ruled out, but we did not find a bility that the reduced oncogenicity of these cells is simply due

10 of 12 | PNAS Mrouj et al. https://doi.org/10.1073/pnas.2026507118 Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis Downloaded by guest on September 26, 2021 to loss of Myc, and again favoring a global effect of Ki-67 loss case, then, paradoxically, it might be advantageous to promote, on the chromatin state. rather than hinder, cell proliferation in order for immunotherapy Our hypothesis is consistent with our previous identification of to be optimally effective. many general chromatin regulators interacting with Ki-67, including components of PRC1 and PRC2, REST, NuRD, NIF1, Materials and Methods NuA4, MLL, SET1, NoRC, and NCOA6 complexes (11). Dis- Details of all materials and methods can be found in SI Appendix. Materials rupting one such interactor, the PRC2 complex, partially, but not include all animals, cell lines, antibodies, plasmids, PCR primers, and re- fully, rescued mesenchymal traits in Ki-67 mutant epithelial agents. Methods include CRISPR-Cas9–mediated genome editing, AOM- cancer cells, consistent with the idea that Ki-67 acts through DSS–mediated colon carcinogenesis, DNA replication assay, mammosphere multiple chromatin regulatory complexes. In accordance, loss of assay, ALDH activity assay, xenografts, visualization of lung metastases, cell −/− PRC2 did not restore tumorigenicity to Mki67 4T1 cells. extracts and western blotting, histology and immunostaining, qRT-PCR, Identifying the exact biochemical mechanisms by which Ki-67 colony formation assay, RNA-sequencing library preparation and sequenc- affects the chromatin state and gene expression will require ing, ChIP-Seq, sequence data processing, gene set enrichment analysis, au- further studies. Ki-67 has no enzymatic activity and appears to be tomated drug library screen, and statistical analysis. a largely intrinsically disordered protein, potentially providing hub-like properties for protein–protein interactions. How this Data Availability. All RNA-Seq and ChIP-Seq raw data have been deposited in confers the ability to adapt to the environment is not clear, but the Gene Expression Omnibus as a SuperSeries on December 13, 2020 (accession no. GSE163114). one can speculate that its binding of a large number of general transcription regulators is involved in maintenance of metastable — ACKNOWLEDGMENTS. We thank D. Grimanelli for initial guidance on states between different transcriptional programs in other analyzing transcriptomes; A. Covinhes and Y. Buscail for immunohistochem- words, transcriptional plasticity. istry development; Montpellier Resources in Imaging for imaging and flow In conclusion, Ki-67, which is universally expressed in prolif- cytometry services; MGX for sequencing services; and P. Jay, D. Santamaria, erating cells, enables multiple steps in carcinogenesis in different M. E. Hochberg, and M. Serrano for comments on the manuscript. K.M and cancer types, which require drastic changes in transcriptional M.S. were funded by the Ligue Nationale Contre le Cancer (LNCC). K.M. and P.S. received funding from World-Wide Cancer Research (WWCR). N.A.-S. programs. Therapeutic targeting of Ki-67 itself will likely be was funded by the University of Montpellier. G.D. and D.C. were funded by challenging since it is an intrinsically disordered protein with no the French National Cancer Institute (INCa). This work was initiated and inherent enzyme activity. However, it could be of therapeutic finalized with support from LNCC Grants EL2013.LNCC/DF and EL2018.LNCC/ benefit to inhibit its effectors in control of cellular adaptation to DF and continued with support from WWCR Grant 16-0006 and INCa Grant the environment. Alternatively, our results also suggest that Ki- PLBIO18-094. The chemical library screen relied on support provided by the CELL BIOLOGY 67 confers an Achilles’ heel to cancer cells, namely their recog- Programme Opérationnel FEDER-FSE 2014-2020 Languedoc Roussillon. The Réseau d’Histologie Expérimentale de Montpellier histology facility was sup- nition by the immune-system. It will be interesting to see if ported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, the nonproliferating cells, which do not express Ki-67, are conse- European Regional Development Foundation, and the Occitanie region quently more resistant to immune-mediated killing. If this is the (FEDER-FSE 2014-2020 Languedoc Roussillon).

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