Pathology - Research and Practice
Yoshizawa, T;Uehara, T;Iwaya, M;Asaka, S;Kobayashi, S;Nakajima, T;Kinugawa, Y;Nagaya, T;Kamakura, M;Shimizu, A;Kubota, K;Notake, T;Masuo, H;Hosoda, K;Sakai, H;Hayashi, H;Umemura, K;Kamachi, A;Goto, T;Tomida, H;Yamazaki, S;Ota, H;Soejima, Y;
| DOI: 10.1016/j.prp.2022.153832
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a known cancer stem cell marker. However, there are no reported analyses of LGR5 mRNA expression in normal liver and liver cancer tissues. Here, we evaluated LGR5 expression by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 25 samples of intrahepatic cholangiocarcinoma (ICC) selected from the medical archives at our hospital. LGR5 expression levels were divided into high and low expression groups by the five-grade scoring system, and clinicopathological features were analyzed. Low LGR5 expression was identified in some normal hepatocytes and bile duct cells. In addition, LGR5 expression was identified in all bile duct cancer samples except one case. Well-differentiated to moderately-differentiated adenocarcinoma tended to show higher LGR5 expression than poorly-differentiated adenocarcinoma (P=0.0561), and the large duct type showed significantly higher LGR5 expression levels than the small duct type (P=0.0225). Patients in the high LGR5 expression group tended to have good overall survival (OS) (P=0.0623). The Cox proportional hazard regression model revealed that the high LGR5 expression group showed independently better OS for ICC (P = 0.0285). High LGR5 expression is possibly a good prognosis factor in ICC. However, the detailed mechanism of LGR5 in this disease remains unclear, and further analysis is warranted.
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.
Norum HJ, Bergström Å, Andersson BA, Kuiper RV, Hoelzl MA, Sørlie T, Toftgård R.
PMID: 25990088 | DOI: canprevres.0090.2015.
LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.
Jang BG, Kim HS, Chang WY, Bae JM, Kim WH, Kang GH.
PMID: 30036518 | DOI: 10.1016/j.ajpath.2018.06.012
We investigated the expression profile of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) during colorectal cancer (CRC) progression and determined the prognostic impact of LGR5 in a large cohort of CRC samples. LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 68% of 788 CRCs and was positively correlated with old age, well-to-moderate differentiation, and nuclear β-catenin expression. Enhanced LGR5 expression remained persistent during the adenoma-carcinoma transition, but markedly declined in the budding cancer cells at the invasive fronts, which was not due to altered Wnt or epithelial to mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS and BRAF mutations. Notably, LGR5 positivity was an independent prognostic marker for better clinical outcomes in CRC patients. LGR5 overexpression attenuated tumor growth by decreasing ERK phosphorylation along with decreased colony formation and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony- forming and migration capacities in LoVo cells. Taken together, our data suggest the suppressive role of LGR5 in CRC progression.
Qin, D;Liu, S;Lu, Y;Yan, Y;Zhang, J;Cao, S;Chen, M;Chen, N;Huang, W;Wang, L;Chen, X;Zhang, L;
PMID: 36168631 | DOI: 10.7150/thno.74194
Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a target gene of Wnt/β-Catenin which plays a vital role in hepatic development and regeneration. However, the regulation of Lgr5 gene and the fate of Lgr5 + cells in hepatic physiology and pathology are little known. This study aims to clarify the effect of metabolic nuclear receptors on Lgr5 + cell fate in liver. Methods: We performed cell experiments with primary hepatocytes, Hep 1-6, Hep G2, and Huh 7 cells, and animal studies with wild-type (WT), farnesoid X receptor (FXR) knockout mice, peroxisome proliferator-activated receptor α (PPARα) knockout mice and Lgr5-CreERT2; Rosa26-mTmG mice. GW4064 and CDCA were used to activate FXR. And GW7647 or Wy14643 was used for PPARα activation. Regulation of Lgr5 by FXR and PPARα was determined by QRT-PCR, western blot (WB) and RNAscope in situ hybridization (ISH) and immunofluorescence (IF), luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) diet was used to induce liver injury. Results: Pharmacologic activation of FXR induced Lgr5 expression, whereas activation of PPARα suppressed Lgr5 expression. Furthermore, FXR and PPARα competed for binding to shared site on Lgr5 promoter with opposite transcriptional outputs. DDC diet triggered the transition of Lgr5 + cells from resting state to proliferation. FXR activation enhanced Lgr5 + cell expansion mainly by symmetric cell division, but PPARα activation prevented Lgr5 + cell proliferation along with asymmetric cell division. Conclusion: Our findings unravel the opposite regulatory effects of FXR and PPARα on Lgr5 + cell fate in liver under physiological and pathological conditions, which will greatly assist novel therapeutic development targeting nuclear receptors.
Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers
Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y
Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.
Shimokawa M, Ohta Y, Nishikori S, Matano M, Takano A, Fujii M, Date S, Sugimoto S, Kanai T, Sato T.
PMID: 28355176 | DOI: 10.1038/nature22081
The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.
Nakajima T, Uehara T, Kobayashi Y, Kinugawa Y, Yamanoi K, Maruyama Y, Suga T, Ota H.
PMID: 30043418 | DOI: 10.1111/pin.12707
LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.
LGR5-Expressing Cells in the Healing Process of Post-ESD Ulcers in Gastric Corpus
Digestive diseases and sciences
Tobe, Y;Uehara, T;Nakajima, T;Iwaya, M;Kobayashi, Y;Kinugawa, Y;Kuraishi, Y;Ota, H;
PMID: 34081250 | DOI: 10.1007/s10620-021-07059-2
LGR5 is a promising stem cell marker in gastric pylorus, but there are few reports on its expression in human gastric corpus.To investigate the involvement of LGR5 expression in gastric corpus ulcer regeneration in humans.LGR5 expression was analyzed in five post-ESD ulcers during the healing process of regenerating epithelial cells of the gastric corpus. LGR5 expression was detected by mRNA in situ hybridization using an RNA scope kit. Immunohistochemistry of MUC6, HIK1083, and pepsinogen 1 (PG1) was performed to identify cell differentiation.We defined MUC6+/HIK1083-/PG1-, MUC6+/HIK1083+/PG1-, MUC6+/HIK1083+/PG1+, MUC6+/HIK1083-/PG1+, and MUC6-/HIK1083-/PG1+cells as pseudopyloric mucosa (PPM) phase 1 (PPM1), PPM phase 2 (PPM2), PPM phase 3 (PPM3), immature chief cells (ICC), and mature chief cells (MCC) in order from the ulcer center, respectively. In the regenerated mucosa around post-ESD ulcers, LGR5 expression was observed throughout the gland in PPM1-PPM3, but it was limited to the bottom of the gland in ICC and MCC. Furthermore, LGR5 expression was not identified in the normal gastric corpus. The H-score of PPM2 was significantly higher than that of PPM3 (P = 0.0313). The H-score of PPM3 was significantly higher than that of ICC (P = 0.0313). The LGR5 H-score was higher at the immature stage, which decreased gradually with progression of the differentiation stage.LGR5 expression appears to contribute to mucosal regeneration in the human gastric corpus. The application of LGR5 expression analysis to mucosal regeneration and fundic gland-type gastric tumors is expected.
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.
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?
Kamakura, M;Uehara, T;Iwaya, M;Asaka, S;Kobayashi, S;Nakajima, T;Kinugawa, Y;Nagaya, T;Yoshizawa, T;Shimizu, A;Ota, H;Umemura, T;
PMID: 35123536 | DOI: 10.1186/s13000-022-01203-w
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a strong cancer stem cell marker in colorectal cancer; however, there are many unclear aspects of LGR5 expression in pancreatic cancer. It has been reported that the interaction between tumor cells and stroma at the fat infiltration site has a significant effect on pancreatic cancer prognosis. Therefore, we report a clinicopathological study of LGR5 expression at the fat invasion front in pancreatic cancer.LGR5 expression was analyzed in 40 pancreatic ductal adenocarcinoma cases with RNAscope, which is a newly developed high-sensitivity in situ hybridization method. Epithelial-mesenchymal transition (EMT) was analyzed by the expression of E-cadherin and vimentin via immunohistochemistry.LGR5-positive dots were identified in all cases, especially with glandular formation. In the fat invasion front, a high histological grade showed significantly reduced LGR5 expression compared with a low histological grade (p=0.0126). LGR5 expression was significantly higher in the non-EMT phenotype group than in EMT phenotype group (p=0.0003). Additionally, LGR5 expression was significantly lower in cases with high vascular invasion than in those with low vascular invasion (p=0.0244).These findings suggest that decreased LGR5 expression in the fat invasion front is associated with more aggressive biological behavior in pancreatic ductal adenocarcinoma, with higher tumor grade, EMT phenotype, and higher vascular invasion.
Pages
