Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

Min J, Vega PN, Engevik AC, Williams JA, Yang Q, Patterson LM, Simmons AJ, Bliton RJ, Betts JW, Lau KS, Magness ST, Goldenring JR, Choi E

Nat Commun. 2019 Dec 5;10(1):5549. PMID:31804471



Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.

Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity

Jin Y, Ibrahim D, Magness ST, Blikslager AT

Am J Physiol Gastrointest Liver Physiol. 2018 Dec 1;315(6):G966-G979. Epub 2018 Oct 4. PMID: 30285466



Adherens junctions (AJs), together with tight junctions (TJs), form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ, membrane-bound E-cadherin binds β-catenin, which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex, chloride channel protein-2 (ClC-2), that we have used to study TJ regulation. In this study, we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation, whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore, the absence of ClC-2 resulted in redistribution of AJ proteins and increased β-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased, associated with β-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of β-catenin-associated signaling. NEW & NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures, including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating β-catenin signaling.

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

Kechele DO, Blue RE, Zwarycz B, Espenschied ST, Mah AT, Siegel MB, Perou CM, Ding S, Magness ST, Lund PK, Caron KM

J Clin Invest. 2017 Feb 1;127(2):593-607.  Epub 2017 Jan 17. PMID: 28094771



Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.

SOX9 Maintains Reserve Stem Cells and Preserves Radio-resistance in Mouse Small Intestine.


Roche KC, Gracz AD, Liu XF, Newton V, Akiyama H, Magness ST

Gastroenterology. 2015 Nov;149(6):1553-1563. Epub 2015 Jul 11. PMID: 26170137



BACKGROUND & AIMS: Reserve intestinal stem cells (rISCs) mediate epithelial regeneration following tissue damage. Unlike active intestinal stem cells (aISCs), rISCs slowly cycle under homeostatic conditions, allowing for their identification with label retention assays. In response to certain epithelial injuries, rISCs convert to an actively dividing state and demonstrate multipotency and self-renewal, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9high) have been associated with rISCs. We investigated the role of SOX9 in maintaining rISCs. METHODS: We performed single-cell analyses to characterize the expression of active and reserve ISC markers in Lgr5high cells (aISC) and Sox9high cells (rISC) isolated from reporter mice by fluorescence-activated cell sorting. We used label-retention assays to determine the proliferative capacity of Sox9high cells. Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radio-resistance of Sox9-expressing cells. Conditional knockout (SOX9cKO) and inducible-conditional (SOX9iKO) knockout mice were used to determine whether SOX9 was required to maintain label-retaining cells (LRCs) and rISC function. RESULTS: Lgr5high and a subset of crypt-based Sox9high cells co-express markers of aISC and rISC (Lgr5, Bmi1, Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration following high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell cycle arrest or DNA repair. CONCLUSIONS: Sox9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.

Sry-box (SOX) transcription factors in gastrointestinal physiology and disease


Gracz AD, Magness ST

Am J Physiol Gastrointest Liver Physiol. 2011 Apr;300(4):G503-15. Epub 2011 Feb 3. Review. PMID: 21995959



The genetic mechanisms underlying tissue maintenance of the gastrointestinal tract are critical for the proper function of the digestive system under normal physiological stress. The identification of transcription factors and related signal transduction pathways that regulate stem cell maintenance and lineage allocation is attractive from a clinical standpoint in that it may provide targets for novel cell- or drug-based therapies. Sox [sex-determining region Y (Sry) box-containing] factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems and might provide valuable insight toward the understanding of these processes in endodermally derived tissues of the gastrointestinal tract. In this review, we focus on the known genetic functions of Sox factors and their roles in epithelial tissues of the esophagus, stomach, intestine, colon, pancreas, and liver. Additionally, we discuss pathological conditions in the gastrointestinal tract that are associated with a dysregulation of Sox factors. Further study of Sox factors and their role in gastrointestinal physiology and pathophysiology may lead to advances that facilitate control of tissue maintenance and development of advanced clinical therapies.

Insulin receptor substrate-1 deficiency promotes apoptosis in the putative intestinal crypt stem cell region, limits Apcmin/+ tumors, and regulates Sox9


Ramocki NM, Wilkins HR, Magness ST, Simmons JG, Scull BP, McNaughton KK, Lund PK

Endocrinology. 2008 Jan;149(1):261-7. Epub 2007 Oct 4. PMID: 17916629



Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and IGFs are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1(-/-), IRS-1(+/-), and IRS-1(+/+) mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apc(min/+) (Min)/beta-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/beta-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1-deficient mice. Tumor load was significantly reduced by 31.2 +/- 14.6% in IRS-1(+/-)/Min and by 64.1 +/- 7.6% in IRS-1(-/-)/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1(+/+)/Min, IRS-1(-/-)/Min mice had fewer Sox9-positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against beta-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/beta-catenin target and putative stem/progenitor cell biomarker.