Journal of Cancer Prevention 2016; 21(4): 279-287
Published online December 30, 2016
© Korean Society of Cancer Prevention
Yoon Jin Choi1, Nayoung Kim1,2, Hye Seung Lee3, Seon Mee Park4, Ji Hyun Park2, Hyuk Yoon1, Cheol Min Shin1, Young Soo Park1, Jin-Wook Kim1, and Dong Ho Lee1
1Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea, 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea, 3Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea, 4Department of Internal Medicine, Chungbuk National University College of Medicine and Medical Research Institute, Cheongju, Korea
Correspondence to :
Nayoung Kim, Department of Internal Medicine, Seoul National University Bundang Hospital, 82 Gumi-ro 173 beon-gil, Bundang-gu, Seongnam 13620, Korea, Tel: +82-31-787-7008, Fax: +82-31-787-4051, E-mail: firstname.lastname@example.org, ORCID: Nayoung Kim, http://orcid.org/0000-0002-9397-0406
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The human leucine-rich repeat-containing G-protein coupled receptor (LGR) 5 and CD44 are one of the candidates for the marker of gastric cancer stem cells. We compared the expressions of two genes among control, dysplasia and cancer groups. We compared the mRNA expression of LGR5, CD44 and CD44v8–10 and immunohistochemistry (IHC) of LGR5 and CD44 in gastric antral mucosa of 45 controls, 36 patients with gastric dysplasia, and 39 patients with early gastric cancer. Additionally, IHC of LGR5 in gastric body mucosa was analyzed. Normal mucosa adjacent to dysplastic or cancer lesions was used for the quantitative real-time–PCR and IHC. Immunoreactivity of LGR5 in base of antral mucosa was higher in non-cancerous tissues of cancer than those of control ( IHC of LGR5 in crypt base and CD44 may be used for gastric CSC markers. LGR5 expression may be associated with the developing of corporal intestinal metaplasia. The expression of CD44v8–10 mRNA would be more suitable for gastric cancer stem cell marker than CD44 or LGR5 mRNA.
The human leucine-rich repeat-containing G-protein coupled receptor (LGR) 5 and CD44 are one of the candidates for the marker of gastric cancer stem cells. We compared the expressions of two genes among control, dysplasia and cancer groups.
We compared the mRNA expression of LGR5, CD44 and CD44v8–10 and immunohistochemistry (IHC) of LGR5 and CD44 in gastric antral mucosa of 45 controls, 36 patients with gastric dysplasia, and 39 patients with early gastric cancer. Additionally, IHC of LGR5 in gastric body mucosa was analyzed. Normal mucosa adjacent to dysplastic or cancer lesions was used for the quantitative real-time–PCR and IHC.
Immunoreactivity of LGR5 in base of antral mucosa was higher in non-cancerous tissues of cancer than those of control (
IHC of LGR5 in crypt base and CD44 may be used for gastric CSC markers. LGR5 expression may be associated with the developing of corporal intestinal metaplasia. The expression of CD44v8–10 mRNA would be more suitable for gastric cancer stem cell marker than CD44 or LGR5 mRNA.
Keywords: LGR5 protein, Gastric cancer, Stem cell
Gastric cancer remains the fourth most common cancer and the second leading cause of cancer-related mortality in the world. The treatment of gastric cancer remains a challenging problem because some patients with advanced gastric cancer who underwent a curative resection develop regional or distant recurrences. The overall 5-year survival rate for gastric cancer patients remains around 20% in the Western countries.1
The cancer stem cell (CSC) hypothesis that CSC may initiate carcinogenesis has emerged. Some data suggest that CSCs are present in many solid tumors and cancer cells are derived from a CSC compartment which undergoes an abnormal replicative process to form a non-stem cell component of the tumor.2 There has been growing evidence that
Unfortunately, CSCs of gastric cancer have not yet been defined and characterized. Various CSC markers for gastric cancer such as CD44, ALDH1, LGR5, and CD166 were reported.5 Although CD44 has been used most frequently as a marker for CSCs in gastric cancer, previous studies have suggested that CD44v8–10, variant form of CD44 was a specific maker for gastric CSCs, but not CD44s.6,7 Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is an adult stem cell marker expressed in the small intestine, colon, stomach, and hair follicles.8 Currently, in vivo lineage tracing revealed that LGR5-postive cells at the base of the pyloric glands were multipotent stem cells that contributed to daily epithelial renewal.9
The technique of sorting cells based on cell surface marker expression and assessing their ability to initiate tumor growth in immunocompromised mice was a common method to investigate any possible existence of solid-organ CSCs. However, the clinical implication of measuring the expression of CD44 and LGR5 in human tissue has not been evaluated. Based on the above findings, we examined the potential application of evaluation of CD44 and LGR5 expression on human gastric mucosa in the normal-dysplasia-carcinoma sequence.
Study subjects were consecutively enrolled at Seoul National University Bundang Hospital from February 2006 to December 2013. Subjects with a current
The study protocol was approved by the Ethics Committee at Seoul National University Bundang Hospital (IRB number B1301/186-111). Written informed consent was obtained from all subjects.
During upper GI endoscopy, specimens for
The first-line therapy for
Total RNA was extracted directly from non-cancerous corporal biopsy specimens with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), as recommended by the manufacturer. Next, 1,000 ng of RNA was reverse transcribed to cDNA with oligo (dT) and Moloney murine leukemia virus reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed in 96-well reaction plates using 2 μL of cDNA in a 20 μL reaction mix containing 2 × SYBR® Premix Ex TaqTM (Takara Bio Inc., Otsu, Japan). Samples were run on StepOnePlus real-time PCR instrument (Applied Biosystems, Foster City, CA, USA), The PCR cycling conditions were as follows: an initial denaturation step for 30 seconds at 95°C and then 40 cycles of denaturation at 95°C (5 seconds) and annealing at 60°C (34 seconds with the final dissociation stage of 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C. The primer sequences for PCR are shown in Table 1. Expression levels of mRNA of the target gene were compared with the endogenous control β-actin using the 2−ΔΔCt method.6
Immunohistochemistry (IHC) of CD44 and LGR5 was also performed. Basically, paraffin-embedded mucosal tissue distant from tumor sites was analyzed. For the evaluation of dysplasia and cancer, the normal mucosa of stomach part where the dysplasia or cancer was located (i.e., antrum or body) was used. Core tissue biopsies (2 mm in diameter) were obtained from paraffin-embedded gastric mucosa. Cores were arranged in recipient paraffin blocks (tissue array blocks) using a trephine. The test procedure used a human control slide for IHC analysis (Superbiochips Laboratories, Seoul, Korea). Antibodies against CD44 (H-CAM) (1:200 dilution; Leica Biosystems, Breckland, UK) and LGR5 (ab75732) (1:30 dilution; Abcam, Cambridge, UK) were used. Staining of sections (4 μm thick) from tissue array blocks was performed using the BenchMark XT staining system and the ultraVIEW universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA). Aberrant expression of CD44 is observed in the membrane and cytoplasm of cancer cells.12 LGR5 was strongly expressed in the membrane of non-neoplastic glands. Immunointensity of LGR5 was evaluated separately at surface mucosa and base of crypt. Scoring for the expression of CD44 and LGR5 was determined using light microscopy, by multiplying the intensity times the area (%) where staining was observed in epithelial glands; the possible scores ranged from 0 to 300.3 Area was defined as a ‘stained cells in a gland/total cells in a gland’. More than 5 glands were evaluated. The intensity of staining was scored as 0, no staining; 1+ faint/barely perceptible partial staining; 2+, weak to moderate staining; 3+, strong staining.13 Tests were repeated three times and mean values were described. Each sample was scored by a blind reviewer.
The χ2 test and Fisher’s exact test were used for the analysis of categorical variables The mRNA levels encoding CD44v8–10 and LGR5 markers were compared between groups using one-way ANOVA followed by Scheffe’s or Tamhane’s tests. For the analysis of changes in histologic grades or mRNA expression after the eradication of
A total of 237 patients in whom
The IHC scores of LGR5 were compared among control, dysplasia and cancer. While the IHC scores of surface of gastric mucosa did not show any differences (Fig. 1A), IHC scores of base of gastric mucosa from non-cancer tissues in cancer subjects were markedly upregulated than those in controls (27.20 ± 4.50 vs. 70.30 ± 9.70,
We then evaluated the effects of
As a conventional marker for the gastric CSC, IHC staining of CD44 was analyzed. The cancer group showed higher IHC score of CD44 compared with the control group (99.41 ± 16.10 vs. 14.30 ± 4.18,
When CD44s mRNA was evaluated, cancer or dysplasia groups showed lower expression of CD44s mRNA than control (0.48 ± 0.14 and 0.24 ± 0.04 vs. 1.55 ± 0.19, all
The CD44 IHC and mRNA expressions of CD44s and CD44v8–10 were compared before and after
IHC score of LGR5 in the body was significantly lower than in the antrum (29.45 ± 4.40 vs. 56.21± 4.21,
To investigate the relationship between LGR5 and CD44, the IHC scores were compared using Spearman’s rho test. The IHC score of LGR5 significantly correlated with the IHC score of CD44 (surface of mucosa: Spearman’s rho = 0.354,
Immunoreactivity of CD44 and LGR5 at mucosal base progressively increased from normal to dysplasia to cancer. Following
American Association of Cancer Research Workshop made a definition of CSC as “cells within a tumor that possess the capacity for self-renewal and that can cause the heterogeneous lineages of cancer cells that constitute the tumor.”14 Even though accumulative evidence supports the existence of CSCs that have the capacity to generate tumor, the stem cell hypothesis in carcinogenesis is under debate. A major obstacle for stem cell filed in solid tumor is that there is no specific tumor marker for CSC especially for stomach cancer.
Currently, in vivo lineage tracing revealed that a group of Lgr5-postive cells at the base of the pyloric glands were multipotent stem cells that contributed to daily epithelial renewal.9 LGR5 was proposed as an early diagnostic marker for gastric CSC, since upregulated expression of LGR5 was correlated with the gastric sequential cascades, “Correa pathway”.15 Based on this observations we hypothesized that LGR5 may have an impact on carcinogenesis in the human stomach. However, investigations of LGR5 expression in human tissues have been limited, and an appropriate histological method to identify LGR5-positive cells in human organs has not been established.16 We tested whether the sequential changes of gastric carcinogenesis, i.e., normal, IM and invasive carcinoma, are associated with an expression of the LGR5+ cells. Although Simon et al.17 reported the LGR5 cells had different spatial distribution according to the ‘precancerous cascade’, we could not discriminate the immunointensity of LGR5 in surface mucosa due to too diffuse and strong periplasmic expression. Therefore, since the usual storage of stem cells of gastric mucosa is crypt base, we measured the LGR5 immunointensity in the base of mucosa. In the present study, non-cancerous tissue of
CD44, most studied, is a cell surface transmembrane glycoprotein encoded by the CD44 gene, a 20-exon DNA segment,18 of which exons 1–5 and 16–20 are spliced together and translated into CD44s, the standard isoform. The variant exons 6–15 can be alternatively spliced and assembled in different combinations from the standard exons to generate other variant protein isoforms (CD44v). Although CD44 has been recognized as a CSC marker in several types of cancer,19 the ubiquitous expression of CD44 in many cell type,20 reduce the usefulness of CD44s as a CSC marker. Moreover, the existence of alternatively spliced variants may be the reason for the conflicting result of CD44, which was implicated in both tumor suppression and progression.21,22 In a genetic mice model for gastric carcinogenesis, the mouse homolog of human CD44v8–10 is expressed in precancerous regions of the stomach, from which tumor-initiating cells are thought to arise, suggesting that CD44v8–10 may be a marker for gastric tumor-initiating cells in the mouse.23
We showed a step-wise increase in CD44 immunostaining in the normal, dysplasia, and cancer sequence (
The present work has several limitations. Since we did not perform functional study for confirming stem cell properties by cell culture or animal models, it is not clear whether the elevated number of CD44 or LGR5-positive epithelial cells reflects true gastric CSCs. Although the downregulation of LGR5 and CD44 IHC was observed after
Nonetheless, this is one of the few studies that evaluated the clinical meaning of LGR5 and CD44 expression in tissue as a potential gastric CSC marker by compare its expression in normal, dysplasia, and cancer groups and before and after
In conclusion, LGR5 and CD44 IHC results could be used as probable gastric CSC markers to evaluate the development of gastric cancer as probable gastric CSCs in tissue. Regarding transcriptional level, CD44v8–10 may be more suitable for CD44 or LGR5 in gastric CSC researches. More studies are needed to identify the association between CD44 IHC and CD44v8–10 mRNA expression.
This work was supported by a National Research Foundation (NRF) of Korea grant for the Global Core Research Center (GCRC) funded by the Korea government (MSIP) (No. 2011-0030001).
Primer sequences for quantitative real-time-PCR.
|Gene name||Primer||Size (bp)|
|h_LGR5_foward||CTC CCA GGT CTG GTG TGT TG||149|
|h_LGR5_reverse||GAG GTC TAG GTA GGA GGT GAA G|
|h_CD44s_forward||TGC CGC TTT GCA GGT GTA T||66|
|h_CD44s_reverse||GGC CTC CGT CCG AGA GA|
|h_CD44v8–10_ forward||AGA ATC CCT GCT ACC AAT ATG GAC TC||334|
|h_CD44v8–10_ reverse||AGG TCA CTG GGA TGA AGG TC|
|h_GAPDH_forward||TGT GGG CAT CAA TGG ATT TGG||115|
|h_GAPDH_reverse||ACA CCA TGT ATT CCG GGT CAA T|
Clinicohistological characteristics of subjects whose antral mucosa were evaluated.
|characteristic||(n = 30)||(n = 21)||cancer (n = 25)|
|Men||12 (40.0)||11 (52.3)||16 (64.0)||0.357|
|Age (yr)||55.6 ± 9.9||60.9 ± 7.5||60.2 ± 10.6||0.104|
|Pepsinogen I/II ratio||2.70 ± 0.26||2.81 ± 0.30||2.59 ± 0.17||0.845|
|Atrophic gastritis||0.82 ± 0.16||1.50 ± 0.20||1.05 ± 0.22||0.077|
|Intestinal metaplasia||0.81 ± 0.15||1.35 ± 0.24||1.68 ± 0.17||0.002*|
Values are presented as number (%) or mean ± SE..
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