Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett's Esophagus.

PLoS One. 2015 May 21;10(5):e0127300.

Jang BG, Lee BL, Kim WH.
PMID: 26015511 | DOI: clincanres.3357.2014.

Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5+ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers-including OLFM4 and EPHB2-are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5+ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett's esophagus (BE)-which is histologically similar to intestinal metaplasia-exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5+ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease.

MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer.

J Pathol.

2017 Sep 09

Baena-Del Valle JA, Zheng Q, Esopi DM, Rubenstein M, Hubbard GK, Moncaliano MC, Hruszkewycz A, Vaghasia A, Yegnasubramanian S, Wheelan SJ, Meeker AK, Heaphy CM, Graham MK, De Marzo AM.
PMID: 28888037 | DOI: 10.1002/path.4980

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition, and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high grade prostatic intraepithelial neoplasia or PIN), and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma, and its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

Restriction of intestinal stem cell expansion and the regenerative response by YAP.

Nature, 493(7430), 106–110.

Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD (2012).
PMID: 23178811 | DOI: 10.1038/nature11693.

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

ARID1A and TERT promoter mutations in dedifferentiated meningioma

Cancer Genetics (2015).

Abedalthagafi MS, Wenya Linda Bi WL, Merrill PH, Gibson WJ, Rose MF, Du Z, Francis JM, Du R, Dunn IF, Ligon AH, Beroukhim R, Santagata S.
PMID: 25963524 | DOI: 10.1016/j.cancergen.2015.03.005

While WHO grade I meningiomas are considered benign, patients with WHO grade III meningiomas have very high mortality. The principles underlying tumor progression in meningioma are largely unknown yet a detailed understanding of these mechanisms will be required for effective management of patients with these high-grade, lethal tumors. We present a case of an intraventricular meningioma that at first presentation displayed remarkable morphologic heterogeneity – comprised of distinct regions independently fulfilling histopathologic criteria for WHO grade I, II and III designations. The lowest-grade regions had classic meningothelial features while the highest grade regions were markedly dedifferentiated. While progression in meningiomas is generally observed during recurrence following radiation and systemic medical therapies the current case offers us a snapshot into histologic progression and intratumor heterogeneity in a native, pre-treatment context. Using whole exome sequencing (WES) and high resolution array comparative genomic hybridization (aCGH) we observe marked genetic heterogeneity between the various areas. Notably, in the higher grade regions we find increased aneuploidy with progressive loss of heterozygosity, the emergence of mutations in the TERT promoter and compromise of ARID1A. These findings provide new insights into intratumoral heterogeneity in the evolution of malignant phenotypes in anaplastic meningiomas and potential pathways of malignant progression.

Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury

Nature.

2018 Apr 04

Lin S, Nascimento EM, Gajera CR, Chen L, Neuhöfer P, Garbuzov A, Wang S, Artandi SE.
PMID: 29618815 | DOI: 10.1038/s41586-018-0004-7

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERTHigh hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERTHigh hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHigh hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHigh hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Towards Goblet Cells

Gastroenterology

2023 Mar 01

He, S;Lei, P;Kang, W;Cheung, P;Xu, T;Mana, M;Park, C;Wang, H;Imada, S;Russell, J;Wang, J;Wang, R;Zhou, Z;Chetal, K;Stas, E;Mohad, V;Bruun-Rasmussen, P;Sadreyev, R;Hodin, R;Zhang, Y;Breault, D;Camargo, F;Yilmaz, Ö;Fredberg, J;Saeidi, N;
| DOI: 10.1053/j.gastro.2023.02.030

Background & aims Fibrosis and tissue stiffening are hallmarks of the inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+ proliferating cells. Conversely, cells expressing the stem cell marker, OLFM4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of OLFM4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs towards goblet cells. Furthermore, analysis of colon samples from murine colitis models and IBD patients demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Acinar cell clonal expansion in pancreas homeostasis and carcinogenesis

Nature

2021 Sep 01

Neuhöfer, P;Roake, CM;Kim, SJ;Lu, RJ;West, RB;Charville, GW;Artandi, SE;
PMID: 34526722 | DOI: 10.1038/s41586-021-03916-2

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide1. Studies in human tissues and in mouse models have suggested that for many cancers, stem cells sustain early mutations driving tumour development2,3. For the pancreas, however, mechanisms underlying cellular renewal and initiation of PDAC remain unresolved. Here, using lineage tracing from the endogenous telomerase reverse transcriptase (Tert) locus, we identify a rare TERT-positive subpopulation of pancreatic acinar cells dispersed throughout the exocrine compartment. During homeostasis, these TERThigh acinar cells renew the pancreas by forming expanding clones of acinar cells, whereas randomly marked acinar cells do not form these clones. Specific expression of mutant Kras in TERThigh acinar cells accelerates acinar clone formation and causes transdifferentiation to ductal pre-invasive pancreatic intraepithelial neoplasms by upregulating Ras-MAPK signalling and activating the downstream kinase ERK (phospho-ERK). In resected human pancreatic neoplasms, we find that foci of phospho-ERK-positive acinar cells are common and frequently contain activating KRAS mutations, suggesting that these acinar regions represent an early cancer precursor lesion. These data support a model in which rare TERThigh acinar cells may sustain KRAS mutations, driving acinar cell expansion and creating a field of aberrant cells initiating pancreatic tumorigenesis.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.

Proceedings of the National Academy of Sciences, 109(2), 466–471.

Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, Su N, Luo Y, Heilshorn SC, Amieva MR, Sangiorgi E, Capecchi MR, Kuo CJ (2012).
PMID: 22190486 | DOI: 10.1073/pnas.1118857109.

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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