Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer

Abstract

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.

Keywords: APC; FAP; Wnt; polyposis; shRNA; tumor regression.

Figure 1. Acute depletion of Apc in the mouse

A. Animal weight during dox treatment and following dox withdrawal at 10 days, normalized to Day 0 within each cohort. Lines represent TG-Ren.713 (black), TG-Apc.3374 (green) and TG-Apc.2235E (blue) mice. B. Immunohistochemical (H&E, Alkaline phosphatase and Lysozyme), immunofluorescent (Ki67 and Keratin 20) stains and in situ hybridizations (Lgr5 and Olfm4) from shRen (control) and shApc intestine following dox treatment for 10 days (left two panels) and withdrawn from dox for 4 days (right panel). C. Quantitative RT-PCR analysis of gene expression in intestinal villi following dox treatment and withdrawal for TG-Apc.3374 mice as indicated. Markers of transgene induction (GFP), Wnt activation (Axin2, Myc) and stem cells (Lgr5, Ascl2) are shown for each time point. Gene expression in day 10-treated TG-Ren.713 is indicated to the right on each plot. D. Western blot of whole cell lysates from intestinal villi following dox treatment and withdrawal at time points indicated, for TG-Ren.713 and TG-APC.3374 mice, probed for Apc, GFP, cMyc and β-Actin, as indicated.

Figure 2. Apc knockdown drives the development of colonic polyps

A. Colon endoscopic images from LSL-rtTA3/shRen/Lgr5-CreER (shRen) or LSL-rtTA3/shApc/Lgr5-CreER (shApc) mice following dox treatment for times indicated, showing gradual polyp development in shApc/Lgr5 animals. B. Immunohistochemical (H&E, Alcian blue), immunofluorescent (GFP/BrdU and Krt20) stains and in situ hybridization (Lgr5) from shRen/Lgr5 and shApc/Lgr5 colons following dox treatment for 10 weeks. C. Hematocrit in peripheral blood of shRen/Lgr5 and shApc/Lgr5 mice over time following 4OHT and dox treatment. D. Kaplan-Meier plot showing survival of dox-treated Apc^Min/+ (red line), Apc^flox/flox/Lgr5 (blue line), shRen/Lgr5 (black line), and shApc/Lgr5 (green line). Lgr5-CreER containing mice were treated with 4OHT at 5-6 weeks of age and all animals (including Apc^Min/+) were treated continually with doxycycline from this time onward. E. Quantification of tumor burden from H&E stained paraffin embedded sections of Apc^flox/flox/Lgr5 and shApc/Lgr5 mice. Tumor area was calculated as a percentage of total tumor burden for each of five intestinal regions representing the duodenum, proximal and distal jejunum, ileum and colon, as indicated. F. Dot plot showing number of macroscopic polyps counted in whole mount colon tissue in Apc^Min/+, Apc^flox/flox/Lgr5, and shApc/Lgr5 mice at necropsy. G. Heatmap representing the top 50 up (red) and down regulated (blue) transcripts in shApc colonic adenomas relative to shRen mucosa (OFF Dox). Transcript abundance (log2 fold change) for each gene is also shown for shApc/Kras tumors, shRen mucosa (ON Dox) and in human Stage I-IV colorectal cancers (Ongen et al., 2014). Genes listed to the right are those among the top 50 deregulated in both the mouse shApc and Human CRC datasets, and those genes in bold are validated by quantitative RT-PCR.

Figure 3. Apc restoration drives adenoma regression

A. Longitudinal colonoscopic images of a dox-treated shApc/Lgr5 mouse at 8 and 15 weeks, and following 2, 8 and 15 weeks of dox withdrawal. B. Immunofluorescent stains for BrdU (green) in colonic 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 (Keratin 20 and Cleaved Caspase 3) stains and in situ hybridization (Lgr5) from shRen/Lgr5 and shApc/Lgr5 colon/adenomas following dox treatment for 12-15 weeks (left two panels) and withdrawal from dox for 4 days and 20 weeks (right two panels). D. Quantitative RT-PCR analysis of Wnt target genes (Axin2, Myc) and stem cell markers (Lgr5, Ascl2) in isolated colonic adenomas (shApc/Lgr5) and normal mucosa (shRen/Lgr5) 15 weeks on dox and 4 days following dox withdrawal, as indicated. Values are normalized to shRen (on dox) for each target. E. Kaplan-Meier plot showing survival of dox-treated shApc/Lgr5 mice (dotted green line, also represented in Figure 2D) and shApc/Lgr5 mice removed from dox treatment between 12-16 weeks (blue line).

Figure 4. Apc restorations re-establishes crypt homeostasis

A. Schematic representation of GFP relabeling experiment. 4OHT treated mice that had been on dox for 20 weeks (green bars) were removed from dox treatment for 2 weeks (grey bar) to promote Apc-driven tumor regression, then refed dox food for 2 days to induce GFP expression. As a control, naïve shApc/Lgr5 mice were treated with 4OHT, and then fed dox food for 2 days to indicate the outcome of Apc shRNA induction for 2 days. B. Immunofluorescent (Krt20/GFP, Muc2/GFP and Ki67/Villin) stains showing naïve shApc/Lgr5 mice treated with 4OHT/dox for 2 days (upper left), and shRen/Lgr5 (left) and shApc/Lgr5 mice (right) pulsed with dox for 2 days after 2 weeks of dox withdrawal. Arrows highlight GFP positive (green) and GFP negative (white) crypts present in the regressing polyp. C. Longitudinal colonoscopic images of a dox-treated shApc/Lgr5 mouse withdrawn and retreated with dox twice to induce tumor regression and regrowth, for times indicated.

Figure 5. Apc restores self-renewal and multi-lineage differentiation

A. Brightfield, epifluorescent, and immunofluorescent images of shApc intestinal organoid cultures treated and withdrawn from dox (0.5μg/ml) as indicated. GFP signal (second row) shows induction of the TRE-regulated GFP-shRNA transgene. Panels below show the induction of markers of differentiation (Krt20, Alk Phos and Muc2) and localization of proliferative cells (EdU) and Paneth cells (Lysozyme) at the base of crypts following Apc restoration (or in No Dox controls). Scale bars represent 50μm. High magnification images of Muc2 positive cells in the villus-like domain and Lysozyme positive cells in the crypt are shown on the lower right. Scale bars are 10μm. B. Western blot of whole cell lysates from shRen and shApc intestinal organoid cultures following dox treatment and withdrawal at time points indicated. C. Graph represents the percentage (%) of undifferentiated spheroids (green) and differentiated, crypt-containing organoids (blue) in dox-treated and dox-withdrawn shRen and shApc cultures.

Figure 6. Kras and p53 mutation drive disease progression in shApc tumors

A. Oncoprint map representing frequency of gene loss (blue bars), mutation (green) and amplification (red) in TCGA analysis of human CRC (www.cbioportal.org). B. Endoscopic images from shRen/Kras/Lgr5 and shApc/Kras/Lgr5 mice, 1 year and 6 weeks post-4OHT, respectively. C. Immunohistochemical (H&E) and immunofluorescent (Ki67, Krt20 and Cleaved-Caspase 3) stains from shRen/Kras, shApc/Kras, shRen/Kras/p53^fl/fl and shApc/Kras/p53^fl/fl colons following dox treatment for 6 weeks (shApc mice) or 20 weeks (shRen mice). Arrows indicate regions of hyperproliferation driven by Kras^G12D, as previously described (Haigis et al., 2008). D. Kaplan-Meier plot showing survival of dox-treated shApc (dotted green line, also represented in Figure 2D), shApc/Kras/Lgr5 (purple line) and shApc/Kras/p53^fl/fl/Lgr5 (orange line), following 4OHT treatment at 5-6 weeks of age. E. Blind-scored histological analysis of intestinal sections from shApc/Lgr5, shApc/Kras/Lgr5 and shApc/Kras/p53^fl/fl/Lgr5 mice, as indicated. Animals were scored based on most advanced disease evident in an H&E section through the entire intestine. F. H&E stains section of tissue obtained sub-cutaneous transplanted shApc and shApc/Kras organoids/spheroids, 7 weeks following transplant.

Figure 7. Apc restoration induces sustained regression of colorectal adenocarcinoma

A. Longitudinal colonoscopic images of dox-treated shApc/Kras and shApc/Kras/p53^fl/fl mice, as indicated, and following 1, 3 and 6 weeks of dox withdrawal. B. Immunohistochemical (H&E) and immunofluorescent stains for Ki67 and Krt20 in shApc/Kras colon tumors, ON Dox (left) or 4 days and 10 weeks following dox withdrawal. Scale bars represent 100μm C. Immunofluorescent images of shApc/Kras intestinal organoid cultures (ON Dox and D4 OFF dox, as indicated) showing markers of proliferation (EdU), differentiation (Krt20, Alk Phos and Muc2) and crypt integrity (Lysozyme). Scale bars represent 50μm. High magnification images of Muc2 and Lysozyme staining in Apc-restored/Kras^G12D organoids are shown below, scale bars represent 10μm. D. Immunohistochemical (H&E) and immunofluorescent stains (GFP/Krt20) of an invasive lesion in an shApc/Kras^G12D/Lgr5 tumor where Apc was restored 14 days before pulsing with dox for 2 days to relabel GFP positive cells sacrifice.