Progastrin stimulates colonic cell proliferation via CCK2R- and β-arrestin-dependent suppression of BMP2

Background & Aims Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R) - partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. Methods We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. Results The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44+, bromodeoxyuridine+, and NUMB+ cells, indicating an increase in symmetric divisions of putative cancer stem cells. Conclusions Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division. © 2013 by the AGA Institute

Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury

WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation

Krt19\u3csup\u3e+\u3c/sup\u3e/Lgr5\u3csup\u3e-\u3c/sup\u3e Cells Are Radioresistant Cancer-Initiating Stem Cells in the Colon and Intestine

Epithelium of the colon and intestine are renewed every 3 days. In the intestine there are at least two principal stem cell pools. The first contains rapid cycling crypt-based columnar (CBC) Lgr5+ cells, and the second is composed of slower cycling Bmi1-expressing cells at the +4 position above the crypt base. In the colon, however, the identification of Lgr5- stem cell pools has proven more challenging. Here, we demonstrate that the intermediate filament keratin-19 (Krt19) marks long-lived, radiation-resistant cells above the crypt base that generate Lgr5+ CBCs in the colon and intestine. In colorectal cancer models, Krt19+ cancer-initiating cells are also radioresistant, while Lgr5+ stem cells are radiosensitive. Moreover, Lgr5+ stem cells are dispensable in both the normal and neoplastic colonic epithelium, as ablation of Lgr5+ stem cells results in their regeneration from Krt19-expressing cells. Thus, Krt19+ stem cells are a discrete target relevant for cancer therapy

Prox1-positive cells monitor and sustain the murine intestinal epithelial cholinergic niche

The enteric neurotransmitter acetylcholine governs important intestinal epithelial secretory and immune functions through its actions on epithelial muscarinic Gq-coupled receptors such as M3R. Its role in the regulation of intestinal stem cell function and differentiation, however, has not been clarified. Here, we find that nonselective muscarinic receptor antagonism in mice as well as epithelial-specific ablation of M3R induces a selective expansion of DCLK1-positive tuft cells, suggesting a model of feedback inhibition. Cholinergic blockade reduces Lgr5-positive intestinal stem cell tracing and cell number. In contrast, Prox1-positive endocrine cells appear as primary sensors of cholinergic blockade inducing the expansion of tuft cells, which adopt an enteroendocrine phenotype and contribute to increased mucosal levels of acetylcholine. This compensatory mechanism is lost with acute irradiation injury, resulting in a paucity of tuft cells and acetylcholine production. Thus, enteroendocrine tuft cells appear essential to maintain epithelial homeostasis following modifications of the cholinergic intestinal niche

Long-lived intestinal tuft cells serve as colon cancer-initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth-sustaining stem cells. DCLK1+ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT-dependent genetic lineage-tracing strategy, we determined that a subpopulation of DCLK1+ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1+ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1+ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate-induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1+ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1+ cells. Thus, our data define an intestinal DCLK1+ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1+ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer

Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer

CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer

BHLHA15-Positive Secretory Precursor Cells Can Give Rise to Tumors in Intestine and Colon in Mice

Background & Aims: The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. Methods: We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apc flox/flox , LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. Results: Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15 + cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15 + secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15 + secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15 + precursors toward those of ISCs. Bhlha15 + enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate–induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. Conclusions: In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5neg or low cell population but was distinct from typical antral Lgr5high stem cells. Treatment with progastrin interconverts Lgr5neg or low CCK2R+ cells into Lgr5high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy

Denervation suppresses gastric tumorigenesis

The nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. We provide evidence that proper innervation is critical at all stages of gastric tumorigenesis. In three separate mouse models of gastric cancer, surgical or pharmacological denervation of the stomach (bilateral or unilateral truncal vagotomy, or local injection of botulinum toxin type A) markedly reduced tumor incidence and progression, but only in the denervated portion of the stomach. Vagotomy or botulinum toxin type A treatment also enhanced the therapeutic effects of systemic chemotherapy and prolonged survival. Denervation-induced suppression of tumorigenesis was associated with inhibition of Wnt signaling and suppression of stem cell expansion. In gastric organoid cultures, neurons stimulated growth in a Wnt-mediated fashion through cholinergic signaling. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic acetylcholine M[subscript 3] receptor suppressed gastric tumorigenesis. In gastric cancer patients, tumor stage correlated with neural density and activated Wnt signaling, whereas vagotomy reduced the risk of gastric cancer. Together, our findings suggest that vagal innervation contributes to gastric tumorigenesis via M[subscript 3] receptor–mediated Wnt signaling in the stem cells, and that denervation might represent a feasible strategy for the control of gastric cancer