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• W1655178001 abstract "•Apc suppression in mouse colon recapitulates the molecular features of human CRC•Apc restoration triggers differentiation and restores crypt homeostasis•CRCs harboring Kras and p53 mutations remain strictly dependent on Apc loss•In vivo validation of the Wnt pathway as an effective therapeutic target in CRC The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC. The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC. Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries, and almost half of the population will develop at least one benign intestinal tumor during their lifetime (Jemal et al., 2011Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J. Clin. 2011; 61: 69-90Crossref PubMed Scopus (30255) Google Scholar). Treatment regimes for advanced CRC involve combination chemotherapies that are toxic and largely ineffective, yet have remained the backbone of therapy over the last decade. Molecularly, the vast majority (∼80%–90%) of colorectal tumors contain inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor (Brannon et al., 2014Brannon A.R. Vakiani E. Sylvester B.E. Scott S.N. McDermott G. Shah R.H. Kania K. Viale A. Oschwald D.M. Vacic V. et al.Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions.Genome Biol. 2014; 15: 454Crossref PubMed Scopus (253) Google Scholar), and individuals with specific germline mutations in APC (familial adenomatous polyposis or FAP) invariably develop colon cancer before the age of 35. Collectively, APC mutant CRC accounts for more than 600,000 deaths annually worldwide, a number greater than KRAS mutant lung or pancreas cancer. Hence, strategies to exploit APC alterations in CRC have broad clinical potential. APC regulates a number of cellular functions, including mitosis, migration, and the maintenance of genome stability (Nelson and Näthke, 2013Nelson S. Näthke I.S. Interactions and functions of the adenomatous polyposis coli (APC) protein at a glance.J. Cell Sci. 2013; 126: 873-877Crossref PubMed Scopus (59) Google Scholar). Most importantly, APC, along with AXIN1 and GSK3β, is part of a multi-protein complex that controls output of the Wnt signaling pathway by regulating the sub-cellular localization and stability of CTNNB1 (β-catenin), a key transcriptional regulator that drives Wnt signaling output. APC inactivation is considered the initiating event in most CRCs, and Apc loss is sufficient to induce benign and dysplastic adenomas in the small and large mouse intestine (Cheung et al., 2010Cheung A.F. Carter A.M. Kostova K.K. Woodruff J.F. Crowley D. Bronson R.T. Haigis K.M. Jacks T. Complete deletion of Apc results in severe polyposis in mice.Oncogene. 2010; 29: 1857-1864Crossref PubMed Scopus (57) Google Scholar, Su et al., 1992Su L.K. Kinzler K.W. Vogelstein B. Preisinger A.C. Moser A.R. Luongo C. Gould K.A. Dove W.F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.Science. 1992; 256: 668-670Crossref PubMed Scopus (1347) Google Scholar). ApcMin (Multiple Intestinal Neoplasia) mice carry a single mutant Apc allele and develop 50–100 benign adenomas in the small intestine by 4–6 months of age, invariably associated with loss of the remaining wild-type gene (Su et al., 1992Su L.K. Kinzler K.W. Vogelstein B. Preisinger A.C. Moser A.R. Luongo C. Gould K.A. Dove W.F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.Science. 1992; 256: 668-670Crossref PubMed Scopus (1347) Google Scholar). Conditional Apc truncation drives a similar neoplastic phenotype, associated with hyperproliferation, reduced multi-lineage differentiation, disrupted tissue structure, and an expansion of intestinal stem cells outside the crypt domain (Barker et al., 2009Barker N. Ridgway R. van Es J. van de Wetering M. Begthel H. van den Born M. Danenberg E. Clarke A. Sansom O. Clevers H. Crypt stem cells as the cells-of-origin of intestinal cancer.Nature. 2009; 457: 608-611Crossref PubMed Scopus (1635) Google Scholar, Sansom et al., 2004Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. et al.Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (659) Google Scholar). Besides APC disruption, CRCs show a high incidence of mutations in KRAS (45%) and TP53 (54%) that cooperate to drive tumor progression (Cancer Genome Atlas Network, 2012Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5904) Google Scholar). Additionally, recent large-scale sequencing efforts have cataloged additional genetic changes that likely influence disease progression (Cancer Genome Atlas Network, 2012Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5904) Google Scholar). Still, little is known about which, if any, of these alterations are required for maintenance of established disease and continued malignant progression. Indeed, it remains unclear whether APC disruption, the predominant CRC-associated event, is required for maintenance of CRC and thus whether hyperactivated Wnt signaling is a viable therapeutic target. To address this question, we generated shRNA transgenic mice that enable conditional and reversible control of Apc expression by TRE-regulated, GFP-linked short-hairpin RNAs (TG-shRNAs) (Dow et al., 2012Dow L.E. Premsrirut P.K. Zuber J. Fellmann C. McJunkin K. Miething C. Park Y. Dickins R.A. Hannon G.J. Lowe S.W. A pipeline for the generation of shRNA transgenic mice.Nat. Protoc. 2012; 7: 374-393Crossref PubMed Scopus (120) Google Scholar, Premsrirut et al., 2011Premsrirut P.K. Dow L.E. Kim S.Y. Camiolo M. Malone C.D. Miething C. Scuoppo C. Zuber J. Dickins R.A. Kogan S.C. et al.A rapid and scalable system for studying gene function in mice using conditional RNA interference.Cell. 2011; 145: 145-158Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In mice that also express a reverse tet-transactivator (rtTA), doxycycline (dox) administration drives GFP expression and Apc silencing, and subsequent dox withdrawal results in restoration of endogenous Apc expression. Our work demonstrates a crucial role for Apc loss in CRC maintenance and reveals an unexpected ability of Apc to re-establish control of crypt homeostasis in animals with hyperproliferative polyps or cancer. Collectively, our results validate the APC/WNT pathway is an attractive target for the treatment of CRC. Acute genetic disruption of Apc in the intestine drives hyperproliferation and expansion of undifferentiated progenitor cells (Sansom et al., 2004Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. et al.Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (659) Google Scholar). This results in the disruption of the crypt-villus axis, whereby stem and progenitor cells, normally restricted to the crypt base, expand and fail to differentiate as they move up the villus (Sansom et al., 2004Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. et al.Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (659) Google Scholar). To assess how shRNA-mediated Apc suppression affects crypt-villus homeostasis, we examined Wnt pathway activation in the intestinal epithelium in multiple Apc shRNA strains (TG-Apc.3374 and TG-Apc.9365) (Premsrirut et al., 2011Premsrirut P.K. Dow L.E. Kim S.Y. Camiolo M. Malone C.D. Miething C. Scuoppo C. Zuber J. Dickins R.A. Kogan S.C. et al.A rapid and scalable system for studying gene function in mice using conditional RNA interference.Cell. 2011; 145: 145-158Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). We observed that only the most potent suppression of Apc (TG-Apc.3374) caused an increase in non-phosphorylated β-catenin in the intestine (Figure S1A). This response was further amplified with a CAGs-rtTA3 transgene, driving greater Apc knockdown and more than 20-fold transcriptional induction of the canonical Wnt target Axin2 (Figures S1A and S1B). To examine the intestinal response to Apc silencing, we treated TG-Apc.3374 (hereafter, shApc) animals with dox and monitored their weight as a surrogate measure of intestinal function. After 6 days of dox treatment shApc animals began to show signs of weight loss relative to neutral TG-shRen.713 (shRen) controls (Zuber et al., 2011Zuber J. McJunkin K. Fellmann C. Dow L.E. Taylor M.J. Hannon G.J. Lowe S.W. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.Nat. Biotechnol. 2011; 29: 79-83Crossref PubMed Scopus (201) Google Scholar), and by day 10, shApc mice were lethargic and moribund (Figure 1A). Depletion of Apc caused a marked expansion of alkaline phosphatase (AP) and keratin 20 (Krt20) negative progenitors and hyperproliferation throughout the crypt-villus axis (Figure 1B). The block in differentiation and expansion of stem and progenitor cells was further confirmed by the identification of lysozyme-positive Paneth cells and Lgr5 and Olfm4-positive stem cells outside their normal position at the base of the crypt (Figure 1B). Apc silencing induced a progressive increase in the expression of canonical Wnt targets, including Axin2 and cMyc, as well as markers of intestinal stem cells Lgr5 and Ascl2 (Figures 1C and 1D). Importantly, a second transgenic strain harboring an independent and potent Apc shRNA (TG-Apc.2235E) produced identical phenotypes (Figure 1A, Figure S1C). These data demonstrate that shRNA-driven Apc silencing can recapitulate the conditional knockout phenotype (Sansom et al., 2004Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. et al.Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (659) Google Scholar). However, in contrast to genetic deletions, shRNAs suppress gene function in trans, and, as such, the endogenous protein can be restored simply by eliminating shRNA expression. To assess whether Apc restoration could rescue the intestinal dysfunction, we treated shApc mice for 10 days and then withdrew dox from the diet. Apc restoration induced a rapid and dramatic phenotypic response, and by 8 days the mice recovered to their original weight (Figure 1A). Within 2 days of dox withdrawal, GFP-shRNA transcription from the TRE promoter was reduced more than 100-fold and by 4 days had returned almost to baseline levels (Figure 1C). Remarkably, 4 days of dox withdrawal was sufficient to restore crypt-villus homeostasis as evidenced by basal localization of proliferative stem and progenitor cells and lysozyme+ Paneth cells, while differentiated markers (AP, Krt20) were strongly expressed throughout the villus (Figure 1B). Accordingly, a decrease in levels of Wnt-responsive mRNAs, as well as Myc protein, were apparent as early as 2 days after dox withdrawal and returned to baseline by 4 days (Figures 1C and 1D). Together these data show that acute Apc suppression recapitulates the phenotypes of Apc deletion and, importantly, that restoration of endogenous Apc expression can recover normal intestinal function. The above results are striking and highlight the utility of inducible shRNA transgenic mice to study the reversibility of loss of function phenotypes. However, these models are not ideal for studying cancer, as tissue-wide Apc suppression produces lethality well before mice develop intestinal tumors. Moreover, tumorigenesis in humans initiates from individual mutated cells surrounded by otherwise normal tissue. To address this, we modeled mosaic Apc loss by combining a stem cell restricted 4-hydroxytamoxifen (4OHT)-inducible CreER strain (Lgr5-GFP-IRES-CreER, hereafter Lgr5-CreER) (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. Clevers H. Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3945) Google Scholar) with a Cre-dependent R26-CAGs-LSL-rtTA3 (LSL-rtTA3) strain that we recently generated (Dow et al., 2014Dow L.E. Nasr Z. Saborowski M. Ebbesen S.H. Manchado E. Tasdemir N. Lee T. Pelletier J. Lowe S.W. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice.PLoS ONE. 2014; 9: e95236Crossref PubMed Scopus (49) Google Scholar). In these mice, a single treatment with 4OHT initiates LoxP-recombination in stem cells of the small and large intestine, inducing long-lived, rtTA3 expression in a small percentage of individual crypts and villi (Figures S2A and S2B). In LSL-rtTA3 / Lgr5-CreER / TG-Ren.713 (shRen/Lgr5) control mice treated with 4OHT and dox we observed GFP induction in individual crypts and villi throughout the length of the small intestine and colon for up to 40 weeks (Figure S2C, data not shown). Of note, the shRNA linked GFP reporter is significantly more abundant than GFP expressed from the Lgr5-GFP-IRES-CreER knockin allele and can be used as a surrogate marker of shRNA expression and Apc knockdown. Next, we generated LSL-rtTA3 / Lgr5-CreER / shApc (shApc/Lgr5) mice and monitored tumor development longitudinally by small animal colonoscopy. Four-to-six weeks following 4OHT/dox treatment, we noted the development of macroscopic polyps in the small intestine and colon (Figure 2A, Figure S3). By 12–16 weeks, shApc/Lgr5 mice developed large colonic polyps that appeared histologically as well-differentiated tubular adenomas. As is characteristic of tumors initiated by mutations in Apc, polyps arising in shApc/Lgr5 animals showed a massive increase in proliferation, marked by BrdU incorporation (Figure 2B). These lesions also carried reduced numbers of Krt20+ enterocytes and Alcian blue+ goblet cells and showed a significant increase in Lgr5+ stem cells outside of their normal position at the crypt base (Figure 2B, Figure S3). Together, these data highlight a block in the production of differentiated cell types and an expansion of the stem and progenitor compartment.Figure S3Apc Restoration Drives Regression of Small Intestinal Adenomas, Related to Figure 3Show full caption(A) Left: Longitudinal colonoscopic images of a dox-treated shApc/Lgr5 mouse, as indicated, and following 4, 8 and 13 weeks of dox withdrawal. Right: Immunohistochemical stains (H&E and Alcian blue) showing a persistent polyp mass in an Apc-restored mouse 15 weeks after dox withdrawal. Although abnormal in structure, the mass consists of “normal” mucosa, predominantly composed of differentiated Goblet cells (Alcian blue positive).(B) Immunofluorescent stains for BrdU (green) in intestinal adenomas either on dox or 1, 2, or 4 days following dox withdrawal, showing progressive decrease in cells incorporating nucleotide.(C) Immunohistochemical (H&E), immunofluorescent (Krt20 and CC3) stains and in situ hybridization (Lgr5) from shRen/Lgr5 and shApc/Lgr5 small intestine following dox treatment for 12–15 weeks (left two panels) and withdrawal from dox for 4 days and 20 weeks (right two panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Left: Longitudinal colonoscopic images of a dox-treated shApc/Lgr5 mouse, as indicated, and following 4, 8 and 13 weeks of dox withdrawal. Right: Immunohistochemical stains (H&E and Alcian blue) showing a persistent polyp mass in an Apc-restored mouse 15 weeks after dox withdrawal. Although abnormal in structure, the mass consists of “normal” mucosa, predominantly composed of differentiated Goblet cells (Alcian blue positive). (B) Immunofluorescent stains for BrdU (green) in intestinal adenomas either on dox or 1, 2, or 4 days following dox withdrawal, showing progressive decrease in cells incorporating nucleotide. (C) Immunohistochemical (H&E), immunofluorescent (Krt20 and CC3) stains and in situ hybridization (Lgr5) from shRen/Lgr5 and shApc/Lgr5 small intestine following dox treatment for 12–15 weeks (left two panels) and withdrawal from dox for 4 days and 20 weeks (right two panels). In line with previous observations in ApcMin animals (Moser et al., 1990Moser A.R. Pitot H.C. Dove W.F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.Science. 1990; 247: 322-324Crossref PubMed Scopus (1309) Google Scholar), shApc/Lgr5 mice treated with 4OHT displayed a progressive decrease in hematocrit, associated with blood loss following polyp development (Figure 2C), and a median survival of 115 days (Figure 2D). However, in contrast to Apcfl/fl/Lgr5 (and ApcMin) animals that developed disease almost exclusively in the small intestine, concentrated in the distal jejunum and ileum (Figure 2E), tumors in shApc/Lgr5 mice arose in the colon and duodenum/proximal jejunum (Figures 2E and 2F), which more closely reflects the anatomy of disease presentation in FAP patients (Sarre et al., 1987Sarre R.G. Frost A.G. Jagelman D.G. Petras R.E. Sivak M.V. McGannon E. Gastric and duodenal polyps in familial adenomatous polyposis: a prospective study of the nature and prevalence of upper gastrointestinal polyps.Gut. 1987; 28: 306-314Crossref PubMed Scopus (171) Google Scholar). This regional difference in tumor distribution was not due to the restricted activation of Cre, or doxycycline treatment, as we observed Cre recombination (GFP induction) throughout the small intestine and colon (Figure S2C), and both ApcMin and Apcfl/fl/Lgr5-CreER mice were treated with dox from 5–6 weeks of age. Regardless of the precise mechanism, the appearance of colorectal disease enabled longitudinal studies of disease progression and/or regression in live mice using small animal endoscopy. To examine the molecular characteristics of colonic adenomas formed in shApc/Lgr5 mice, we performed gene expression analysis by RNAseq and identified 3,225 (1,339 up, 1,886 down) genes that were significantly deregulated (2-fold or greater) relative to shRen/Lgr5 control mucosa. This expression signature was enriched for both up and downregulated transcripts identified in human colorectal cancers (Stages I-IV, FDR < .001) (Figure 2G, Figure S2D) (Ongen et al., 2014Ongen H. Andersen C.L. Bramsen J.B. Oster B. Rasmussen M.H. Ferreira P.G. Sandoval J. Vidal E. Whiffin N. Planchon A. et al.Putative cis-regulatory drivers in colorectal cancer.Nature. 2014; 512: 87-90PubMed Google Scholar) as well as human colon adenomas (FDR < .001) (Figure S2D) (Sabates-Bellver et al., 2007Sabates-Bellver J. Van der Flier L.G. de Palo M. Cattaneo E. Maake C. Rehrauer H. Laczko E. Kurowski M.A. Bujnicki J.M. Menigatti M. et al.Transcriptome profile of human colorectal adenomas.Mol. Cancer Res. 2007; 5: 1263-1275Crossref PubMed Scopus (385) Google Scholar). shApc/Lgr5 tumors also contained an overrepresentation of transcripts found in purified Lgr5+ stem cells and enterocyte progenitors (FDR < .001) and an underrepresentation of genes exclusively expressed in secretory progenitors or enterocytes (Figure S2D and Table S1) (Kim et al., 2014Kim T.H. Li F. Ferreiro-Neira I. Ho L.L. Luyten A. Nalapareddy K. Long H. Verzi M. Shivdasani R.A. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity.Nature. 2014; 506: 511-515Crossref PubMed Scopus (172) Google Scholar). Further, shApc/Lgr5 colon tumors showed a decrease in Krt20 (Figure 2B) and Slc26a3 expression (Figure 2G, bottom), which together, define a two-gene classifier that is associated with poor prognosis in human CRC (Dalerba et al., 2011Dalerba P. Kalisky T. Sahoo D. Rajendran P.S. Rothenberg M.E. Leyrat A.A. Sim S. Okamoto J. Johnston D.M. Qian D. et al.Single-cell dissection of transcriptional heterogeneity in human colon tumors.Nat. Biotechnol. 2011; 29: 1120-1127Crossref PubMed Scopus (539) Google Scholar). Thus, the gene expression profile of shApc/Lgr5 tumors is consistent with our histological analyses and indicates that Apc suppression impairs differentiation and drives the expansion of progenitor cells. In all, the histological, anatomical, and molecular characteristics of colon adenomas formed in shApc/Lgr5 mice suggest they accurately reflect the early stages of human colorectal cancer. We next asked whether sustained Apc loss was essential for disease maintenance by restoring endogenous Apc protein expression in established polyps of shApc/Lgr5 mice. The tumor response to dox withdrawal was striking: in the colon, longitudinal endoscopic imaging of dox-withdrawn animals showed rapid regression of polyps over many weeks (Figure 3A, Figure S3A). Small intestinal polyps regressed even more rapidly, and indeed, no macroscopic polyps were visible in the small intestine after two weeks of Apc restoration (Figure S3C). In some cases, we noted the persistence of small polyp-like masses visible in the colon for weeks following dox withdrawal (Figure S3A, arrows). While abnormal in structure, these masses were composed of histologically normal, differentiated epithelium (Figure S3A, right), perhaps retained due to irreversible changes in tissue structure or fibrosis during tumor growth. As expected, the marked response to Apc reactivation was associated with a corresponding decrease in Wnt target gene expression (Figure 3D). However, in contrast to studies showing forced overexpression of APC in tumor cells can promote apoptosis (Morin et al., 1996Morin P.J. Vogelstein B. Kinzler K.W. Apoptosis and APC in colorectal tumorigenesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 7950-7954Crossref PubMed Scopus (445) Google Scholar), endogenous Apc produced no increase in apoptotic cell death as measured by histology and lack of cleaved-Caspase 3 staining (Figure 3C, Figure S3C). Instead, Apc restoration was associated with rapid cell-cycle arrest and massive differentiation. Specifically, within 4 days following dox withdrawal, almost all tumor cells had ceased proliferation (Figure 3B, Figure S3B), showed a dramatic increase in Krt20 expression, and a decrease in Lgr5+ stem cells within the polyp (Figure 3C, Figure S3C). The tumor suppressive effect of Apc was potent, and indeed, we have never observed relapsed disease in the small intestine or colon in mice monitored up to 6 months following Apc restoration (n = 13, Figure 3E). The rapid differentiation response of cells within adenomas prompted us to explore if any Apc-restored cells remained at the site of the regressed adenoma or if they were eliminated as differentiated epithelial tissue. In our model, bright GFP expression marks Cre-recombined cells that express a dox-dependent shRNA, however, following dox withdrawal, GFP expression is lost. To identify any remaining tumor cells 2 weeks following Apc restoration, we pulsed mice with dox for 2 days (Figure 4A). Importantly, 2 days is sufficient time to induce GFP expression and thus “lineage trace” Cre-recombined cells, but not long enough to produce a hyperproliferative response to Apc silencing (Figure 4B, “shApc - naive label”). GFP re-labeling in control (shRen/Lgr5) mice revealed entirely GFP-positive crypts, indicating the presence of long-lived, Cre-recombined stem cells that contribute continually to the normal mucosa (Figure 4B, “shRen re-label”). In regressing adenomas, we noted similar GFP “ribbons” extending from the crypt base to the outer differentiated epithelium (Figure 4B, “shApc re-label”). GFP-positive regions appeared histologically similar to GFP-negative neighboring crypts, containing markers of differentiated enterocytes (Krt20 and Villin) and Goblet cells (Muc2 and Alcian blue) (Figure 4B, Figure S4).Figure S4Apc Restoration Re-establishes Goblet Cell Differentiation, Related to Figure 4Show full captionImmunohistochemical (H&E and Alcian blue) stains showing an shApc/Lgr5 polyp pulsed with dox for 2 days after 2 weeks of dox withdrawal. Krt20/GFP stained image of the same polyp is shown in Figure 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunohistochemical (H&E and Alcian blue) stains showing an shApc/Lgr5 polyp pulsed with dox for 2 days after 2 weeks of dox withdrawal. Krt20/GFP stained image of the same polyp is shown in Figure 4. Apc-restored cells retained within regressed tumors maintained the potential for tumor growth and 10 weeks following re-silencing of Apc, regressed polyps had regrown to their original size (Figure 4C). We repeated the cycle, this time maintaining the animals off dox for 15 weeks, and again all tumors showed regression and subsequent regrowth at their original sites (Figure 4C). Thus, Apc restoration in colorectal polyps not only triggers differentiation, but also restores normal homeostasis in crypt based columnar cells, even after multiple rounds of tumorigenic growth and regression. The in vivo experiments described above provide a clear demonstration that Apc restored cells can re-establish normal behavior; however, closely following cell fate and differentiation capacity longitudinally in a complex tissue is challenging. Ex vivo intestinal crypt culture (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4123) Google Scholar) provides a means to directly interrogate cellular response to Apc in the absence of a complex microenvironment. To ask whether Apc-restored cells could reestablish the balance of self-renewal and multi-lineage differentiation without cues from surrounding normal tissue, we derived small intestinal crypt cultures from shApc mice and examined their behavior following the addition and withdrawal of dox. Consistent with studies using Apc mutant organoids (Sato et al., 2011Sato T. Stange D.E. Ferrante M. Vries R.G. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2093) Google Scholar, Dow et al., 2015Dow L.E. Fisher J. O’Rourke K.P. Muley A. Kastenhuber E.R. Livshits G. Tschaharganeh D.F. Socci N.D. Lowe S.W. Inducible in vivo genome editing with CRISPR-Cas9.Nat. Biotechnol. 2015; (2015 Apr;33(4):390–4)PubMed Google Scholar), shRNA-mediated gene silencing of Apc triggered a transition to hyperproliferative “spheroids” (Figures 5A and 5B ). Like shApc-driven adenomas, spheroids showed a significant increase in proliferation, a block in differentiation (marked by loss of Krt20, Alk Phos and Muc2-positive cells) and loss of crypt-like projections containing Paneth cells (Figure 5A). Restoration of Apc expression in this setting (Figure 5B) induced a rapid phenotypic reversion, and by 4 days following dox withdrawal greater than 90% of the culture s" @default.
• W1655178001 created "2016-06-24" @default.
• W1655178001 creator A5012566985 @default.
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• W1655178001 date "2015-06-01" @default.
• W1655178001 modified "2023-10-16" @default.
• W1655178001 title "Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer" @default.
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