Nakajima T, Uehara T, Maruyama Y, Iwaya M, Kobayashi Y, Ota H.
PMID: 27593551 | DOI: 10.1111/pin.12451
Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a putative intestinal stem cell marker that is also expressed in various tumors. To analyze its pathological characteristics in mucosal gastric signet-ring cell carcinoma (SRCC), we investigated Lgr5 expression in 35 intramucosal gastric SRCC patients using RNAscope, a newly developed RNA in situ hybridization technique. Lgr5 expression in individual tumor cells was scored semi-quantitatively from 0 to 400. Ki67 was also examined by immunohistochemistry, with a linear arrangement of Ki67-expressing cells present in 20 of 35 cases. This area of Ki67-expressing cells was topographically divided into upper, middle, and lower regions. All cases with linear Ki67 expression patterns also had Lgr5-positive cells arranged in a linear fashion in the lower area-which was distinct from the area of high Ki67 expression. The rate of Ki67 positivity in Lgr5-positive cells was significantly lower than that of Lgr5-negative cells in areas of high Ki67 expression. In intramucosal SRCC, the low mitotic activity of Lgr5-positive cells suggests that they may represent cancer stem cells as seen in other types of stomach carcinomas. Intramucosal SRCC may therefore contain stem cells expressing Lgr5 in the lower area of the lamina propria, akin to normal gastric pyloric mucosa.
Ren, W;Ma, Z;Wang, L;Feng, X;Yu, H;Yu, Y;
PMID: 35966594 | DOI: 10.7150/thno.60636
Olfactory sensory neurons (OSNs) located in the olfactory epithelium (OE) detect thousands of volatile environmental odors to form the sense of smell. OSNs are generated from basal cells, which show the characteristics of progenitor/stem cells. In the mammalian OE, persistent neurogenesis occurs during lifetime, providing a unique model to study the tissue turnover and fate determination of stem cells. Methods: Immunohistochemical analysis and RNAscope in situ hybridization indicated the localization of leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) in the intact and injured OE. Lineage tracing was conducted to analyze the dynamic role of Lgr5+ cells in the OE homeostasis and regeneration. We also used DTR-driven genetic depletion of Lgr5+ cells and lentivirus-mediated Lgr5 downregulation to demonstrate the essential role of Lgr5+ cells in the OE regeneration. Results: We show that Lgr5 marks horizontal basal cells (HBCs) in the OE of adults but not newborns. We revisit the role of Lgr5+ cells in the OE homeostasis and regeneration, and find that Lgr5+ cells participate in the OE homeostasis from neonatal to one-month-old age, as well as in the OE regeneration post injury. During the OE regeneration, Lgr5 is transiently expressed in apical supporting cells, immature neurons, and mature sensory neurons. The Lgr5+ cells become or generate HBCs in the regenerated OE. DTR-driven cell depletion shows that Lgr5+ cells are not necessary in the adult OE homeostasis, but required in the recovery of OE from injury. Lgr5 down-regulation by lentiviral infection also demonstrates the essential role of Lgr5 expression in the OE regeneration. Conclusion: Our study elucidates the role of Lgr5+ cells in the OE homeostasis and regeneration, potentially providing a candidate to cell-based therapy against olfactory dysfunction.
Proceedings of the National Academy of Sciences of the United States of America
Xue, Y;San Luis, B;Dress, RJ;Murad, KBA;Ginhoux, F;Barker, N;Lane, D;
PMID: 36940336 | DOI: 10.1073/pnas.2219978120
We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.
Walters, BW;Tan, TJ;Tan, CT;Dube, CT;Lee, KT;Koh, J;Ong, YHB;Tan, VXH;Jahan, FRS;Lim, XN;Wan, Y;Lim, CY;
PMID: 37259855 | DOI: 10.1242/jcs.260723
The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.
Jang BG, Lee C, Kim HS, Shin MS, Cheon MS, Kim JW, Kim WH.
PMID: 28070642 | DOI: 10.1007/s00428-016-2061-3
Mammalian epidermis, which is composed of hair follicles, sebaceous glands, and interfollicular epidermis, is maintained by discrete stem cells. In vivo lineage tracing demonstrated that murine LGR5 cells are mainly responsible for hair follicle regeneration whereas LGR6 cells generate sebaceous glands and interfollicular epidermis. However, little is known about their expression in the human skin tumors. In this study, we investigated the expression profile of LGR5 and LGR6 in a variety of human skin tumors including basaloid tumors with follicular differentiation (94 basal cell carcinomas, 18 trichoepitheliomas, 3 basaloid follicular hamartomas, and 12 pilomatricomas) and tumors with ductal differentiation (7 eccrine poromas, 8 hidradenomas, and 5 spiradenomas). LGR5 expression was highest in basal cell carcinomas (BCCs) followed by trichoepitheliomas (TEs) and basaloid follicular hamartomas. LGR6 had the same expression pattern as LGR5, even though its expression was lower. Interestingly, LGR6 expression was detected in stromal cells around the tumor and papillary mesenchymal bodies of TEs but not in stromal cells of BCCs, suggesting different characteristics of tumor-associated fibroblasts between TEs and BCCs. It was unexpected to find that pilomatricomas exclusively expressed LGR6, and its expression was limited to the basaloid cells. Notably, LGR6-positive cells were observed in sweat gland ductal cells in normal skin. This might explain, in part, the finding that LGR6 expression was relatively higher in basaloid tumors with ductal differentiation than in those with follicular differentiation. In particular, spiradenomas displayed the same distribution pattern of LGR6 as normal sweat glands, suggesting the possibility of LGR6-positive cells as tumor stem cells. In conclusion, we documented the different expression patterns of stem cell markers, LGR5 and LGR6 in various skin tumors. These data may provide important insights to understand the origin and development of basaloid skin tumors.
Teng CS, Ting MC, Farmer DT, Brockop M, Maxson RE, Crump JG.
PMID: 30375332 | DOI: 10.7554/eLife.37024
Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.
Nakajima T, Uehara T, Iwaya M, Kobayashi Y, Maruyama Y, Ota H
PMID: 32293346 | DOI: 10.1186/s12885-020-06791-8
BACKGROUND:
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a promising intestinal stem cell and carcinoma stem cell marker. We examined the relationship between mismatch repair (MMR) protein deficiency and LGR5 expression in poorly differentiated (PD) colorectal carcinoma (CRC).
METHODS:
In 29 cases of PD-CRC, deficiencies in MMR proteins (MLH1, PMS2, MSH2, MSH6) and ?-catenin expression were identified by immunohistochemistry (IHC). LGR5 expression was examined by the RNAscope assay in tissue microarrays.
RESULTS:
LGR5 H-scores in MMR-deficient (MMR-D) cases were significantly lower than those in MMR-proficient (MMR-P) cases (P?=?0.0033). Nuclear ?-catenin IHC scores in MMR-D cases were significantly lower than those in MMR-P cases (P?=?0.0024). In all cases, there was a positive correlation between LGR5 H-score and nuclear ?-catenin IHC score (r?=?0.6796, P?
Kim, H;Lee, DH;Park, E;Myung, JK;Park, JH;Kim, DI;Kim, SI;Lee, M;Kim, Y;Park, CM;Hyun, CL;Maeng, YH;Lee, C;Jang, B;
PMID: 35778589 | DOI: 10.1038/s41598-022-15234-2
Lgr5 has been identified as a marker of the stem/progenitor cells in the murine ovary and oviduct by lineage tracing. However, little is known regarding LGR5 expression or its functional significance in human ovary tissues. Here, using RNA in situ hybridization and/or immunohistochemistry, we thoroughly investigated LGR5 expression in normal human ovaries, fallopian tubes and various ovarian tumors. We discovered that LGR5 expression is negligible in the human ovary surface epithelium, whereas ovarian stromal cells normally express low levels of LGR5. Remarkably, fallopian tube epithelium, inclusion cysts and serous cystadenomas with a Müllerian phenotype expressed high levels of LGR5, and LGR5 expression was restricted to PAX8+/FOXJ1- secretory cells of the tubal epithelium. Strong stromal LGR5 expression without epithelial LGR5 expression was consistently observed in the path from serous cystadenoma to serous borderline tumor to low grade serous carcinoma (LGSC). Unlike LGSC, high grade serous carcinoma (HGSC), clear cell carcinoma, endometrioid carcinomas displayed various epithelial-stromal LGR5 expression. Notably, high levels of LGR5 expression were observed in serous tubal intraepithelial carcinoma, which slightly declined in invasive HGSC. LGR5 expression was significantly associated with improved progression-free survival in HGSC patients. Moreover, in vitro assays demonstrated that LGR5 expression suppressed tumor proliferation and migratory capabilities. Taken together, these findings indicate a tumor-suppressive role for LGR5 in the progression of HGSC.
Virchows Arch. 2015 Jun 13.
Olfactomedin 4 (OLFM4) has been demonstrated to be upregulated in various cancers and involved in many cellular processes such as cell adhesion, apoptosis, and cell proliferation. In gastric cancer, clinicopathological relevance of OLFM4 expression has been reported. However, there are few studies showing how expression of OLFM4 evolves during multistep gastric carcinogenesis. In this study, we investigated OLFM4 expression during gastric carcinogenesis using RNA in situ hybridization (ISH). We found that OLFM4 expression is absent in normal gastric mucosa, begins to appear at the isthmus region in gastric glands in chronic gastritis, and is remarkably increased in intestinal metaplasia (IM). Interestingly, gastric-type glands around IM frequently expressed OLFM4 before CDX2 was expressed, suggesting that OLFM4 might be involved in regulating CDX2 expression. However, overexpression of OLFM4 failed to induce CDX2 transcription. All gastric adenomas were strongly positive for OLFM4. OLFM4 expression was higher in intestinal type, well to moderately differentiated and early-stage adenocarcinomas, and decreased in poorly differentiated and advanced-stage gastric cancer (GC). Although OLFM4 expression had no prognostic value for GC overall (P = 0.441), it was associated with poor survival of GC in stage II, III, and IV (P = 0.018), suggesting that OLFM4 expression has prognostic significance for late-stage GC. Our findings suggest that OLFM4 is not only involved in early stages of gastric carcinogenesis but also a useful prognostic marker for advanced GC, which is encouraging for further studies exploring OLFM4 as a potential target for therapy of GC.
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.
Jang BG, Lee BL, Kim WH. (2013).
PMID: 24340024 | DOI: 10.1371/journal.pone.0082390.
Lgr5 was identified as a promising gastrointestinal tract stem cell marker in mice. Lineage tracing indicates that Lgr5(+) cells may not only be the cells responsible for the origin of tumors; they may also be the so-called cancer stem cells. In the present study, we investigated the presence of Lgr5(+) cells and their biological significance in normal human gastric mucosa and gastric tumors. RNAscope, a newly developed RNA in situ hybridization technique, specifically labeled Lgr5(+) cells at the basal glands of the gastric antrum. Notably, the number of Lgr5(+) cells was remarkably increased in intestinal metaplasia. In total, 76% of gastric adenomas and 43% of early gastric carcinomas were positive for LGR5. Lgr5(+) cells were found more frequently in low-grade tumors with active Wnt signaling and an intestinal gland type, suggesting that LGR5 is likely involved in the very early stages of Wnt-driven tumorigenesis in the stomach. Interestingly, similar to stem cells in normal tissues, Lgr5(+) cells were often restricted to the base of the tumor glands, and such Lgr5(+) restriction was associated with high levels of intestinal stem cell markers such as EPHB2, OLFM4, and ASCL2. Thus, our findings show that Lgr5(+) cells are present at the base of the antral glands in the human stomach and that this cell population significantly expands in intestinal metaplasias. Furthermore, Lgr5(+) cells are seen in a large number of gastric tumors ; their frequent basal arrangements and coexpression of ISC markers support the idea that Lgr5(+) cells act as stem cells during the early stage of intestinal-type gastric tumorigenesis.
Sci Rep. 2015 Mar 2;5:8654.
Baker AM, Graham TA, Elia G, Wright NA, Rodriguez-Justo M.
PMID: 25728748 | DOI: 10.1038/srep08654
LGR5 is known to be a stem cell marker in the murine small intestine and colon, however the localization of LGR5 in human adenoma samples has not been examined in detail, and previous studies have been limited by the lack of specific antibodies. Here we used in situ hybridization to specifically examine LGR5 mRNA expression in a panel of human adenoma and carcinoma samples (n = 66). We found that a small number of cells express LGR5 at the base of normal colonic crypts. We then showed that conventional adenomas widely express high levels of LGR5, and there is no evidence of stereotypic cellular hierarchy. In contrast, serrated lesions display basal localization of LGR5, and the cellular hierarchy resembles that of a normal crypt. Moreover, ectopic crypts found in traditional serrated adenomas show basal LGR5 mRNA, indicating that they replicate the stem cell organization of normal crypts with the development of a cellular hierarchy. These data imply differences in the stem cell dynamics between the serrated and conventional pathways of colorectal carcinogenesis. Furthermore we noted high LGR5 expression in invading cells, with later development of a stem cell niche in adenocarcinomas of all stages.
