ZEB1's role as a determinant of worse cancer survival depends on the joint coexpression of the Wnt antagonist DKK1

ZEB1 has an independent prognostic value for decreased survival in a number of cancers.14 Since ZEB1 directly activates several Wnt target genes, we hypothesised that ZEB1 would inhibit the expression of Wnt antagonists and decided to explore first whether ZEB1 and Wnt antagonists have opposing effects on survival of patients with CRC.

Examination of the relapse-free survival associated with ZEB1 and Wnt antagonists in gene expression arrays of CRCs18 ,19 revealed that, as expected, high expression of ZEB1 correlated with poorer survival. In turn, expression of Wnt antagonists was either associated with better prognosis (sFRP3) or had no significant association with survival (see online supplementary table S1). Unexpectedly, we found that DKK1 and sFRP4 were associated with poorer prognosis, indicating a potential tumour-promoting role of these Wnt antagonists in CRCs. Given that DKK1, like ZEB1, is induced by canonical Wnt signalling,3 we investigated whether the joint expression of DKK1 and ZEB1 affects overall survival in CRCs. Interestingly, using survival data for 928 patients from published databases (see Methods), we found that patients displaying high expression of both DKK1 and ZEB1 had a lower survival rate than those with high levels of ZEB1 but low DKK1 (figure 1A). In other words, the maximum effect of ZEB1 as a predictor of reduced survival requires high levels of DKK1. This suggests that, at least for some of its tumour-promoting functions, ZEB1 depends on DKK1 expression.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

ZEB1 and DKK1 expression associate in colorectal carcinomas (CRCs) where the maximum effect of ZEB1 as a factor of poorer survival depends on its simultaneous expression with DKK1. (A) Patients with the highest expression of ZEB1 and DKK1 (red line) have shorter relapse-free survival than those with high expression of ZEB1 but low expression of DKK1 (yellow line) that in turn have worse survival than those with high DKK1 but low ZEB1 (green line) or that display low levels of both genes (blue line). The survival probability of patients with CRC was represented in a Kaplan-Meier plot according to their median expression of ZEB1 and DKK1 (see Methods). (B) Expression of ZEB1 and DKK1 determines overall cell viability in CRC cells. ZEB1 and DKK1 were knocked down in SW480 cells by transient transfection of 100 nM of specific small interfering RNA (siRNAs) against ZEB1 (siZEB1-A) and/or DKK1 (siDKK1) or transfected with 100–200 nM of a control siRNA (siCtl). Cell viability was determined at 96 hours by an MTT assay. (C) ZEB1 and DKK1 expression associate in CRCs. Expression of ZEB1—segregated by the median—in gene arrays of CRCs (see Methods) correlated positively with DKK1 levels. (D) ZEB1 and DKK1 expression correlate in a panel of CRC cell lines. SW480, COLO320, SW620, DLD1 and LS174T CRC cells were assessed for ZEB1 and DKK1 mRNA levels by quantitative real-time PCR with respect to GAPDH.

In light of these results, we next investigated whether downregulation of ZEB1 and/or DKK1 affects overall cell viability of SW480 cells, a commonly used Wnt-active CRC cell line, which harbour a mutation in APC at 1338 bp that results in a stop codon and a truncated protein, methylated p16^INK4A (CDKN2A), phosphorylated Rb protein and mutant TP53 (R273H and P309S). Compared with an siRNA control (siCtl), knockdown of either ZEB1 or DKK1 with specific siRNAs (siZEB1 and siDKK1, respectively) reduced overall cell viability in SW480 CRC cells (figure 1B and online supplementary figure S1A). Simultaneous interference of ZEB1 and DKK1 did not have an additional effect over ZEB1 knockdown alone suggesting that in cell viability—although not in relapse-free survival—DKK1 may act in the same functional pathway as ZEB1 (figure 1B). We next tested whether recombinant human DKK1 (rhDKK1) was able to revert the effect of siDKK1 on cell viability. Despite the fact that SW480 cells have a truncated APC, they are still responsive to Wnt ligands and antagonists.20 As control we used recombinant human DKK3 (rhDKK3), a protein homologous to DKK1 that through a different receptor and mechanism also reduces nuclear β-catenin in SW480 cells and, as other Wnt antagonists, can either activate or repress Wnt-mediated signalling depending on the cell type.21 ,22 We found that although both rhDKK1 and rhDKK3 displayed similar effectiveness blocking Wnt signalling in SW480 cells, only rhDKK1 reverted the effect of siDKK1 on cell viability (see online supplementary figures S1B and S1C).

We then examined whether ZEB1 and DKK1 were correlated in primary CRCs. Examination of 1557 cases from expression arrays of CRCs (see supplementary figure S1D). This positive correlation between ZEB1 and DKK1 was also observed in a panel of CRC cell lines (figure 1D). SW620 cells originated from CRC metastasis in the same patient than SW480 cells and although they carry identical mutations they have epigenetic differences. Expression of DKK1 did not associate with ZEB2, a second member of the ZEB family and highly homologous to ZEB123 (see online supplementary figure S1E). Altogether, these results indicate that ZEB1 and DKK1 are coexpressed in CRCs, where they jointly determine a worse prognosis.

ZEB1 transcriptionally activates DKK1 expression

The above data prompted us to investigate whether ZEB1 may activate DKK1 expression. We first examined whether deletion of the Zeb1 gene alters Dkk1 expression in the intestinal tract of Zeb1-deficient mice. Compared with wild-type littermates, Dkk1 expression was reduced in the intestinal tract of Zeb1 (+/−) mice—Zeb1 (−/−) mice die perinatally24—corroborating the in vivo association between both genes (figure 2A).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

ZEB1 transcriptionally activates DKK1 expression by direct binding to its promoter. (A) In vivo expression of Dkk1 depends on Zeb1. mRNA levels for Zeb1 and Dkk1 in small intestine and colon from Zeb1 (+/+) and (+/−) mice aged 3 months. Results shown are the average of four mice for each genotype. (B) Knockdown of endogenous ZEB1 downregulates endogenous DKK1 mRNA in colorectal carcinoma (CRC) cells. Left panel: SW480 cells were transiently transfected with 100 nM of siRNAs against ZEB1 (siZEB1-A and siZEB1-B) or siCtl. Relative mRNA levels of ZEB1 and DKK1 were determined by quantitative real-time (qRT)-PCR. Right panel: as in left panel but using SW480 cells stably infected with an shRNA control (shCtl) or two independent sets of shRNAs against ZEB1 (shZEB1-A and shZEB1-B). (C) Knockdown of endogenous ZEB1 protein downregulates endogenous DKK1 protein in CRC cells. As in (B), SW480 cells were transiently (siZEB1-A, left panel) or stably (shZEB1-A, right panel) knocked down for ZEB1. Cell lysates were then blotted for DKK1 (clone 2A5) and ZEB1 (H-102) along with α-tubulin (B5-1-2) as loading control. (D) ZEB1 binds to the DKK1 promoter. ChIP assays using DNA from SW480 cells immunoprecipitated without antibody (Ab) or with Abs against ZEB1 (E-20X) or goat IgG control. Regions from the DKK1 promoter—either lacking consensus binding sites for ZEB1 (non-binding site (NBS) −757/−631 bp) or containing a consensus ZEB1 binding site at −490 bp—or from the GAPDH promoter were amplified by qRT-PCR. For each promoter fragment, the condition without Ab was equalled to 100. (E) Knockdown of ZEB1 downregulates DKK1 transcription. Left panel: SW480 cells transiently knocked down for ZEB1 (siZEB1-A and siZEB1-B) or transfected with siCtl were co-transfected with 0.25 μg of either wild-type DKK1 promoter (DKK1_wt) or a version of it where the ZEB1 binding site at −490 bp has been mutated (DKK1_ZEB1mut). In all panels of this figure, transcription is assessed as relative luciferase units(RLU). Right panel: as in left panel but using SW480 cells stably interfered for ZEB1 (shZEB1-A and shZEB1-B) or infected with an shRNA control (shCtl). (F) Overexpression of ZEB1 activates DKK1 transcription. As in (E), SW480 cells were transfected with either DKK1_wt or DKK1_ZEB1mut but were then cotransfected with 0.25 μg of an empty expression vector or the corresponding equal molar amount of the same vector encoding full-length ZEB1. (G) Transcriptional activation of the DKK1 promoter by ZEB1 involves p300. SW480 cells were transfected with 0.25 μg of DKK1_wt promoter along with 0.25 μg of an empty expression vector or the corresponding equal molar amount of full-length ZEB1 or a version of ZEB1 lacking the N-terminal region (ZEB1ΔNterm) in the presence or absence of 0.5 μg of VP16-p300. (H) p300 binds to the DKK1 promoter in a ZEB1-dependent manner. ChIP assays using DNA from SW480 cells immunoprecipitated without Ab or with Abs against p300 (N-15) or rabbit IgG serum control. The NBS region used in (D) was included as control. For each promoter fragment, the condition without Ab was equalled to 100. (I) ZEB1 and TCF4 cooperate in the regulation of DKK1. As in (E), SW480 cells were transfected with 0.25 μg of either DKK1_wt or DKK1_ZEB1mut reporters and 0.25 μg of the expression vectors for ZEB1 and/or TCF4 (denoted by ‘+’) or equimolar amounts of their corresponding empty vectors (denoted by ‘−’).

We then examined the effect on endogenous DKK1 expression of knocking down endogenous ZEB1 in three CRC cell lines expressing high levels of ZEB1. Compared with siCtl, transient knockdown of ZEB1 with two specific siRNAs (siZEB1-A and siZEB1-B) resulted in the downregulation of endogenous DKK1 mRNA levels (figure 2B, left panel, and online supplementary figures S2A and S2B). Similar downregulation of DKK1 was observed when ZEB1 was stably knocked down in SW480 cells using two different shRNAs (to generate SW480-shZEB1-A and SW480-shZEB1-B cells) and compared with cells transfected with an shRNA control (SW480-shCtl cells) (figure 2B, right panel). In contrast, knockdown of ZEB2 in ZEB2-positive COLO320 CRC cells did not affect DKK1 mRNA levels (see online supplementary figure S2C). Positive regulation of DKK1 by ZEB1 was also examined at the protein level. Compared with control siRNA or shRNA, expression of DKK1 protein was downregulated upon transient and stable knockdown of ZEB1 in SW480 CRC cells (figure 2C, left and right panels, respectively). Altogether, these results indicate that ZEB1 induces DKK1 expression in CRC cell lines.

ZEB1 binds to a subset of E-box and E-box-like sequences in the regulatory regions of its target genes.10 We therefore investigated whether ZEB1-mediated induction of DKK1 occurs at the transcriptional level. Examination of the first 1 kb of the human DKK1 promoter revealed the existence of several ZEB1 consensus binding sites including two high-affinity sequences at −490 and −880 bp. Accordingly, the transcriptional activity of serial deletions of the DKK1 promoter increased when the site at −490 bp was included (see online supplementary figure S2D). The ability of endogenous ZEB1 to directly bind to the DKK1 promoter was tested for the site at −490 bp by ChIP assay. An antibody against ZEB1, but not its respective matched specie IgG control, immunoprecipitated a region of the human DKK1 promoter containing the site at −490 bp, but not a region lacking consensus binding sites for ZEB1 (figure 2D). ZEB1 also failed to bind to the GAPDH promoter, whose expression is not regulated by ZEB1 (figure 2D).

To test the functionality of the ZEB1 binding site at −490 bp, this sequence was mutated within the −535 bp DKK1 promoter reporter to a sequence known not to bind ZEB1 and the resulting mutant DKK1 promoter (DKK1_ZEB1mut) was compared with the wild-type DKK1 promoter (DKK1_wt) for its regulation by ZEB1. Mutation of the ZEB1 site in DKK1_wt promoter reduced its basal activity in SW480 and SW620 CRC cells (figure 2E and online supplementary figure S2E). Transient and stable knockdown of ZEB1 in both CRC cell lines resulted in a downregulation of the activity of DKK1_wt but not of DKK1_ZEB1mut promoter (figure 2E, left and right panels, respectively, and online supplementary figure S2E). We also examined the response of the wild-type and mutant versions of the DKK1 promoter to ZEB1 overexpression in SW480 and SW620 cells. While exogenous ZEB1 further activated transcription of the DKK1_wt promoter, it had no effect on DKK1_ZEB1mut promoter (figure 2F and online supplementary figure S2F).

ZEB1 activates transcription through recruitment of the p300 histone acetyltransferase to its N-terminal region.12 ,13 Overexpression of ZEB1 upregulates Wnt target genes in a p300-dependent manner in cells with active Wnt signalling, but not in cells where this pathway is inactive.15 Therefore, we investigated the involvement of p300 in ZEB1-mediated activation of DKK1—that as noted earlier is a direct Wnt target—in SW480 and SW620 cells, two Wnt-active CRC cell lines that therefore express high levels of ZEB1 (figure 1D). A version of p300 where its cDNA (EP300) is fused to the activation domain of the herpes simplex virus VP16 protein (VP16-p300) enhanced ZEB1-induced activation of DKK1_wt promoter (figure 2G). In contrast, deletion of the N-terminal region of ZEB1 (ZEB1ΔNterm) eliminated most of the transcriptional activation effect of ZEB1 on the DKK1_wt promoter (figure 2G). The recruitment of p300 by full-length ZEB1 was confirmed by a ChIP assay where a p300 antibody, but not a matched specie IgG control, precipitated the fragment of the DKK1 promoter harbouring the ZEB1 binding site at −490 bp, but not a fragment of this promoter lacking ZEB1 binding sites (figure 2H).

ZEB1 and TCF4 mutually modulate their transcriptional activities in the regulation of selected Wnt target genes,15 and we found here that both transcription factors cooperate in the regulation of the DKK1 promoter in SW480 and SW620 CRC cells (figure 2I and online supplementary figure S2G). Altogether, the above results demonstrate that ZEB1 binds to the DKK1 promoter to directly drive its transcription through a mechanism involving p300 and in cooperation with TCF4.

ZEB1, through its induction of DKK1, inhibits senescence-associated genes

The 1557 cases of CRC in figures 1C and online supplementary figure S1D were classified in four cohorts based on their expression of ZEB1 and DKK1 above or below the upper quartile. Analysis of the gene signature of these cohorts revealed that, compared with CRCs where both ZEB1 and DKK1 were above the upper quartile (cohort 1), 429 genes were upregulated when the expression of either ZEB1 or DKK1 was below that quartile (cohorts 2 and 3, respectively) (see online supplementary table S2). The expression of these genes was further increased when both ZEB1 and DKK1 levels were in the three lower quartiles (cohort 4). Interestingly, within this subset of genes upregulated when ZEB1 and DKK1 expression was low, around 10% of them were associated with senescence, suggesting that ZEB1 and DKK1 may cooperate in the repression of cellular senescence (figure 3A).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

ZEB1 and DKK1 cooperate in repressing a senescence-associated gene signature. (A) Heat map of senescence-associated genes upregulated by low levels of ZEB1 and/or DKK1. A total of 1157 colorectal carcinomas (CRCs) from gene arrays (see Methods) were classified in four cohorts according to their levels of ZEB1 and/or DKK1 expression. Cohort 1: expression of both genes in the upper quartile; cohort 2: ZEB1 in upper quartile, DKK1 below Q4; cohort 3: DKK1 in the upper quartile, ZEB1 below Q4; cohort 4: both genes below Q4. (B) ZEB1 and DKK1 repress senescence-associated genes. Stably interfered/transfected SW480 cell lines—namely, SW480-shCtl+vector, SW480-shZEB1-A+vector, SW480-shZEB1-A+DKK1, SW480-shZEB1-B+vector and SW480-shZEB1-B+DKK1—were assessed for relative mRNA levels of selected senescence-associated genes by quantitative real-time (qRT)-PCR. Data are represented as the difference in relative mRNA levels between each of the cell lines and shCtl-infected cells. (C) ZEB1 represses CDKN2A and CDKN2B independently of DKK1. Stable SW480 cells in (B) were assessed for relative mRNA levels of CDKN2A (p16^INK4a) and CDKN2B (p15^INK4b) by qRT-PCR. Data are represented as the difference in relative mRNA levels between each cell line and shCtl-infected cells.

We therefore validated the potential regulation by ZEB1 and/or DKK1 of some of these senescence-related genes. To that effect, SW480-shCtl, SW480-shZEB1-A and SW480-shZEB1-B cells used in previous figures were stably transfected with an expression vector for DKK1 (to generate SW480-shCtl+DKK1, SW480-shZEB1-A+DKK1 and SW480-shZEB1-B+DKK1 cell lines, respectively) or their corresponding empty vector equivalents (to generate SW480-shCtl+vector, SW480-shZEB1-A+vector and SW480-shZEB1-B+vector cell lines, respectively) (see online supplementary figure S3A for ZEB1 and DKK1 levels in these stable cell lines).

The upregulation of senescence-associated genes in CRCs with low expression of ZEB1 and/or DKK1 (figure 3A) suggested that ZEB1 and DKK1 are repressing their expression. Using these stable cell lines, we validated the regulation by ZEB1 and/or DKK1 of several of these senescence-associated genes. Namely, histone variant macroH2A1 (encoded by H2AFY)—an integral component of SAHF and whose knockdown blocks SAHF formation and cellular senescence,6 GLB1—that encodes lysosomal β-galactosidase and whose mRNA levels as well as enzymatic activity at pH 6.0 increase in senescent cells, commonly referred as SA β-gal activity25 ,26—RPL5, EIF3K, EHMT2 and AATF.27–30 Indeed, we found that mRNA levels for all these genes increased in SW480-shZEB1-A and SW480-shZEB1-B cells with respect to SW480-shCtl cells (figure 3B). In turn, overexpression of DKK1 reverted the effect of ZEB1 knockdown (figure 3B).

Cell cycle inhibitors p16^INK4A (CDKN2A) and p15^INK4b (CDKN2B) are upstream of Rb1 and accumulate in senescent cells.5 Although CDKN2A is methylated in SW480 cells, expression of CDKN2A and CDKN2B was upregulated in SW480 cells knocked down for ZEB1 (SW480-shZEB1-A+vector and SW480-shZEB1-B+vector) compared with SW480-shCtl+vector cells (figure 3C). In that line, CDKN2A and CDKN2B are also repressed by ZEB1 in lung epithelial cells and mouse embryo fibroblasts (MEFs).13 ,31 In contrast, overexpression of DKK1 in SW480-shZEB1-A+DKK1 and SW480-shZEB1-B+DKK1 cells failed to revert this increase, suggesting that CDKN2A and CDKN2B are regulated by ZEB1 but not by DKK1.

ZEB1, through its induction of DKK1, inhibits SAHF formation and cell senescence

Downregulation of Wnt3a is both necessary and sufficient for the translocation of the chaperone HIRA to nuclear bodies and accelerates the assembly of SAHF, which are required to trigger senescence.9 Since ZEB1 is both a target and an effector of Wnt signalling, we first tested whether ZEB1 could mediate Wnt3a-induced inhibition of senescence in SW480 CRC cells. As noted earlier, SW480 cells harbour a stop codon mutation in APC. Nevertheless, truncated forms of the APC protein can still bind β-catenin and cells carrying them are responsive to Wnt ligands.20 SW480-shCtl and SW480-shZEB1-A cells were cultured in the presence or absence of Wnt3a and their senescence was assessed by staining for SA β-gal activity.25 We found that ZEB1 knockdown increased the number of SA β-gal-positive cells and that Wnt3a inhibited basal SA β-gal activity in SW480-shCtl cells but not in SW480-shZEB1-A cells (figure 4A). ZEB1 interference yielded similar results in SW620 CRC cells treated with Wnt3a (see online supplementary figure S4A). Activation of Wnt signalling by Wnt3a was confirmed by the upregulation of Wnt targets ZEB1, DKK1 and AXIN2 (see online supplementary figure S4B). These results indicate that ZEB1 mediates the Wnt-induced inhibition of senescence and that ZEB1 knockdown triggers senescence in CRC cells.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

ZEB1, through its induction of DKK1, inhibits senescence-associated heterochromatin foci (SAHF) formation and cell senescence. (A) ZEB1 mediates Wnt-induced inhibition of cellular senescence. SW480-shCtl and SW480-shZEB1-A cells were cultured for 72 hours in the presence of conditioned medium from L cells stably transfected with a vector encoding recombinant Wnt3a (L-Wnt3a cm) or its corresponding empty vector (L-Ctl cm). Senescence was assessed by staining for senescent associated β-galactosidase (SA β-gal). Representative pictures are displayed in the left panel and quantification of the relative number of SA β-gal-positive cells from four independent experiments is shown in the right panel, where the first condition was arbitrarily set to 100. (B) ZEB1 and DKK1 inhibit senescence in colorectal carcinoma (CRC) cells. SW480 cells transiently knocked down with siZEB1-A and/or siDKK1 were assessed for SA β-gal staining. As in (A), representative pictures are shown in the left panel and quantification of SA β-gal-positive cells in the right panel. (C) Blockade of DKK1 induces senescence. SW480 cells were incubated with either Dulbecco's Modified Eagle Medium (no antibody (Ab)), 5 μg/mL of goat IgG serum or 5 μg/mL of a goat blocking anti-DKK1 Ab (clone AF-1096). SA β-gal-positive cells were quantified after 24 hours. Representative pictures are shown in online supplementary figure S4D. (D) Right panel: quantification of SA β-gal-positive cells in the following stable cell lines: SW480-shCtl+vector, SW480-shCtl+DKK1, SW480-shZEB1-A+vector, SW480-shZEB1-A+DKK1, SW480-shZEB1-B+vector and SW480-shZEB1-B+DKK1. Representative pictures for the first four cell lines are shown in the left panel. (E) Induction of senescence by shZEB1 is reverted by rhDKK1, but not rhDKK3. SW480-shCtl and SW480-shZEB1-A cells were cultured in complete medium alone (untreated) or supplemented with 200 ng/mL of either rhDKK1 or rhDKK3. SA β-gal-positive cells were quantified after 24 hours with the first condition arbitrarily set to 100. Representative pictures are shown in online supplementary figure S4E. (F) ZEB1 and DKK1 inhibit formation of SAHF in CRC cells. SW480 cells were transiently transfected with 100 nM of siZEB1-A and/or siDKK1 or 100–200 nM of siCtl. Cells were stained with DAPI (blue) and an Ab against macroH2A1 (clone 4827) to assess for nuclear staining and SAHF formation, respectively. Representative merged pictures are shown here, while single staining captures are shown in online supplementary figure S4F. Scale bars represent 10 μm. (G) Quantification of the relative expression of DAPI and macroH2A1 in (F). RGB profiles display relative levels of macroH2A1 (green) or DAPI (blue) across a cell section—as marked by a white line in panel (F).

We next investigated whether DKK1 also inhibits senescence in cancer cells. SW480 cells were transiently interfered with siCtl, specific siRNAs for either ZEB1 (siZEB1-A) or DKK1 (siDKK1) or a combination of siZEB1-A and siDKK1 and then examined for SA β-gal staining. Compared to siCtl, single interference of either ZEB1 or DKK1 resulted in an increase in the number of SA β-gal-positive cells (figure 4B). Induction of senescence by siDKK1 was reverted by rhDKK1, but not by rhDKK3 (see online supplementary figure S4C). These results indicate that inhibition of senescence by DKK1 occurs independently of its role as a Wnt antagonist since both rhDKK1 and rhDKK3 were equally efficient blocking Wnt signalling (ref 22 and online supplementary figure S1B). The number of senescent cells following joint knockdown of both ZEB1 and DKK1 was similar to that observed with just single ZEB1 knockdown (figure 4B), thus suggesting that DKK1 inhibits senescence through the same functional pathway as that by ZEB1. Lastly, the induction of senescence by siDKK1 was corroborated with an anti-DKK1 blocking antibody (figures 4C and online supplementary figure S4D).

We then examined whether exogenous overexpression of DKK1 could revert the senescence induced by ZEB1 knockdown. To that effect, the stable SW480 cells used in figures 3B,C were assessed for senescence by SA β-gal staining. Like in transient interference, stable knockdown of ZEB1 increased the number of SA β-gal-positive cells that, interestingly, was reverted by overexpression of DKK1 (figure 4D). The increase in SA β-gal-positive cells induced by shZEB1 was reverted by rhDKK1 but not by rhDKK3 (figure 4E and online supplementary figure S4E). Once again, the failure of DKK3 to inhibit senescence indicates that DKK1 mediates ZEB1-induced inhibition of senescence through a mechanism that is independent of its role as Wnt antagonist. It is worth noting that since SW480 cells are Wnt-active and express high levels of ZEB1, exogenous DKK1 also inhibited basal levels of senescence in shCtl cells. The above data allow us to conclude that inhibition of senescence by ZEB1 is mediated, at least in part, by activating DKK1 expression.

Lastly, we examined whether manipulation of ZEB1 and/or DKK1 expression alters SAHF formation triggered by macroH2A1. We found that knockdown of ZEB1 or DKK1 increased the assembly of SAHF (figures 4F,G and online supplementary figure S4F). In parallel with figure 4B, simultaneous knockdown of ZEB1 and DKK1 did not increase SAHF formation vis-à-vis single ZEB1 knockdown suggesting, once again, that DKK1 may exert its effects on senescence through the same functional pathway than ZEB1.

Inhibition of senescence by ZEB1 requires repression of H2AFY

Contrary to ZEB1, macroH2A1 suppresses tumour initiation and progression and associates with better survival in multiple types of cancer.32 Therefore, out of all the genes validated in figure 3B, we focused on H2AFY. First, we confirmed the inhibition of macroH2A1 by ZEB1 and DKK1 at the protein level. Knockdown of ZEB1 and DKK1—with siZEB1-A and siDKK1—upregulated macroH2A1 protein expression (figure 5A). Repression of macroH2A1 by ZEB1 was also tested in vivo. Compared with their wild-type littermates, expression of H2afy was upregulated in the intestine of Zeb1 (+/−) mice (figure 5B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5

Inhibition of senescence by ZEB1 and/or DKK1 depends on the induction of CTBP to repress H2AFY. (A) ZEB1 and DKK1 inhibit macroH2A1 expression. SW480 cells were transiently interfered with siCtl, siZEB1-A or siDKK1. Cell lysates were then blotted for macroH2A1 (clone 4827), ZEB1 (H-102) and DKK1 (2A5) along with α-tubulin (B5-1-2) as loading control. (B) In vivo expression of macroH2A1 depends on ZEB1. H2afy mRNA levels in the small intestine of Zeb1 (+/+) and Zeb1 (+/−) mice aged 3 months. See figure 2A for comparison of Zeb1 and Dkk1 levels in both genotypes. (C) ZEB1 binds to the H2AFY promoter. Quantitative real-time (qRT)-PCR amplification of fragments of the H2AFY—either containing the consensus ZEB1 binding site at −1059 bp or with no binding sites (NBS) for ZEB1—and GAPDH promoters that have been immunoprecipitated in ChIP assays from SW480 cells without antibody (Ab) or with Abs against ZEB1 (E-20X) or goat IgG serum control. The condition without Ab was equalled to 100. (D) Inhibition of senescence by ZEB1 and DKK1 depends on their repression of macroH2A1. Stable SW480-shCtl+vector, SW480-shCtl+DKK1, SW480-shZEB1-A+vector and SW480-shZEB1-A+DKK1 cell lines were transiently interfered with either a siRNA specific for H2AFY (siH2AFY) or siCtl. H2AFY knockdown efficiency in each condition is shown in online supplementary figure S5B. Representative pictures of senescent associated β-galactosidase (SA β-gal)-positive cells are shown here and quantified in online supplementary figure S5C. (E) ZEB1 represses H2AFY through CtBP. SW480 cells were interfered for siCtl or a siRNA against CTBP (siCTBP) and H2AFY mRNA determined by qRT-PCR. (F) Repression of H2AFY by ZEB1 depends on CtBP. SW480 cells were transfected with an expression vector encoding full-length ZEB1, ZEB1-CID_mut or the corresponding empty vector. H2AFY mRNA was determined by qRT-PCR. (G) DKK1 induces CTBP mRNA and protein expression. Left panel: CTBP mRNA levels from SW480-shCtl+vector and SW480-shCtl+DKK1 cells were assessed by qRT-PCR. Right panel: Lysates from these cells were blotted for CtBP (E12), DKK1 (2A5) and ZEB1 (H-102) along with α-tubulin (B5-1-2). (H) rhDKK1 but not rhDKK3 induces CTBP mRNA. SW480 cells were cultured during 24 hours with complete medium alone (untreated) or supplemented with 200 ng/mL of either rhDKK1 or rhDKK3 before CTBP mRNA expression was assessed by qRT-PCR. (I) DKK1 enhances ZEB1's repressor activity; 0.25 μg of expression vectors for Gal4's DNA binding domain (Gal4) or equal molar amounts of Gal4-ZEB1-CID or Gal4-ZEB1-CID_mut were transfected in SW480-shCtl+vector or SW480-shCtl+DKK1 cells along with 0.25 μg of a Gal4×5-SV40-luciferase reporter. (J) Inhibition of CtBP induces senescence in colorectal carcinoma (CRC) cells. SW480 cells were culture during 24 hours in the presence or absence of 10 mM of the CtBP inhibitor methylthiobutyrate (MTOB) and stained for SA β-gal. Quantification of SA β-gal-positive cells is shown in online supplementary figure S5D. DMEM, Dulbecco's Modified Eagle Medium; RLU, relative luciferase units.

Next, we investigated whether inhibition of H2AFY by ZEB1 involves direct binding of ZEB1 to the H2AFY promoter. We identified two high-affinity binding sites for ZEB1 at −1059 and −1687 bp and tested the ability of the former to recruit ZEB1 in ChIP assays (figure 5C and online supplementary figure S5A). An antibody against ZEB1, but not its corresponding matched specie IgG control, immunoprecipitated a region of the H2AFY promoter containing this binding site but not a region lacking consensus binding sites for ZEB1 (figure 5C). The GAPDH promoter, which is not regulated by ZEB1, did not recruit ZEB1.

Repression of H2AFY by ZEB1 and DKK1 prompted us to investigate whether inhibition of senescence by ZEB1 and DKK1 depends on macroH2A1. The stable SW480 cell lines used in figures 3B,C—with ZEB1 and DKK1 expression knocked down or overexpressed, respectively—were now transiently transfected with siCtl or a siRNA against H2AFY (siH2AFY) and tested for SA β-gal staining (see online supplementary figure S5B for H2AFY knockdown efficiency). As shown in figure 5D and online supplementary figure S5C, siH2AFY inhibited both basal SA β-gal staining and the senescence induced by ZEB1 knockdown. Once again, DKK1 overexpression reverted the senescence induced by shZEB1, which was further reduced by siH2AFY. These results indicate that the ability of ZEB1 and DKK1 to repress senescence depends on their repression effect on H2AFY.

ZEB1 represses H2AFY expression and senescence through the subsequent induction of DKK1, mutant p53, Mdm2 and CtBP

ZEB1 represses gene expression through the recruitment of non-DNA binding corepressors, chiefly CtBP.11 ,13 ,33 Therefore, we first checked whether CtBP represses H2AFY expression. Indeed, as shown in figure 5E, transient knockdown of CtBP with a specific siRNA (siCtBP) upregulated H2AFY mRNA levels. Furthermore, mutation of the CtBP binding sites in ZEB1 (ZEB1-CID_mut) hampered ZEB1's repression of H2AFY (figure 5F).

The ability of DKK1 to mediate ZEB1 inhibition of H2AFY prompted us to investigate whether DKK1 could induce CtBP expression. Indeed, overexpression of DKK1 upregulated CtBP expression at the mRNA and protein levels (figure 5G and online supplementary figure S5D). Likewise, addition of rhDKK1, but not rhDKK3, upregulated CTBP expression (figure 5H). We then examined whether the increased CtBP expression induced by DKK1 enhances ZEB1 repressor activity. The region of ZEB1 containing the CtBP interacting domain (CID), either wild-type or mutated to a sequence unable to bind ZEB1, was fused to the DNA binding domain of the yeast protein Gal4 to create Gal4-ZEB1-CID and Gal4-ZEB1-CID_mut. Both constructs were then cotransfected with a luciferase reporter containing UAS DNA binding sites and driven by the SV40 promoter into SW480 cells stably expressing DKK1 (SW480+DKK1 cells) or its corresponding empty vector (SW480+vector cells). DKK1 overexpression greatly enhanced the ability of ZEB1 to repress transcription (figure 5I).

CtBP binding to 2-keto-4-methylthiobutyrate (MTOB), an intermediate in the methionine salvage pathway, relieves CtBP-mediated transcriptional repression in breast cancer cells.34 We found that blocking of CtBP activity in SW480 cells by MTOB induced senescence (figure 5J and online supplementary figure S5E) and upregulated H2AFY (see online supplementary figure S5F). Altogether, these results indicate that DKK1 cooperates with ZEB1 in the repression of H2AFY and senescence by upregulating CtBP.

Mdm2 is an E3 ubiquitin-ligase best known for targeting the p53 tumour suppressor for proteasomal degradation, although Mdm2 also promotes tumour progression through transcriptional regulation and histone modification in a p53-independent manner.35–37 Induction of senescence is in fact one of the key mechanisms through which p53 suppresses tumourigenesis and, consequently, Mdm2 antagonists like nutlin-3a trigger senescence.38 ,39 Of note, Mdm2 binds to and cooperates with CtBP to repress transcription.40 This evidence prompted us to investigate whether mutant p53 and/or Mdm2 are involved in the CtBP-mediated repression of H2AFY and senescence by ZEB1 and DKK1.

It is now well established that mutations in TP53 not simply eliminate wild-type TP53 tumour suppressor functions but also confer cancer cells with oncogenic hallmarks to promote tumour progression such as overcoming of senescence.41 ,42 Wild-type and mutant p53 proteins differ in their target genes and also in the direction (eg, activation or repression) and degree (eg, deficient or enhanced transcription) of their regulation.43 Interestingly, mutant p53—including some variants with mutations in the DNA binding domain—retain the ability to bind and transcriptionally activate the MDM2 promoter.44 ,45 Thus, some mutant TP53 cells, like SW480 CRC cells, still express MDM2.46

We found that overexpression of DKK1 or addition of rhDKK1, but not rhDKK3, in mutant SW480 cells upregulated mutant TP53 and MDM2 expression in these cells (figure 6A, B). In turn, DKK1 knockdown downregulated both mutant TP53 and MDM2 (figure 6C) and this downregulation was reverted by addition of rhDKK1, but not of rhDKK3 (see online supplementary figure S6A). The downstream effect of DKK1 via mutant p53 was corroborated by the knockdown of the latter with a specific siRNA (siTP53). siTP53, but not siCtl, downregulated mRNA expression levels of MDM2 and CTBP while increasing those of H2AFY (figure 6D).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6

ZEB1 represses H2AFY through the subsequent induction of DKK1, mutant p53, Mdm2 and CtBP. (A) DKK1 induces TP53 and MDM2. mRNA levels for both genes were determined by quantitative real-time (qRT)-PCR in SW480-shCtl+vector and SW480-shCtl+DKK1 cells. (B) rhDKK1 but not rhDKK3 induces mutant TP53 and MDM2 mRNA. Mutant TP53 SW480 cells were cultured during 24 hours in complete medium alone (untreated) or supplemented with 200 ng/mL of either rhDKK1 or rhDKK3. TP53 and MDM2 mRNA expression was assessed by qRT-PCR. (C) Knockdown of DKK1 downregulates TP53 and MDM2. SW480 cells were interfered for siCtl or siDKK1 and TP53 and MDM2 mRNA levels assessed by qRT-PCR. (D) Mutant TP53 activates MDM2 and CTBP and represses H2AFY. SW480 cells were interfered for siCtl or a siRNA against TP53 (siTP53) and mRNA levels of mutant TP53, MDM2, CTBP and H2AFY were assessed by qRT-PCR. (E) MDM2 represses CTBP and subsequently induces H2AFY. SW480 cells were interfered for siCtl or a siRNA against MDM2 (siMDM2) and mRNA levels for CTBP (left panel) and H2AFY (right panel) were assessed by qRT-PCR. (F) MDM2 is downstream of DKK1 and induction of CTBP by DKK1 requires of MDM2. SW480 cells were interfered with 100 nM siCtl or siMDM2 and then transfected with either 1 μg of an expression plasmid encoding DKK1 or an equal molar amount of its corresponding empty vector. DKK1, MDM2 and CtBP mRNA levels were assessed by qRT-PCR.

Interestingly, wild-type and mutant p53 have been described to have opposite functions with regard to EMT.47 Wild-type p53 inhibits the EMT by direct transactivation of miR-200c, which targets ZEB1 mRNA for decay. Meanwhile, mutant p53 proteins induce an EMT.47 Accordingly, we found that siTP53 downregulated ZEB1 in SW480 cells (figure 6D).

MDM2 expression was also knocked down with a specific siRNA (siMDM2). siMDM2 downregulated CtBP mRNA and protein levels (figure 6E and online supplementary figure S6B) and increased H2AFY mRNA expression (figure 6E). MDM2 knockdown was also used to confirm the sequencing of the regulatory cascade from DKK1 to CtBP. SW480 cells interfered with siCtl or siMDM2 were transfected with an expression plasmid for DKK1 or its corresponding empty vector. As shown in figure 6F, MDM2 knockdown blocked the upregulation of CTBP by DKK1 as shown in figure 5G. Altogether, the above results indicate that in mutant TP53 cells DKK1 cooperates with ZEB1 in the repression of H2AFY through the subsequent induction of mutants TP53, MDM2 and CTBP.

Downregulation of Zeb1 is sufficient to trigger in vivo senescence, reduce tumour load and improve survival in a mouse model of colon cancer

ZEB1 is not expressed in the normal colonic mucosa or by well-differentiated cancer cells in the tumour centre of CRCs. Instead, ZEB1 is found in dedifferentiated malignant cells at the CRC invasive front.14 ,48 ZEB1 is also expressed by stromal cells in both normal colorectal mucosa and CRCs.10 ,14 ,48 In contrast, macroH2A1 is strongly expressed in normal colonic mucosa, but not in invasive CRCs (ref. 49, and online supplementary figure S7A). In light of this reverse pattern of expression, we tested the correlation of ZEB1, DKK1 and macroH2A1 at the invasive front of sporadic CRCs. Evaluation of ZEB1 and DKK1 staining in a tissue microarray of 53 primary human CRCs stages I–IV revealed a positive correlation between both proteins (Spearman's ρ=0.46). Conversely, macroH2A1 expression displayed an inverse correlation with ZEB1 and DKK1 (figures 7A, B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 7

Downregulation of Zeb1 induces senescence in vivo and reduces tumour load in a Zeb1-deficient mice. (A) ZEB1 and DKK1 inversely correlate with macroH2A1 in the invasive front of colorectal carcinomas (CRCs). A tissue array of primary CRCs was stained for ZEB1 (H-102), DKK1 (clone 2A5) and macroH2A1 (gift from M Buschbeck, see online supplementary methods). CRC cells at the tumour front were identified by their expression of pan-cytokeratin (IR053). Representative pictures of the tumour front are shown. Magnification 400×. (B) Quantification of the staining in (A). Samples were scored for the number of positively stained CRC cells at the invasive front as per the scale described in online supplementary information. (C) Scheme of the mouse model of CRC used in (D). Zeb1 (+/+) and Zeb1 (+/−) mice were treated azoxymethane (AOM) and cycles of dextran sodium sulfate (DSS) as detailed in the Methods section. (D) Downregulation of Zeb1 triggers in vivo senescence in CRC tumours. Frozen sections of CRC tumours from Zeb1 (+/+) and Zeb1 (+/−) mice treated as in (C) were stained for senescent associated β-galactosidase (SA β-gal) and counterstained with Fast Red. (E) Downregulation of Zeb1 triggers in vivo senescence-associated heterochromatin foci (SAHF) assembly in CRC tumours. As in (D), but formalin-fixed paraffin-embedded (FFPE) tumour samples were stained for macroH2A1 and DAPI as in figure 4F. Merged pictures are shown here and individual staining are displayed in online supplementary figure S7B. Scale bars represent 2 μm. (F) Zeb1 (+/−) mice developed lower number of tumours and survived in greater number to the end of protocol than Zeb1 (+/+) counterparts. Twenty-three Zeb1 (+/+) mice and 19 Zeb1 (+/−) mice were subjected to the protocol described in (C). At the end of the procedure (day 70), mice from both genotypes were examined for the number of colorectal tumours (upper left panel). A representative picture of colorectal tumours in Zeb1 (+/+) and Zeb1 (+/−) is shown (lower panel). The percentage of Zeb1 (+/−) mice that reached day 70 was larger than for Zeb1 (+/+) mice (upper right panel). (G) FFPE tumour samples as in (E) were stained for ZEB1 (H-102) and macroH2A1 (ab37264). Magnification 400×.

We then examined whether expression of ZEB1 in CRCs represses senescence in vivo. To that effect, Zeb1 (+/+) and Zeb1 (+/−) mice were used in a well-established mouse model of CRC induced by administration of azoxymethane and cycles of dextran sodium sulfate (reviewed in ref. 50) (figure 7C). Colorectal tumours collected at the end of the protocol (day 70) were examined for cellular senescence. Interestingly, we found that tumours from wild-type mice barely had senescent cells, whereas those from Zeb1-deficient ones displayed larger number of SA β-gal-positive cells (figure 7D). Likewise, SAHF were only observed in tumours from Zeb1 (+/−) mice but not in those from Zeb1 (+/+) mice (figure 7E and online supplementary figure S7B). Given the role of senescence as a tumour suppressor programme, we examined mice from both genotypes for the total number of colonic tumours. In line with the above results, Zeb1 (+/−) mice developed a lower number of tumours than Zeb1 (+/+) mice and the percentage of Zeb1 (+/−) mice that survived until the end of the protocol was also higher than among Zeb1 (+/+) mice (figure 7F).

Colorectal tumours from Zeb1 (+/+) and (+/−) mice were also stained for macroH2A1. ZEB1 was strongly expressed by cancer epithelial cells in colorectal tumours from Zeb1 (+/+) mice but only a few epithelial cells were stained for macroH2A1 (figure 7G). In contrast, in the tumours of Zeb1 (+/−) mice, expression of ZEB1 was greatly reduced in the malignant epithelial cells—ZEB1 was mostly confined to a few stromal cells—while they displayed strong expression of macroH2A1. In addition, tumours in Zeb1 (+/−) mice displayed neoplastic glands with partially preserved goblet cells and polarisation and scarce signs of mitotic activity (figure 7G). In contrast, the tumours arising in Zeb1 (+/+) mice showed altered tubular structures with almost complete loss of polarisation and atypical nuclei and mitosis homogeneously distributed throughout the gland, even reaching the lumen (figure 7G). Altogether, these data indicate that expression of ZEB1 in CRCs represses senescence. Importantly, these results also show that partial downregulation of Zeb1, even to only heterozygous levels, is sufficient to induce macroH2A1 expression and SAHF formation, to trigger in vivo cancer cell senescence, to reduce tumour load and to confer a more differentiated histological pattern.

The tumourigenic capacity of ZEB1 critically depends on its repression of H2AFY

Next, we wondered whether ZEB1's role promoting tumourigenesis requires in vivo repression of macroH2A1. The SW480-shCtl and SW480-shZEB1-A cells used above were now stably cotransfected with retroviral shRNA vector against H2AFY (pSUPER-shH2AFY) or its corresponding shRNA control (pSUPER-shCtl) to generate the following cell lines: SW480-shCtl/shCtl, SW480-shZEB1-A/shCtl, SW480-shCtl/shH2AFY and SW480-shZEB1-A/shH2AFY (see protein levels of macroH2A1 and ZEB1 in these cell lines in online supplementary figure S8A). The four cell lines were then used in a xenograft mouse model by inoculation in 10-week-old SCID mice and tumour growth was monitored for up to 23 days. As expected, single knockdown of ZEB1 (SW480-shZEB1-A/shCtl cells, blue line in figure 8A, B and online supplementary figure S8B) drastically reduced tumour growth. Interestingly, when cells were simultaneously knocked down for both ZEB1 and H2AFY (SW480-shZEB1-A/shH2AFY cells, green line in figures 8A, B and online supplementary figure S8B), the ability of shZEB1 to reduce tumourigenesis was reversed. Lastly, single knockdown of H2AFY (SW480-shCtl/shH2AFY cells, red line in figure 8A, B and online supplementary figure S8B) accelerated tumour growth compared with control cells (SW480-shCtl/shCtl cells, yellow line in figure 8A, B and online supplementary figure S8B). Overall, these results indicate that in vivo expression of macroH2A1 inversely correlates with ZEB1 (figure 7A, G) and ZEB1 induces tumourigenesis through repression of macroH2A1 (figure 8A).

• Download figure
• Open in new tab
• Download powerpoint

Figure 8

ZEB1-induced tumourigenesis and ZEB1's role as determinant of worse survival depends on its repression of macroH2A1 and senescence. (A) H2AFY knockdown reverses the inhibitory effect on tumourigenesis of ZEB1 knockdown. SW480 shCtl/shCtl, SW480 shZEB1-A/shCtl, SW480 shCtl/shH2AFY and SW480 shZEB1-A/shH2AFY stable cell lines were injected into SCID mice (seven to eight mice per cell line) and tumour development was monitored over time as described in the Methods section. A representative tumour for each cell line is shown with pictures of all tumours displayed in online supplementary figure S8B. See main text for details and discussion. (B) As in (A) but tumour volume was visualised by the uptake of fluorescent-labelled 2-deoxy glucose. Colour scale represents relative fluorescence units (RFU) with respect to an area where there was no tumour. A representative mouse for each condition is shown. (C) The maximum effect of ZEB1 as a factor of worse survival in colorectal carcinomas (CRCs) depends on low expression of H2AFY. The relapse-free survival probability of patients with CRC was represented in a Kaplan-Meier plot segregated by the median expression of ZEB1 and H2AFY. (D) As in (C) but for the GLB1 gene instead of H2AFY. (E) Schematic model of the molecular mechanism by which ZEB1 promotes tumourigenesis by repressing senescence in cancer cells. ZEB1 inhibits senescence and tumourigenesis and determines worse survival through subsequent activation of DKK1, mutant p53, Mdm2 and CtBP, and repression of macroH2A1. The dashed line refers to the induction of DKK1 by Wnt signalling as described in3 and the induction of ZEB1 by mutant p53.47 See main text for details. RFU, relative fluorescence units; SAHF, senescence-associated heterochromatin foci.

It is worth remarking here that there was no statistical difference between the tumourigenic capacity of SW480-shZEB1-A/shH2AFY and SW480-shCtl/shH2AFY cells (green line vs red line in figure 8A) or between SW480-shCtl/shCtl and SW480-shZEB1-A/shH2AFY cells (yellow line vs green line in figure 8A). Importantly, the lack of significant difference between these experimental condition pairs suggests that macroH2A1 is a key ZEB1 target in tumourigenesis and also that most of ZEB1's role in tumourigenesis and tumour progression is contingent on its repressor effect on macroH2A1.

ZEB1's role as determinant of worse cancer survival depends on its inhibition of H2AFY and senescence

In vivo evidence in human and mouse tumours indicates that senescence is associated with lower malignancy, slower tumour growth and, consequently, better clinical prognosis.5 Therefore, we tested whether the worse survival effect of ZEB1 in CRCs depends on its inhibition of senescence.

Altogether, the results shown above indicate that ZEB1 inhibits senescence in CRCs by induction of DKK1 and repression of macroH2A1. We therefore first examined whether expression of H2AFY modulates the effect of ZEB1 on the prognosis of patients with CRC. Analysis of the 928 human CRCs used in figure 1A revealed that those cases displaying high expression of ZEB1 and low expression of H2AFY have lower survival probability than those with high levels of ZEB1 but high levels of H2AFY (yellow and red lines in figure 8C). This implies that the maximum effect of ZEB1 as a predictor of poorer survival in CRC requires of low levels of H2AFY or that, in other words, high levels of ZEB1 are not sufficient to determine worse survival if not accompanied by low levels of H2AFY. Similar results were found for the effect of H2AFY expression on the survival probability determined by DKK1: high levels of DKK1 only determined maximum levels of reduced survival when expression of H2AFY was low (see online supplementary figure S8C). Of note, overall survival depends on multiple variables beyond tumour growth thus contributing to explain that H2AFY knockdown promoted tumour growth in the xenograft mouse model independently of ZEB1 levels (red and green lines in figure 8A), while lower overall survival in patients with CRC associated with low levels of H2AFY is worsened by high levels of ZEB1 (red and green lines in figure 8C). From all these data it could be therefore concluded that, at least for some of its tumour-promoting and worse-survival functions, ZEB1 depends on its activation effect on DKK1 (figure 1A) and also on its repression of H2AFY (figure 8C).

Likewise, we also tested whether expression of GLB1, encoding for SA β-gal and whose mRNA levels increase in senescent cells,26 alters the predictor value of ZEB1 in CRC survival. Indeed, among patients with CRC with high levels of ZEB1, low levels of GLB1 determined poorer survival (figure 8D). Altogether, these results indicate that ZEB1 promotes tumour progression and determines worse prognosis in patients with CRC, at least in part, through its effect as an inhibitor of senescence.