Both alterations to the epigenome and loss of polarity have been linked to cancer initiation, progression, and metastasis. It has previously been demonstrated that loss of the epigenetic reader protein Kaiso suppresses intestinal tumorigenesis in the Apc+/min mouse model, in which altered polarity plays a key role. Thus, we investigated the link between Kaiso deficiency, polarity, and suppression of intestinal tumorigenesis. We used Kaiso-deficient mice to conditionally delete Apc within the intestinal epithelia and demonstrated upregulation of the spindle polarity genes Dlg1 and Dlgap1. To understand the role of Dlg1, we generated Villin-creApc+/minDlg1flx/flx Kaiso−/y mice to analyze gene expression, survival, tumor burden, and spindle orientation. In vivo analysis of the Dlg1-deficient intestine revealed improper orientation of mitotic spindles and a decreased rate of cellular migration. Loss of Dlg1 decreased survival in Apc+/min mice, validating its role as a tumor suppressor in the intestine. Significantly, the increased survival of Apc+/minKaisoy/− mice was shown to be dependent on Dlg1 expression. Taken together, these data indicate that maintenance of spindle polarity in the intestinal crypt requires appropriate regulation of Dlg1 expression. As Dlg1 loss leads to incorrect spindle orientation and a delay in cells transiting the intestinal crypt. We propose that the delayed exit from the crypt increase the window in which spontaneous mutations can become fixed, producing a “tumor-permissive” environment, without an increase in mutation rate.

Implications:

Loss of mitotic spindle polarity delays the exit of cells from the intestinal crypt and promotes a tumorigenic environment.

Recent work recognizing the importance of the link between polarity and epigenetic pathways in cancer onset and progression (1) has highlighted a need to increase our understanding of these relationships. DNA methylation of CpG dinucleotides is a fundamental epigenetic modification. Within gene promoters' methylation leads to transcriptional silencing and is exploited by a range of different cancer types, including colorectal cancer, to silence tumor suppressor genes (2, 3). There is now a growing body of evidence that aberrant epigenetic silencing of polarity genes influences tumorigenesis in a variety of settings (4–6). Mechanistically, methylated CpG dinucleotides are recognized and bound to by proteins from the methyl binding family, which then recruit transcriptional repressor complexes (7). These proteins play a key role in disease by identifying tumor suppressor genes that have been aberrantly methylated (2, 3, 8). The methyl binding protein Kaiso, a POZ/BTB (Broad Complex Tramtrak, Bric a brac/pox virus, and zinc finger) family zinc finger transcription factor protein, has been shown to play a key role in intestinal tumorigenesis. POZ/BTB proteins are a well conserved protein family (9), which have been indicated to play a role in both development and cancer (10, 11). Despite the requirement of Kaiso for amphibian development (11), Kaiso null mice are viable and do not show any notable abnormalities in either development or reproduction (12). In cancer cells, Kaiso appears to have a pro-tumorigenic function. Its deficiency relieved repression of tumor suppressor and DNA repair genes (13, 14) in colon cancer. Although its nuclear localization is indicative of higher grade and metastatic breast and prostate cancers (15). In addition to its roles as a transcriptional repressor, it can also bind to δ-Catenin and behave as a transcriptional activator (16, 17). Further, in vivo studies have shown that Kaiso overexpression promotes intestinal inflammation and intestinal tumorigenesis (18, 19), whereas its absence increased survival of Apc+/min mice by delaying the onset of intestinal tumorigenesis (12). The Apc+/min mouse is a model of the human disease familial adenomatous polyposis (FAP). In both Apc+/min mice and patients with FAP, mutation of the wild-type Apc allele (or ‘second hit’) is required for adenoma formation (20). APC mutation and subsequent activation of the canonical Wnt pathway is an early, if not the first step, in the development of more than 80% of all sporadic colorectal cancers (21, 22). Because of its role in mediating epigenetic silencing of tumor suppressor gene in human colorectal cancer (13) and the low frequency of mutations observed in the disease (0% observed in 72 cases; refs. 23, 24), Kaiso represents a potentially druggable target. However, the mechanism of intestinal tumor suppression due to Kaiso deficiency in vivo has yet to be explored. As the dividing cells in the precancerous and cancerous intestinal tissue of Apc+/min mice display alterations to the spindle polarity (25), we have investigated whether alterations to spindle polarity play a role in the intestinal tumor suppression observed in the Apc+/minKaisoy/− mouse model. We have demonstrated that the polarity and tumor suppressor gene Dlg1 is mis-regulated in the absence of Kaiso. Dlg1 forms part of the Scribble/Lgl/Dlg polarity complex, which has been shown to play an important role in the maintenance of epithelial integrity (26). Mutations in this complex associated with the progression of a range of different epithelial cancer types (27–29) and mutations within the Dlg family have been observed in 12 of 72 (16.7%) of patients with colorectal cancer (23, 24). We further demonstrate that the absence of Dlg1 alters spindle polarity, delays migration, and promotes tumorigenesis and progression in the Apc+/min and Apc+/minKaisoy/− setting. Taken together, this increases the evidence demonstrating the importance of epigenetic regulation in maintenance of spindle polarity in intestinal homeostasis and disease.

Mice

All animal procedures were conducted in accordance with institutional animal care guidelines and UK Home Office regulations. Mice were maintained in a SPF barrier facility in conventional open top cages on Eco-Pure Chips 6 Premium bedding (Datesand) under a 12-hour light cycle, with IPS 5008 diet (Labdiet-IPS Ltd.) provided for nutritional support. To enrich environment, we provided irradiated sunflower seeds (at weaning only), Techniplast mouse houses (Techniplast) and small chewsticks (Labdiet-IPS Ltd.). All mice were from a mixed background and were homozygous with respect to the C57Bl/6 Pla2g2a (also called Mom-1) allele. Experimental animals were between 10 and 15 weeks old with siblings used as controls. The alleles for the Ah-cre (30), Apcflx (31), Apc+/min (32), Villin-Cre (33), Dlg1flx (34), Kaisoy/− (12) have been described previously. Genotyping conditions available upon request. Induction of the Ah-cre transgene was performed by administering three intraperitoneal injections of β-naphthoflavone (BNF; Sigma) at 80 mg/kg in a 24-hour period. For BrdU labeling, mice were injected intraperitoneally with 0.15 mL of BrdU. Following cre induction loxP flanked alleles are referred to as deleted in intestinal epithelial cells (ΔIEC).

Microarray expression analysis

Ten-week-old male mice were used for the array analysis. Three mice for each of the desired genotypes were used. Three-centimeter portions of the SI located 5 cm from the stomach were placed in RNAlater (after removing any mesentery and ensuring that no Peyer's patches were present). The tissue was then homogenized in Trizol reagent and RNA extracted using standard phenol–chloroform methodologies. Samples were sent to the Molecular Biology Core facility at the Cancer Research UK Molecular Biology Core Facility at The University of Manchester. Where biotinylated target cRNA was generated and hybridized to Affymetrix Mouse430A_2 gene expression chips. The raw data from these microarrays is available at http://www.ebi.ac.uk/arrayexpress/. The data were normalized and analyzed to generate differential expression tables using the AffylmGUI package for linear modeling of microarray data (35).

Tissue isolation, reporter visualization, IHC

Mice were euthanized at specified time points and the small intestine (SI) removed and flushed with water. Intestines were dissected as follows: The proximal 7 cm was mounted, fixed overnight in 10% formalin, and paraffin embedded. The following 3 cm was opened and placed into RNA later (Sigma), ensuring that all mesentery and Peyers patches were removed. The following 5 cm was divided into 1-cm lengths, bundled using surgical tape, and then fixed in 4% formaldehyde at 4°C for no more than 24 hours before processing into wax blocks by conventional means. Section were cut at 5 μm thickness, dewaxed, and rehydrated into PBS. Staining was performed using the Envision+ mouse or Rabbit Kit (Dako; Agilent Ltd.) according to manufacturer's instructions. To identify cells which had lost Dlg1 we stained using a validated rabbit polyclonal anti-Dlg1 antibody (Catalog No. #STJ111281; St John's Antibodies) at 1:200. Cells which had incorporated BRDU were identified using an anti-BrdU antibody conjugate at 1:50, cells positive for DNA damage marker γH2AX were identified using anti-γH2AX (Upstate 05636) Cellular analysis was performed on >25 whole crypts from at least three mice of each genotype.

Cellular analysis

Cellular analysis was performed on >25 whole crypts from at least three mice of each genotype. Apoptotic and mitotic index were scored from hematoxylin-and-eosin (H&E)-stained sections as previously described (36). The cells between the base of the crypt and the junction with the villus was designated as the proliferative zone. For migration analysis, mice of 60 to 80 days of age were given an intraperitoneal injection of BrdU 2 or 24 hours prior to culling and dissection. IHC analysis for BrdU incorporation was performed on formalin-fixed small intestinal rolls, and the number BrdU-positive cells and their location (with 0 being the bottom of the crypt) was measured on 50 half-crypts per mouse, minimum of four mice. Statistical analysis of the cumulative frequency of positive cells was performed using a two-tailed Kolmogorov–Smirnov test, on graphs P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0 0.001. Measurement of mitotic angle was performed on H&E slides stained sections. The angles of 200 mitosis in which the two spindle poles were easily observed were measured per mouse, with the angle of a direct line drawn between the two spindle poles and the line of the basement membrane measured. Measurements of 0° to 45° and 135° to 180° were classified as “planar,” being parallel to the basement membrane, whereas measurements of 45° to 135° were classified as being “Apico-basal,” being perpendicular to the basement membrane and dividing in the orientation of apico-basal polarity. The Chi-squared test was performed to assess statistical differences between the two genotypes, on graphs P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed to assess Kaiso binding to the promoters of Dlgap1 and Dlg1 in murine samples from aged Apc+/min mice, using polyps and adjacent normal tissue from the SI and the large intestine. Anti-Kaiso antibody (sc-23871) was used to identify DNA binding partners of Kaiso, and primers for Dlgap1 and Dlg1 were used to confirm presence of these genes (primers detailed in Supplementary Fig. S2).

siRNA against Apc mRNA was purchased from Bioneer. The Apc siRNA (sense 5′-GCAGGCGUAGAGUAUUCAU-3′, antisense 5′-AUGAAUACUCUACGCCUGC-3′) was transfected into CT-26 cells using Lipofectamine RNAiMax (Invitrogen), and knockdown levels of were confirmed using qRT-PCR (available upon request). CT-26 cells at passage 86 were purchased from the Korean Cell Line Bank and certified mycoplasma free and verified by STR profiling. The cells were expanded to create a cell stock and frozen. For each experiment, cells were thawed and used at <3 passages or within 2 weeks. After transfection, the cells were harvested, and ChIP analysis performed.

qRT-PCR analysis

The following methods were all performed according to manufacturer's instructions unless otherwise stated. For analysis of gene expression in the intestine epithelia, three to five mice from each control and experimental group were harvested. RNA was extracted from a 0.5 cm portion of SI taken ∼5 cm distally from the stomach and stored at −80 in RNAlater. Total RNA was extracted using the RNeasy Kit (Qiagen) and DNase treated using the Turbo DNase Kit (Ambion). Complimentary DNA (cDNA) was transcribed from 1 μg of RNA using random hexamers (Promega) and Superscript III (Invitrogen) kits. For relative quantitation, all samples were run in duplicate on the StepOnePlus PCR machine using Fast Sybr Green master mix (Applied Biosystems). The threshold cycle (Ct) values of each gene analyzed were normalized to a reference gene. For expression analysis (qRT-PCR) Ct values were normalized against the ActB gene. Differences between groups were assessed using the 2-ΔΔCT method [47]. Two-tailed Mann–Whitney U tests were performed on the ΔCt values to determine significance (P ≤ 0.05) differences between groups. Oligonucleotide sequences are available upon request.

Survival

A minimum of 14 mice per cohort were used to assess survival. Mice were aged until they showed symptoms of intestinal tumor burden (pale feet, bloating, prolapse or piloerection). Survival data were analyzed using the Kaplan–Meier test. If not indicated otherwise, the statistical mean is presented, and error bars represent standard deviation, on graphs P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Tumor burden and grading

Tumor burden was measured at point of death with the number of macroscopic lesions and their size from fresh tissue. Grading was performed microscopically using H&E slides of SI taken from aged mice, tumors were microscopically identified as; single crypt lesion (grade 1), microadenoma (grade 2), adenoma (grade 3), adenocarcinoma with stromal invasion (grade 4), and adenocarcinoma with smooth muscle invasion (grade 5).

Intestinal organoid culture

Intestinal organoid culture was performed using the method described by Sato and colleagues (37). Organoids were grown in Nunc LabTek Chamber Well slides for whole mount immunofluorescence. Organoids were fixed overnight in 4% PFA, then permeabilized using 1% Triton-X100 for 1 hour at 37°C. Alexa Fluor Phalloidin at 1:150 and neomarkers anti-lysozyme (Rb-327-A1) at 1:300 were used as primary antibodies.

Apc-deficient SI crypts are resistant to Kaiso deficiency

The delayed onset of adenomas in the Apc+/minKaisoy/− mouse is potentially due to a role for Kaiso in the Wnt signaling pathway. To investigate the direct effects of Kaiso status upon gene expression and Wnt signaling, we analyzed the phenotype of a conditional deletion of Apc in the context of Kaiso deficiency. Kaisoy/-deficient mice were crossed with mice carrying an Ah-cre transgene and a loxP-flanked Apc allele, following induction with BNF Apc is deleted in the SI crypts. We generated and compared cohorts of Ah-cre+ Kaiso+/+ (WT), Ah-cre+ Kaiso−/y (Kaiso−/y), Ah-cre+ Apcflx/flx Kaiso+/+(ApcΔIEC/ΔIEC), and Ah-cre+ Apcflx/flx Kaisoy/− (ApcΔIEC/ΔIECKaiso−/y) mice. At 4 days post-induction (d.p.i.), comparing WT and Kaiso−/y indicated the absence of Kaiso does not grossly alter the crypts (Fig. 1A and B). Similarly, the ApcΔIEC/ΔIEC mice, which are characterized by enlarged, aberrant crypts (Fig. 1C; as previously reported; ref. 38), appeared unaltered in ApcΔIEC/ΔIECKaisoy/− setting (Fig. 1D). To examine cell division and migration, mice were killed 2 or 24 hours after injection with BrdU. BrdU is incorporated into the DNA of cells at S phase and is bioavailable for less than 2 hours. Therefore, by killing mice at 2 and 24 hours after exposure, we could track the movement of BrdU-positive cells from the proliferation zone in the crypt on to the villus by IHC. The position of BrdU-positive cells was plotted as the cumulative frequency of cells 2 and 24 hours after labeling (Fig. 1E–H). Migration patterns of the SI were not significantly altered by the absence of Kaiso; however, the data indicated a trend towards an increase in the speed of migration of Kaiso-deficient cells from the proliferative zone onto the villi. In ApcΔIEC/ΔIEC mice, cell proliferation occurs independently of position and migration of cells along the crypt–villus axis is perturbed (Fig. 1F), as previously reported (27), the additional loss of Kaiso failed to alter this phenotype (Fig. 1F).

Figure 1.

Loss of Kaiso does not alter the gross phenotype of WT and Apc-deficient SI crypts. H&E staining showing no difference between WT (A) and Kaiso−/y (B) or Ah-cre ApcΔIEC/ΔIEC (C) and Ah-creApcΔIEC/ΔIEC Kaiso−/y (D) SI, note the enlarged crypts in the ApcΔIEC/ΔIEC mice are retained in ApcΔIEC/ΔIECKaiso−/y ([indicates height of crypts). Scale bars represent 50 μm. E and F, Mice were injected with BrdU to mark cells in S phase at 2 hours and track migration onto villi 24 hours later. E, WT 2h (), Kaiso−/y 2h (), WT 24h (), Kaiso−/y 24h () and (F) ApcΔIEC/ΔIEC 2h (), ApcΔIEC/ΔIEC Kaiso−/y 2h (), ApcΔIEC/ΔIEC 24h (), ApcΔIEC/ΔIEC Kaiso−/y 24h () BrdU-positive cells indicated no alteration to the position and size of the proliferative zones or rate of migration (absent in the ApcΔIEC/ΔIEC SI), n ≥ 4, with representative images of 24 hours BrdU in WT and Kaiso−/y SI (G and H), scale bar represents 50 μm. I, The absence of Kaiso does not alter the normal position of Paneth cells or rescue their mislocalization in the ApcΔIEC/ΔIEC SI; WT (), Kaiso−/y (), ApcΔIEC/ΔIEC (), and ApcΔIEC/ΔIEC Kaiso−/y (), n ≥ 4. J, Relative expression analysis indicated no significant alteration to Wnt target genes in the absence of Kaiso (WT-black, Kaiso−/y-white, ApcΔIEC/ΔIEC -dark gray, and ApcΔIEC/ΔIEC Kaiso−/y-light gray), error bars indicate SD, n ≥ 4.

Figure 1.

Loss of Kaiso does not alter the gross phenotype of WT and Apc-deficient SI crypts. H&E staining showing no difference between WT (A) and Kaiso−/y (B) or Ah-cre ApcΔIEC/ΔIEC (C) and Ah-creApcΔIEC/ΔIEC Kaiso−/y (D) SI, note the enlarged crypts in the ApcΔIEC/ΔIEC mice are retained in ApcΔIEC/ΔIECKaiso−/y ([indicates height of crypts). Scale bars represent 50 μm. E and F, Mice were injected with BrdU to mark cells in S phase at 2 hours and track migration onto villi 24 hours later. E, WT 2h (), Kaiso−/y 2h (), WT 24h (), Kaiso−/y 24h () and (F) ApcΔIEC/ΔIEC 2h (), ApcΔIEC/ΔIEC Kaiso−/y 2h (), ApcΔIEC/ΔIEC 24h (), ApcΔIEC/ΔIEC Kaiso−/y 24h () BrdU-positive cells indicated no alteration to the position and size of the proliferative zones or rate of migration (absent in the ApcΔIEC/ΔIEC SI), n ≥ 4, with representative images of 24 hours BrdU in WT and Kaiso−/y SI (G and H), scale bar represents 50 μm. I, The absence of Kaiso does not alter the normal position of Paneth cells or rescue their mislocalization in the ApcΔIEC/ΔIEC SI; WT (), Kaiso−/y (), ApcΔIEC/ΔIEC (), and ApcΔIEC/ΔIEC Kaiso−/y (), n ≥ 4. J, Relative expression analysis indicated no significant alteration to Wnt target genes in the absence of Kaiso (WT-black, Kaiso−/y-white, ApcΔIEC/ΔIEC -dark gray, and ApcΔIEC/ΔIEC Kaiso−/y-light gray), error bars indicate SD, n ≥ 4.

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Further, it has been previously shown that Paneth cell localization is altered in the induced ApcΔIEC/ΔIEC SI (27), the additional loss of Kaiso had no effect on this phenotype (Fig. 1I). Finally, analysis of Wnt target genes demonstrated that the increase in Wnt target gene expression synonymous with Apc deletion was not significantly altered in the absence of Kaiso (Fig. 1J; Supplementary Fig. S1). In conclusion, the lack of any gross phenotype in either the Kaisoy/ or the ApcΔIEC/ΔIECKaisoy/− mice suggested that repression of intestinal tumorigenesis in the Apc+/minKaisoy/− mice is Wnt independent.

The polarity genes Dlg1 and Dlgap1 are upregulated within the Kaiso-deficient SI

As Kaiso deficiency is well tolerated by the intestine, we aimed to identify novel roles for Kaiso in the normal and Apc-deficient intestine which may influence tumorigenesis. To identify genes mis-regulated in the absence of Kaiso, we generated microarray gene expression profiles on SI from WT and Kaiso−/y mice (raw data are available from https://www.ebi.ac.uk/arrayexpress/ express under accession number E-MTAB-6009). After normalization genes differentially expressed were identified and ranked according to their statistical significance (Supplementary Table S1). As expected, the genes Kaiso and Hprt which were deleted in the generation of the model were highly downregulated and the most significantly altered. As Kaiso is a mediator of epigenetic transcriptional repression, we identified the genes that were upregulated by its loss and candidates for a role in tumorigenesis. The analysis identified the gene Discs Large Homologue Associated Protein 1 (Dlgap1; Supplementary Table S1), which was significantly 4.9-fold upregulated in the Kaiso−/y intestine. Dlgap1 is a highly conserved protein, which interacts with the guanylate kinase-like domain of the important cancer regulatory protein Discs Large Homolog 1 (Dlg1), a binding partner of the key Wnt regulator Apc. To support the evidence that these genes are regulated by Kaiso, we analyzed whether the sequence of the promoter regions of these genes contained the Kaiso binding sequences TCCTGCNA (minimum binding sequence of CTGCNA) and CGCG (39). In silico analysis of the 5kb promoter regions upstream of the Dlg1 and Dlgap1 ATG start codons confirmed the presence of five minimum Kaiso binding motifs and one complete Kaiso binding motif in Dlg1 and one minimum Kaiso binding motif in the Dlgap1 promoter region, indicating its potential to regulate expression of these genes (Supplementary Fig. S2). To confirm the upregulation of gene expression, we used qRT-PCR analysis to demonstrate a significant 1.5- and 3.3-fold upregulation of Dlgap1 and Dlg1 expression, respectively (Fig. 2A), in the Kaiso-deficient intestine. This was further confirmed by IHC which showed a dramatic increase in Dlg1 protein levels within the intestinal epithelium of Kaiso-deficient mice (Fig. 1B and C). Dlg1 is important in maintenance of spindle polarity and is a binding partner of Apc. As spindle polarity is altered in the Apc+/min intestine (25), the upregulation of Dlg1 suggested that it may play a functional role in suppression of intestinal tumorigenesis and the increased survival of Apcmin Kaisoy/− mice. To confirm a direct role for Kaiso to regulate Dlg1 and Dlgap1 expression, ChIP was utilized to pull-down DNA associated with the Kaiso protein in both normal and polyp tissue from the small and large intestine of Apcmin mice. Kaiso binding to either Dlgap1 or Dlg1 was not observed in 10 normal small intestinal samples or 5 normal large intestinal samples (Fig. 2D). In contrast within Apcmin polyps, Dlgap1 binding was observed in 3/10 small intestinal tumors and 3/5 colonic polyps, with Dlg1 binding observed in 1/10 small intestinal tumors and 3/5 colonic polyps (Fig. 2D). These data indicate that Kaiso has the ability to bind to Dlgap1 and Dlg1, but does so only an Apc-deficient environment. To confirm this finding, ChIP was also used to assess Kaiso binding to Dlgap and Dlg1 in the mouse colon carcinoma cell line CT-26 in which Apc is intact. Using siRNA methods to generate a greater than 8-fold knock-down Apc (Fig. 2E), demonstrated that binding of Kaiso to the promoters of Dlgap1 and Dlg1 was significantly increased in the in an Apc-deficient setting (Fig. 2F). Taken together these data indicate that regulation of expression of Dlgap1 and Dlg1 in the normal intestine is most likely indirect; however, in a tumor permissive environment, Kaiso can directly bind to the promoters and regulate expression.

Figure 2.

Upregulation of Dlg1 and Dlgap1 in the Kaiso-deficient intestine. A, qRT-PCR analysis indicating downregulation of Kaiso and upregulation of Dlg1 and Dlgap1 in the Kaiso−/y intestine (WT, black bars; Kaiso−/y, gray bars), error bars indicate standard deviation, n ≥ 4. IHC comparing WT (B) to Kaiso−/y SI (C) demonstrating an increase in Dlg1 staining in the intestinal epithelial of the Kaiso-deficient intestine, scale bar represents 100 μm. ChiP analysis was used to assess the ability of Kaiso to bind Dlgap1 and Dlg1 in murine SI, large intestine, small intestinal polyps, and large intestinal polyps (D), Apc SiRNA enabled a more than eight-fold knockdown of Apc within the murine cell line CT-26 (E), and ChIP analysis was used to assess the ability of Kaiso to bind to Dlgap1 and Dlg1 when CT-26 cells were treated with either control scrambled siRNA (light gray) or Apc siRNA (dark gray) (F). Error bars represent SD.

Figure 2.

Upregulation of Dlg1 and Dlgap1 in the Kaiso-deficient intestine. A, qRT-PCR analysis indicating downregulation of Kaiso and upregulation of Dlg1 and Dlgap1 in the Kaiso−/y intestine (WT, black bars; Kaiso−/y, gray bars), error bars indicate standard deviation, n ≥ 4. IHC comparing WT (B) to Kaiso−/y SI (C) demonstrating an increase in Dlg1 staining in the intestinal epithelial of the Kaiso-deficient intestine, scale bar represents 100 μm. ChiP analysis was used to assess the ability of Kaiso to bind Dlgap1 and Dlg1 in murine SI, large intestine, small intestinal polyps, and large intestinal polyps (D), Apc SiRNA enabled a more than eight-fold knockdown of Apc within the murine cell line CT-26 (E), and ChIP analysis was used to assess the ability of Kaiso to bind to Dlgap1 and Dlg1 when CT-26 cells were treated with either control scrambled siRNA (light gray) or Apc siRNA (dark gray) (F). Error bars represent SD.

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Dlg1 loss disrupts cell division polarity and migration rates in intestine

Dlg1 is known to regulate cell polarity and is a key part of the scribble/dlg/lgl polarity complex (40) and the Apc destruction complex. To investigate the role of Dlg1 in the intestine, we analyzed villin-creDlg1flx/flx mice (34), in which exon 4 of the Dlg1 gene is flanked by LoxP and driven by endogenous expression of Cre from the Villin promoter to initiate loss of Dlg1 at embryonic E9 within the intestinal epithelium (41). Survival analysis was performed by aging cohorts of villin-cre Dlg1+/+ (WT) and villin-cre Dlg1flx/flx (Dlg1ΔIEC/ΔIEC) mice. Overall there was no significant difference in survival between WT and Dlg1ΔIEC/ΔIEC and at death the intestinal epithelium of Dlg1ΔIEC/ΔIEC mice appeared functionally normal (Fig. 3A). Histologic assessment of Dlg1ΔIEC/ΔIEC intestinal tissue revealed that Dlg1 deficiency does not alter crypt length (number of cells), apoptosis (number of apoptotic bodies), or proliferation (Supplementary Fig. S3A, S3B, and S3C). In summary, it appears that Dlg1 loss is well tolerated by the intestinal epithelium and alone is not sufficient to induce colorectal tumorigenesis. As Dlg1 is known to form part of the important Scribble/Lgl/Dlg polarity complex, we next explored the role of these gene in the Dlg1-deficient intestine. Expression qRT-PCR analysis of the polarity genes associated with Dlg1 indicated a significant reduction in expression of the Scribble (Scrib1) gene, which forms part of the polarity complex; Fig. 3B). The lack of any gross phenotype as a result of loss of Dlg1 and significant reduction in Scrib expression suggested that other cues from the extracellular matrix (ECM) or redundancy amongst Dlg family members may compensate for the absence of Dlg1 in order to maintain the correct architecture of the intestinal epithelia in vivo (42). However, further investigation revealed no expressional compensation by other Dlg family member genes (Supplementary Fig. S4), indicating that extremely low levels of Dlg1 expression as observed in the Dlg1ΔIEC/ΔIEC model are sufficient to maintain intestinal crypt morphology.

Figure 3.

Absence of Dlg1 is tolerated by the SI but disrupts mitotic spindle orientation and migration. A, Kaplan–Meier survival analysis of aged WT () and vilCre-Dlg1ΔIEC/ΔIEC (…) cohorts indicates no difference in overall survival. B,Scrib is significantly (Mann–Whitney; P = 0.002) downregulated in the Dlg1ΔIEC/ΔIEC intestine, error bars indicate SD, n ≥ 4. C and D, Whole mount immunofluorescence of WT (C) and Dlg1ΔIEC/ΔIEC (D) organoids demonstrating the maintenance of cellular polarity (green-phalloidin, red-lysozyme expression, and blue-DAPI). Analysis of mitotic angles in WT (E) and Dlg1ΔIEC/ΔIEC (F) indicates a significant decrease in divisions occurring in a planar orientation (white segments; E, right panel red circle indicating planal mitosis) and an increase in apico-basal divisions (gray segments; F, right panel red circle indicating apico-basal mitosis) as a result of Dlg1 deficiency, n ≥ 6. G, Comparison of the position of BrdU+ cells at 2 hours () and 24 hours (…) in WT (black) and Dlg1ΔIEC/ΔIEC (gray) indicating a delay in migration in the Dlg1ΔIEC/ΔIEC crypt (Kolmogorov–Smirnov test P < 0.0001), with representative images of 24 hours BrdU, n ≥ 4 (H and I).

Figure 3.

Absence of Dlg1 is tolerated by the SI but disrupts mitotic spindle orientation and migration. A, Kaplan–Meier survival analysis of aged WT () and vilCre-Dlg1ΔIEC/ΔIEC (…) cohorts indicates no difference in overall survival. B,Scrib is significantly (Mann–Whitney; P = 0.002) downregulated in the Dlg1ΔIEC/ΔIEC intestine, error bars indicate SD, n ≥ 4. C and D, Whole mount immunofluorescence of WT (C) and Dlg1ΔIEC/ΔIEC (D) organoids demonstrating the maintenance of cellular polarity (green-phalloidin, red-lysozyme expression, and blue-DAPI). Analysis of mitotic angles in WT (E) and Dlg1ΔIEC/ΔIEC (F) indicates a significant decrease in divisions occurring in a planar orientation (white segments; E, right panel red circle indicating planal mitosis) and an increase in apico-basal divisions (gray segments; F, right panel red circle indicating apico-basal mitosis) as a result of Dlg1 deficiency, n ≥ 6. G, Comparison of the position of BrdU+ cells at 2 hours () and 24 hours (…) in WT (black) and Dlg1ΔIEC/ΔIEC (gray) indicating a delay in migration in the Dlg1ΔIEC/ΔIEC crypt (Kolmogorov–Smirnov test P < 0.0001), with representative images of 24 hours BrdU, n ≥ 4 (H and I).

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To investigate the role of Dlg1 loss on polarity, specifically within the intestinal epithelium in the absence of ECM cues, small intestinal organoid cultures were made from both WT and Dlg1ΔIEC/ΔIEC tissue. Whole mount immunofluorescence for the marker of the apical tip/brush border of IECs revealed no obvious cell polarity defects as a result of Dlg1 deficiency within the intestinal epithelium (Fig. 3C and D). As well as playing a key role in maintaining normal apical basal polarity Dlg1 has a role in spindle orientation during mitosis. Within the WT crypt 86% of cell divisions occurred in a “planar” orientation, with mitotic spindles running parallel to the basement membrane while the remaining 14% of mitotic spindles occurred in a “apico-basal” direction (Fig. 3E). Analysis of the Dlg1ΔIEC/ΔIEC intestine demonstrated a significant shift in spindle orientation to 79% planar to 21% apico-basal (Fig. 3F; Chi-squared test P = 0.0437). As planar cell divisions are in line with normal migration along the crypt–villus axis any alterations may lead to delayed exit of cells from the crypt. Further BrdU analysis at a 2-hour time point indicated that Dlg1 deficiency did not alter the position of cells undergoing mitosis (Fig. 3G). However, at 24h the Dlg1-deficient BrdU+ cells were significantly lower down the crypt–villus axis compared with wild-type controls (Fig. 3G–I; Kolmogorov–Smirnov P < 0.0001). Potentially, the delay in Dlg1-deficient cells exiting the crypt may impact on tumorigenesis as it could increase the time for oncogenic mutations to become fixed, creating a tumor-permissive environment. Levels of cell DNA mutation within the intestine were assessed by qRT-PCR for a range of DNA repair pathway genes, and the protein localization of the DNA damage marker γH2AX was assessed by IHC and counted, showing no difference (Supplementary Fig. S5).This was expected as it is not thought that the rate of mutation is controlled by Dlg1, simply the rate at which mutated cells migrate.

Dlg1 loss promotes intestinal tumorigenesis in Apc+/min mice

To explore the role of Dlg1 in intestinal tumorigenesis, VillinCre+ Dlg1flx/flx mutant mice were crossed with Apc+/min mice to generate Apc+/min and Apc+/min VillinCre+ Dlg1fl/fl (ApcminDlg1ΔIEC/ΔIEC) cohorts. To confirm our previous finding for the role of Dlg1 in spindle orientation, we repeated the analysis on the normal crypts in these mice. As predicted we observed a significant increase in apico-basal mitotic spindles from 14% in WT (Fig. 3E) to 19% in Apc+/min (Fig. 4A; Chi-squared test P < 0.001) to 32% in Apc+/minDlg1ΔIEC/ΔIEC mice (Fig. 4B; Chi-squared test P > 0.001). The mice were aged and harvested until they reached a humane endpoint indicating an intestinal tumor burden. Survival analysis indicated a significant decrease in survival of ApcminDlg1ΔIEC/ΔIEC in comparison to Apc+/min mice [Fig. 4C, log-rank(Mantel–Cox) P = 0.0309]. At point of harvest there was no significant difference to the tumor number or median tumor burden between the cohorts, indicating mice were analyzed at equivalent stages (Fig. 4D and E). However, it was noted that even though it did not achieve statistical significance the median number of tumors in the SI increased from 17 in the Apc+/min mice to 30 in the ApcminDlg1ΔIEC/ΔIEC cohort (Fig. 4D), with the ApcminDlg1ΔIEC/ΔIEC mice showing a significant increase in inter-mouse variability. To understand why ApcminDlg1ΔIEC/ΔIEC reached the endpoint sooner than Apc+/min mice, we examined the grade of tumors in the mice, as loss of spindle polarity is associated with EMT and progression. Microscopic grading of the observed lesions identified a significant increase in the transition of single crypt lesions to more advance tumor types, including adenocarcinomas with stromal and smooth muscle invasion rarely observed in the Apcmin mice (Fig. 4F and G). In conclusion, the data indicate that that Dlg1 acts as a tumor suppressor in the murine intestine by preventing loss of spindle polarity, supporting cell migration rates and preventing progression of lesions.

Figure 4.

Dlg1 is a tumor suppressor in the murine intestine. Analysis of mitotic spindle angles in crypts of Apc+/min (A) and Apc+/minDlg1ΔIEC/ΔIEC (B) crypts indicates loss of Dlg1 leads to a significant decrease in the proportion of cells dividing in a normal planar orientation (Chi-squared test P > 0.001; gray). C, Kaplan–Meier analysis demonstrating a significant reduction in overall survival of Apc+/minDlg1ΔIEC/ΔIEC (black line) mice compared with Apc+/min [gray line; log-rank (Mantel–Cox) P = 0.0309]. The total number of tumors (D) and their burden (E) were not significantly altered in Apc+/minDlg1ΔIEC/ΔIEC mice, although they both displayed an increased inter mouse variability (Levene's test for equality of variances P = 0.018). F, Lesions observed within Apc+/minDlg1ΔIEC/ΔIEC (black bars) SI at endpoint were significantly more severe than those observed in Apc+/min (gray bars, error bars indicate standard deviation; Chi-squared test). SI, stromal invasion; SMI, smooth muscle invasion, n ≥ 28 lesions counted from four mice. G, Lesion from an Apc+/minDlg1ΔIEC/ΔIEC mouse indicating progression of tumor to adenocarcinoma with submucosal () and SMI (), scale bar represents 500 μm.

Figure 4.

Dlg1 is a tumor suppressor in the murine intestine. Analysis of mitotic spindle angles in crypts of Apc+/min (A) and Apc+/minDlg1ΔIEC/ΔIEC (B) crypts indicates loss of Dlg1 leads to a significant decrease in the proportion of cells dividing in a normal planar orientation (Chi-squared test P > 0.001; gray). C, Kaplan–Meier analysis demonstrating a significant reduction in overall survival of Apc+/minDlg1ΔIEC/ΔIEC (black line) mice compared with Apc+/min [gray line; log-rank (Mantel–Cox) P = 0.0309]. The total number of tumors (D) and their burden (E) were not significantly altered in Apc+/minDlg1ΔIEC/ΔIEC mice, although they both displayed an increased inter mouse variability (Levene's test for equality of variances P = 0.018). F, Lesions observed within Apc+/minDlg1ΔIEC/ΔIEC (black bars) SI at endpoint were significantly more severe than those observed in Apc+/min (gray bars, error bars indicate standard deviation; Chi-squared test). SI, stromal invasion; SMI, smooth muscle invasion, n ≥ 28 lesions counted from four mice. G, Lesion from an Apc+/minDlg1ΔIEC/ΔIEC mouse indicating progression of tumor to adenocarcinoma with submucosal () and SMI (), scale bar represents 500 μm.

Close modal

Tumor suppression due to Kaiso deficiency is Dlg1 dependent

Previously we reported that loss of Kaiso suppressed tumorigenesis in the Apc+/min model. Here we have identified that Dlg1 is upregulated in the Kaiso null intestinal epithelium and acts as a tumor suppressor in the Apc+/min model. To assess if the increased survival of Apcmin Kaisoy/− mice is dependent on the presence of a functional Dlg1 gene and/or related to spindle polarity, we generated Apcmin Kaisoy/−Dlg1ΔIEC/ΔIEC mice. Analysis of spindle polarity indicated that the proportion of aberrantly orientated spindles was significantly reduced from 19% in the Apc+/min mice to 12% in the Apcmin Kaiso−/− (Fig. 5A and B; Chi-squared test P < 0.001). A similar level to the WT setting (Fig. 3E) and a potential reason for the subtle increase in cell migration from villus to crypt (Fig. 1E). We next examined the Apc+/min Kaiso−/−Dlg1flx/flx mice and demonstrated that the improvement in percentage of mis-orientated divisions due to Kaiso loss was lost with the additional absence of Dlg1 (Fig. 5A and B; Chi-squared test P < 0.001). Significantly, there was no difference in the percentage of aberrant mitosis in the apico-basal orientation between the ApcminDlg1ΔIEC/ΔIEC and Apcmin Kaisoy/−Dlg1ΔIEC/ΔIEC intestines (Fig. 5A), and no change in total levels of cell proliferation between the cohorts (Fig. 5C). To investigate whether the improvement in the proportion of mitotic spindles in the planar orientated in Apc+/min Kaisoy/− intestine was important in the tumor suppression, we generated cohorts of Apcmin Kaisoy/−Dlg1ΔIEC/ΔIEC mice and aged until humane endpoint was reached. As previously observed, the loss of Kaiso increased survival (Fig. 5D; Apc+/min Kaisoy/− mice) were only aged to 200 days to confirm increased survival and then sacrificed. As reported earlier (Fig. 4C), there is a significant decrease in survival of Apcmin Dlg1ΔIEC/ΔIEC, which was not rescued by the additional loss of Kaiso (Fig. 5D). Further, the increased aggression of lesions observed in ApcminDlg1ΔIEC/ΔIEC again presented in the Apcmin Kaisoy/−Dlg1ΔIEC/ΔIEC (Fig. 5E). In conclusion, these data suggest that the tumor resistance observed in the intestine ApcminKaisoy− mice requires expression of the Dlg1 gene. We propose that the appropriate expression of Dlg1 prevents mitosis occurring at an aberrant apico-basal orientation, maintains the rates of cell migration onto the villus, and therefore may decrease the window of opportunity for tumorigenesis (Fig. 6).

Figure 5.

The Kaiso-deficient intestine requires an intact Dlg1 to manifest its tumor resistance. A, The proportion of aberrantly orientated mitotic spindles is significantly reduced in the Apc+/minKaisoy/− but increased in the Apc+/minDlg1ΔIEC/ΔIEC, and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC, when compared with Apc+/min intestine. Statistical analyis were performed using Mann–Whitney, * indicates P ≤ 0.05, n ≥ 6. B, Representative images of Apc+/min, Apc+/minDlg1ΔIEC/ΔIEC, Apc+/minKaisoy/−, and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC intestine, with indicated aberrant apico-basally orientated cell divisions (←) and planar cell divisions (↑). Scale bars represent 100 μm. BrdU analysis (C) indicated there were no differences in total levels of proliferation within the intestine, n ≥ 4. D, Kaplan–Meier survival analysis demonstrating a decreased survival of Apc+/minΔIEC/ΔIEC (gray dotted line) and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC [gray solid line; log-rank (Mantel–Cox) P = 0.0441] compared with Apc+/min (black solid) and Apc+/minKaisoy/− (black dashed) mice, vertical mark indicate mouse disease-free survival, n ≥ 9. E, Adenocarcinoma with submucosal invasion (←) (from an Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC mouse, scale bar represents 500 μm.

Figure 5.

The Kaiso-deficient intestine requires an intact Dlg1 to manifest its tumor resistance. A, The proportion of aberrantly orientated mitotic spindles is significantly reduced in the Apc+/minKaisoy/− but increased in the Apc+/minDlg1ΔIEC/ΔIEC, and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC, when compared with Apc+/min intestine. Statistical analyis were performed using Mann–Whitney, * indicates P ≤ 0.05, n ≥ 6. B, Representative images of Apc+/min, Apc+/minDlg1ΔIEC/ΔIEC, Apc+/minKaisoy/−, and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC intestine, with indicated aberrant apico-basally orientated cell divisions (←) and planar cell divisions (↑). Scale bars represent 100 μm. BrdU analysis (C) indicated there were no differences in total levels of proliferation within the intestine, n ≥ 4. D, Kaplan–Meier survival analysis demonstrating a decreased survival of Apc+/minΔIEC/ΔIEC (gray dotted line) and Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC [gray solid line; log-rank (Mantel–Cox) P = 0.0441] compared with Apc+/min (black solid) and Apc+/minKaisoy/− (black dashed) mice, vertical mark indicate mouse disease-free survival, n ≥ 9. E, Adenocarcinoma with submucosal invasion (←) (from an Apc+/minKaisoy/−Dlg1ΔIEC/ΔIEC mouse, scale bar represents 500 μm.

Close modal
Figure 6.

Model of tumor suppression by Dlg1. A, In the normal intestine Dlg1 plays an important role in maintaining the planal orientation of mitosis to ensure that division occurs in the same direction as migration. B, In the Dlg1-deficient intestine alteration to the angles of mitosis conflict with the direction of cell migration leading to longer transit times from the crypt-base to the villus. Potentially increasing the window of opportunity for fixation of a mutant cell, which may then migrate in multiple directions and increasing the chance of invasion into the submucosa.

Figure 6.

Model of tumor suppression by Dlg1. A, In the normal intestine Dlg1 plays an important role in maintaining the planal orientation of mitosis to ensure that division occurs in the same direction as migration. B, In the Dlg1-deficient intestine alteration to the angles of mitosis conflict with the direction of cell migration leading to longer transit times from the crypt-base to the villus. Potentially increasing the window of opportunity for fixation of a mutant cell, which may then migrate in multiple directions and increasing the chance of invasion into the submucosa.

Close modal

In summary, here we have demonstrated that in the absence of the epigenetic regulator Kaiso there is an increase in expression of the polarity proteins Dlg1 and Dlgap1. The absence of Dlg1 results in an increase in the number of baso-lateral cell divisions within the intestinal crypt and a decrease in cell migration rates. On the Apc+/min background, the absence of Dlg1 decrease survival due to an increase in the number and aggressiveness of tumors. Further, the loss of Dlg1 overcomes the suppression of intestinal tumorigenesis observed in the Apc+/minKaiso−/y mouse model. Suggesting that the mechanisms that maintain planal mitotic orientation and migration rates have important antitumorigenic roles. Although there are many potential mechanisms for the control of migration rates along the crypt–villus axis, including cell:cell signaling and negative draw of cells through the sloughing of cells at the villus tip, there is much evidence to support the concept that mitosis supports normal cellular migration along the crypt–villus axis through mitotic pressure (43, 44). This leads to the assumption that the angle of mitosis within the crypt could play a part in normal migration patterns. The rapid cell migration along the crypt–villus axis is one of the driving factors which controls the quick turn over of cells within the intestinal epithelium and is essential to minimize the risk of tumorigenesis. It achieves this by flushing undetected mutant cells from the epithelium before they can disrupt intestinal homeostasis. Through delayed migration it can be hypothesized that Apc+/min cells which have lost the wild type copy of Apc (through random mutations, loss of heterozygosity or epigenetic silencing) may remain within the intestinal epithelium for a longer period of time, increasing the opportunity for lesion formation. An alternative hypothesis is that the increased crypt residence time of a cell increases the likelihood that it will accumulate additional oncogenic mutations, for example K-ras, Smad4, or p53. This may account for the increased aggressiveness of the tumors observed in the Apc+/min Dlg1−/− model. The role of cell migration speed within the intestinal epithelium in creating a tumor permissive environment has been hypothesized elsewhere (45, 46), and in fact many previously established chemo-preventative agents play a role in increasing cellular migration (47), which may contribute to their efficacy in delaying tumor onset.

Normal planar cell divisions within the crypt can support mitotic pressure for migration to occur in a straight line, the quickest route from crypt base to villus tip, whereas apico-basal divisions could logically result in “side-to-side” cell movement and “upward,” which could be responsible for the increased migration time observed in Dlg1ΔIEC/ΔIEC mice (Fig. 6). This has been previously shown in astrocyte cells in which deficiency of the Dlg1:Dlgap1 complex, also identified in this study, leads to inefficient migration (40). Further, although severely abnormal angles of mitosis may lead to delamination of cells they would normally undergo apoptosis. Any further suppression of apoptosis leads to formation of disorganized masses of aberrant cells with characteristics of tumorigenesis and EMT, potentially accounting for the increase in more advanced lesion observed here. However, it must be noted that during mitosis within the intestinal crypt, cells step out of the epithelial sheet but maintain contact with surrounding cells and divide more luminally before the daughter cells return to the intestinal epithelium proper. It is possible that the orientation of cell division makes less difference to the final position of the daughter cells within the intestinal epithelium than the process by which cells return to the epithelial sheet.

Previous reports have linked epigenetic modification to altered expression of genes associated with cell polarity (48), thereby enabling transcriptional repressors such as Kaiso to regulate cell polarity. However, further work using chromatin immunoprecipitation techniques is required to confirm that these genes are direct targets of Kaiso. The work we present here supports the idea that transcriptional repression of polarity associated genes plays a role in tumorigenesis and tumor invasion (49), and that inhibition of such epigenetic regulators may be of therapeutic value. However, it should be noted that the phenotype associated with the loss of Kaiso may also reflect yet unidentified changes in genes associated with its transcriptional activation functions. At the very least, identification and exploration of the targets of such epigenetic transcriptional regulators which may influence cell polarity could prove to be of value to the field of cancer research. As Kaiso inhibition has been suggested as a potential therapeutic strategy for the treatment of colorectal cancer, and inhibitors for this transcriptional repressor are currently being investigated (50). The work presented here indicates that the tumor-suppressive effect of Kaiso loss is limited to systems in which functional Dlg1 is present at the initiation stage of a tumor. It remains to be determined whether targeting Kaiso in a higher-grade tumor will have any beneficial effect and whether it would be dependent on Dlg1 remaining unmutated within the patient's tumor. As such, the efficacy of such a treatment would be limited to lower grade tumors where Dlg1 remains functional, and tumor resistance to treatment could develop through additional mutations in Dlg1.

No potential conflicts of interest were disclosed.

Conception and design: W. Swat, L. Parry

Development of methodology: M.A. Young, W. Swat, L. Parry

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.A. Young, S. May, A. Damo, L. Parry

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.A. Young, Y.S. Yoon, M.-W. Hur, L. Parry

Writing, review, and/or revision of the manuscript: M.A. Young, L. Parry

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.A. Young, S. May, Y.S. Yoon, L. Parry

Study supervision: M.A. Young, M.-W. Hur, L. Parry

The authors are grateful to Elaine Taylor, Matthew Zverev, and Derek Scarborough for technical support. L. Parry would like to thank David & Deborah Philpott and Liam Hurley for assistance with space. This work was supported by Cancer Research UK (to M. Young; L. Parry; program grant C1295/A15937); Moorhouse Foundation award (to S. May); Cardiff University, School of Bioscience Seedcorn Award (to M. Young); Cardiff University Fellowship (to L. Parry).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data