Expression Profile of LGR5 and Its Prognostic Significance in Colorectal Cancer Progression
2018; Elsevier BV; Volume: 188; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2018.06.012
ISSN1525-2191
AutoresBo Gun Jang, Hye Sung Kim, Weon Young Chang, Jeong Mo Bae, Woo Ho Kim, Gyeong Hoon Kang,
Tópico(s)Metastasis and carcinoma case studies
ResumoWe 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 older age, moderately to well-differentiated cells, 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 wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial–mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. 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 a suppressive role of LGR5 in CRC progression. 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 older age, moderately to well-differentiated cells, 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 wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial–mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. 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 a suppressive role of LGR5 in CRC progression. Leucine-rich, repeat-containing, G-protein–coupled receptor 5 (LGR5), a wingless-type mouse mammary tumor virus integration site family (Wnt) target gene that functions as a receptor for Wnt agonist R-spondins (RSPOs), is an adult stem cell marker in the intestines, hair follicles, liver, and ovary, as revealed by in vivo lineage tracing.1Barker N. Van Es J.H. Kuipers J. Kujala P. Van Den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3943) Google Scholar, 2Glinka A. Dolde C. Kirsch N. Huang Y.L. Kazanskaya O. Ingelfinger D. Boutros M. Cruciat C.M. Niehrs C. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling.EMBO Rep. 2011; 12: 1055-1061Crossref PubMed Scopus (412) Google Scholar, 3de Lau W. Barker N. Low T.Y. Koo B.-K. Li V.S. Teunissen H. Kujala P. Haegebarth A. Peters P.J. van de Wetering M. 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Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells.Stem Cells. 2012; 30: 2378-2386Crossref PubMed Scopus (198) Google Scholar More recently, it was demonstrated that selective LGR5+ cell ablation resulted in the inhibition of primary tumor growth or regression of tumors,18Shimokawa M. Ohta Y. Nishikori S. Matano M. Takano A. Fujii M. Date S. Sugimoto S. Kanai T. Sato T. Visualization and targeting of LGR5+ human colon cancer stem cells.Nature. 2017; 545: 187-192Crossref PubMed Scopus (415) Google Scholar, 19de Sousa e Melo F. Kurtova A.V. Harnoss J.M. Kljavin N. Hoeck J.D. Hung J. Anderson J.E. Storm E.E. Modrusan Z. Koeppen H. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer.Nature. 2017; 543: 676-680Crossref PubMed Scopus (442) Google Scholar and that LGR5+ cells were important in the formation and maintenance of liver metastasis of CRCs.19de Sousa e Melo F. Kurtova A.V. Harnoss J.M. Kljavin N. Hoeck J.D. Hung J. Anderson J.E. 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Significant correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy.J Cancer Res Clin Oncol. 2013; 139: 131-138Crossref PubMed Scopus (29) Google Scholar and some meta-analyses have concluded that LGR5 is an undesirable prognostic factor in CRC patients.25Chen Q. Zhang X. Li W.-M. Ji Y.-Q. Cao H.-Z. Zheng P. Prognostic value of LGR5 in colorectal cancer: a meta-analysis.PLoS One. 2014; 9: e107013Crossref PubMed Scopus (25) Google Scholar, 26Han Y. Xue X. Jiang M. Guo X. Li P. Liu F. Yuan B. Shen Y. Guo X. Zhi Q. LGR5, a relevant marker of cancer stem cells, indicates a poor prognosis in colorectal cancer patients: a meta-analysis.Clin Res Hepatol Gastroenterol. 2015; 39: 267-273Crossref PubMed Scopus (32) Google Scholar, 27Jiang Y. Li W. He X. Zhang H. Jiang F. Chen Z. Lgr5 expression is a valuable prognostic factor for colorectal cancer: evidence from a meta-analysis.BMC Cancer. 2016; 16: 12Crossref PubMed Scopus (25) Google Scholar However, these studies used immunohistochemistry analysis to evaluate the expression of LGR5 and it is well known that there is no antibody that specifically marks LGR5+ cells at the intestinal stem cell niche, thus casting great doubt on the reliability of these results. In contrast, RNA in situ hybridization (ISH) clearly demonstrated LGR5-expressing cells at the crypt bases and has been recognized as the most reliable method for detecting LGR5 on formalin-fixed, paraffin-embedded (FFPE) tissues obtained from CRC patients.11Uchida H. Yamazaki K. Fukuma M. Yamada T. Hayashida T. Hasegawa H. Kitajima M. Kitagawa Y. Sakamoto M. Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in colorectal cancer.Cancer Sci. 2010; 101: 1731-1737Crossref PubMed Scopus (127) Google Scholar, 13Baker A.-M. Graham T.A. Elia G. Wright N.A. Rodriguez-Justo M. Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.Sci Rep. 2015; 5: 8654Crossref PubMed Scopus (77) Google Scholar, 28Jang B.G. Kim H.S. Kim K.J. Rhee Y.Y. Kim W.H. Kang G.H. Distribution of intestinal stem cell markers in colorectal precancerous lesions.Histopathology. 2016; 68: 567-577Crossref PubMed Scopus (23) Google Scholar Ziskin et al29Ziskin J.L. Dunlap D. Yaylaoglu M. Fodor I.K. Forrest W.F. Patel R. Ge N. Hutchins G.G. Pine J.K. Quirke P. In situ validation of an intestinal stem cell signature in colorectal cancer.Gut. 2012; 62: 1012-1023Crossref PubMed Scopus (84) Google Scholar used ISH to explore the prognostic value of LGR5 in a large-scale series of CRCs and showed that LGR5 was not associated with poor clinical outcomes. Therefore, the prognostic significance of LGR5 remains controversial, and studies with a large number of CRC samples using an RNA ISH are necessary. In this study, we examined the expression of LGR5 in a large cohort of human CRC samples using RNA ISH and assessed its prognostic significance. Furthermore, we analyzed changes in the expression of LGR5 during CRC progression: adenoma–carcinoma transition, tumor budding, and lymph node metastasis. The functional implications of LGR5 in cancer growth and migration were also explored in CRC cell lines. CRC samples were collected from 1133 patients who underwent surgical resection at Seoul National University Hospital (Seoul, Republic of Korea) between 2004 and 2008. In total, 785 patients were treated with one of the following systemic chemotherapy regimens: FL (5-fluorouracil and leucovorin), FOLFOX (oxaliplatin, leucovorin, and 5-fluorouracil), XELOX (oxaliplatin and capecitabine), FOLFIRI (irinotecan, leucovorin, and 5-fluorouracil), capecitabine (Xeloda), and others. Among them, 109 patients also received radiation therapy. Clinicopathologic data, including patient age, sex, tumor size, location, histologic type, evidence of lymphovascular invasion, cancer stage as defined by the American Joint Committee on Cancer/Union Internationale Contre le Cancer, seventh edition,30Sobin L.H. Compton C.C. TNM seventh edition: what's new, what's changed: communication from the International Union Against Cancer and the American Joint Committee on Cancer.Cancer. 2010; 116: 5336-5339Crossref PubMed Scopus (411) Google Scholar time of death, tumor recurrence, and follow-up time were obtained by reviewing the clinical and pathologic reports. Tumor differentiation was determined using a three-tier grading system as described in the World Health Organization's Classification of Tumours of the Digestive System.31Bosman F.T. Carneiro F. Hruban R.H. Theise N.D. WHO classification of tumours of the digestive system. World Health Organization, Geneva, Switzerland2010Google Scholar Tumor budding was defined as a single tumor cell or a group of less than five tumor cells at the invasive fronts. A total of 206 CRC samples were obtained from the patients at Jeju National University Hospital (Jeju, Republic of Korea); 24 cases were CRCs arising from adenomas, and 182 cases were conventional CRCs, the clinicopathologic characteristics of which are shown in Supplemental Tables S1 and S2, respectively. The histopathologic features of the CRCs were evaluated by two gastrointestinal pathologists (J.M.B. and G.H.K.). In addition, 32 paired, fresh-frozen CRC tissue samples and matched normal tissue samples were provided by the Jeju National University Hospital Biobank, a member of the National Biobank of Korea, for which informed consent was obtained from all subjects. All procedures were performed in accordance with the Declaration of Helsinki of 1964 and later versions, and the study protocol was approved by the institutional review boards of Seoul National University Hospital and Jeju National University Hospital. FFPE tissue samples of 1133 primary CRCs were retrieved from pathology archives. Genomic DNA isolation from all FFPE tissues was performed as follows: tumor spots (tumor cells >70% of the selected area) were microdissected using a surgical blade from 10 μm–thick, unstained tissues. Dissected tumor tissues were incubated for digestion in lysis buffer [100 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA (pH 8.0), 1 mg/mL proteinase K, and 0.05 mg/mL tRNA] at 55°C for 2 days, followed by a 95°C incubation for 10 minutes to inactivate proteinase K. The extracted DNA was stored at −20°C until used for molecular studies including analyses for KRAS/BRAF mutations, microsatellite instability (MSI), and CpG island methylation phenotype (CIMP). All of 1133 CRCs were subjected to MSI analysis using fluorescent multiplex PCR methods with five National Cancer Institute–recommended microsatellite markers (BAT25, BAT26, D5S346, D17S250, and D2S123).32Boland C.R. Thibodeau S.N. Hamilton S.R. Sidransky D. Eshleman J.R. Burt R.W. Meltzer S.J. Rodriguez-Bigas M.A. Fodde R. Ranzani G.N. Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar The MSI status of each CRC case was classified as one of three types: MSI-high indicates two or more unstable markers among the five markers; MSI-low indicates one unstable marker among the five markers; and MSI-stable indicates no unstable marker among the five markers. DNA analysis for the determination of CIMP status was performed as previously described.33Kim J.H. Kim K.-J. Rhee Y.-Y. Oh S. Cho N.-Y. Lee H.S. Kang G.H. Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer.Mod Pathol. 2014; 27: 443-453Crossref PubMed Scopus (26) Google Scholar Sodium bisulfite modification of genomic DNA samples was conducted in all 1133 CRC tissues. The quantitative measurement of the promoter CpG island methylation of eight CIMP marker genes [MLH1, NEUROG1, CRABP1, CACNA1G, CDKN2A (p16), IGF2, SOCS1, and RUNX3] was conducted using the methylation-specific real-time PCR method (MethyLight assay; Qiagen, Valencia, CA). A CIMP-high tumor was defined as having five or more hypermethylated markers; a CIMP-low tumor, as having one to four hypermethylated markers; and a CIMP-negative tumor, as having no hypermethylated markers. A hypermethylated CpG island locus was identified as having a percentage of methylated reference value of >4. The MethyLight analysis of each CIMP marker gene was repeated three times independently, and when two or more of the three experiments showed a percentage of the methylated reference value of >4, the final determination of the promoter hypermethylation was made. KRAS/BRAF mutation analysis was performed as previously described.33Kim J.H. Kim K.-J. Rhee Y.-Y. Oh S. Cho N.-Y. Lee H.S. Kang G.H. Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer.Mod Pathol. 2014; 27: 443-453Crossref PubMed Scopus (26) Google Scholar Mutations in KRAS codons 12 and 13 and BRAF codon 600 were detected using PCR–restriction fragment length polymorphism and direct-sequencing techniques. Among the 788 CRCs, 39 and 81 samples were excluded from the KRAS and BRAF mutation analyses, respectively, due to insufficient DNA amounts. Twenty tissue microarrays containing 1133 CRCs from Seoul National University Hospital were generated as previously described.33Kim J.H. Kim K.-J. Rhee Y.-Y. Oh S. Cho N.-Y. Lee H.S. Kang G.H. Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer.Mod Pathol. 2014; 27: 443-453Crossref PubMed Scopus (26) Google Scholar Briefly, through a histologic examination, representative tumor portions including >70% of the cell population were marked in each case. In CRCs from Seoul National University Hospital, core-tissue biopsy samples (2 mm in diameter) were obtained from each FFPE CRC (donor blocks) and arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus (SuperBioChips Laboratories, Seoul, Republic of Korea). In CRCs from Jeju National University Hospital, two tissue microarrays containing 24 pairs of adenoma and carcinoma portions and 15 tissue microarrays containing 182 cases of CRCs were constructed with cores 4 mm in diameter. For ulcerofungating cancers, both superficial and invasive areas were included, and, if present, metastatic cancers of the lymph node were also included. Immunohistochemistry analysis was performed on 4-μm tissue microarray sections using a Ventana BenchMark XT Staining system (Leica Microsystems, Wetzlar, Germany) according to the manufacturer's instructions. The primary antibodies used were anti–β-catenin (catalog number 17C2; Novocastra Laboratories, Newcastle, UK; 1:800), E-cadherin (catalog number 35; BD Biosciences, San Jose, CA, 1:800), and vimentin (catalog number V9; Thermo Fisher Scientific, Waltham, MA; 1:1500). Nuclear β-catenin staining was considered as positive when >10% of the tumor cell nuclei were strongly stained for β-catenin. RNA ISH was performed using the RNAScope FFPE assay kit (Advanced Cell Diagnostics, Hayward, CA) as previously described.28Jang B.G. Kim H.S. Kim K.J. Rhee Y.Y. Kim W.H. Kang G.H. Distribution of intestinal stem cell markers in colorectal precancerous lesions.Histopathology. 2016; 68: 567-577Crossref PubMed Scopus (23) Google Scholar Briefly, 4-μm FFPE tissue sections were pretreated with heat and protease digestion followed by hybridization with the probe. Then, a horseradish peroxidase–based signal-amplification system was hybridized to a probe before color development with 3,3′-diaminobenzeidine tetrahydrochloride. The housekeeping gene ubiquitin C and the bacterial gene DapB served as a positive and negative control, respectively. CRCs with ubiquitin C easily visible under a ×10 objective lens were considered to be adequate according to the manufacturer's recommendation. Finally, 788 CRCs were adequate for the analysis. Positive staining was indicated by brown punctate dots in the nucleus and/or cytoplasm. LGR5, SNAIL, SLUG, and ZEB1 expression levels were quantified according to the manufacturer's scoring guideline: 0 indicates no staining or less than one dot per cell; 1 indicates 1 to 3 dots per cell (visible at original magnification ×20 to ×40); 2 indicates 4 to 10 dots per cell and no or very few dot clusters (visible at ×20 to ×40); 3 indicates >10 dots per cell and 10 dots per cell and >10% positive cells with dot clusters (visible at ×20). Scoring was done by two pathologists (B.G.J. and G.H.K.) independently. In the case of conflicting results, the case was reviewed and discussed until a consensus was reached. LGR5 status was considered as positive if the ISH score was >2. Total RNA was extracted from 32 paired fresh-frozen CRC samples and noncancerous colon tissue samples using TRIzol reagent (InvitroGen, Carlsbad, CA). RNA (1 to 2 μg) was subjected to reverse transcription with oligo-dT primers and the GoScript reverse-transcription system (Promega, Madison, WI). cDNA was subsequently used to perform real-time PCR with Premix EX Taq (Takara Bio, Shiga, Japan) following the manufacturer's instructions, and the cycling conditions were as follows: initial denaturation for 30 seconds at 95°C, followed by 40 cycles of 95°C for 1 second and 60°C for 5 seconds in a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA). The TaqMan gene expression assays were used as follows: Hs00173664_m1 (LGR5), Hs00270888_s1 (ASCL2), Hs00197437 (OLFM4), Hs00362096-m1 (EPHB2), Hs02379687_s1 (CD24), Hs01075684_m1 (CD44), Hs01009250_m1 (PROM1/CD133), Hs00233455_m1 (ALCAM/CD166), and Hs0275899_g1 (GAPDH). GAPDH served as the endogenous control. Ten human CRC cell lines (DLD-1, HT29, SW620, HCT116, HCT15, LoVo, SW480, KM12C, KM12L4, and KM12SM) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in RPMI1640, minimal essential medium, Dulbecco's modified Eagle's medium, or L15 medium (Welgene, Daegu, Republic of Korea) containing 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin (Gibco) and maintained at 37°C in a humidified incubator with 5% CO2. Anti-LGR5 and anti–β-actin antibodies were purchased from Abcam and Sigma, respectively. Anti-vimentin antibodies, anti-mouse IgG–horseradish peroxidase, and anti-rabbit IgG–horseradish peroxidase antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The anti-SNAIL (zinc finger protein SNAI1), anti-SLUG (zinc finger protein SNAI2), anti-ZEB1 (zinc finger E-box-binding homeobox 1), anti–E-cadherin, anti–claudin-1, anti-ERK, anti-AKT, anti–β-catenin, anti–phospho (p)-ERK, and anti-pAKT antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Recombinant human RSPO2 and transforming growth factor (TGF)-β1 were purchased from PeproTech (Rocky Hill, NJ). Cellular proteins were extracted from colon cancer cells in lysis buffer (iNtRON Biotechnology, Seongnam, Republic of Korea) and protein levels were measured with the BCA Protein Assay Kit (Pierce, Rockford, IL). Cell lysates were separated on 10% SDS-polyacrylamide gel and transferred electrophoretically to PVDF Membrane (EMD Millipore Corporation, Bedford, MA). After transfer, PVDF membranes were blocked with 5% nonfat dry milk in Tris-buffered saline–Tween-20 for 1 hour. After extensive washes in 0.05% Tris-buffered saline–Tween-20, the membranes were incubated with a primary antibody. After overnight incubation at 4°C and washing with Tris-buffered saline–Tween-20, blots were incubated for 1 hour at room temperature with a secondary antibody. The target proteins were detected using the chemiluminescent reagents and visualized in the Alliance Mini HD9 chemiluminescence documentation system (Uvitec, Cambridge, UK). Full-length cDNA encoding LGR5 (pEX-LGR5) was purchased from GeneCopoeia (Rockville, MD) and cDNA encoding SNAIL (pCMV6-SNAIL), SLUG (pCMV6-SLUG), and ZEB1 (pCMV6-ZEB1) were from OriGene (Rockville, MD). LGR5 siRNA pool was purchased from Dharmacon (Lafayette, CO). Cells were seeded at 1 × 106 cells/well in 6-well plate after transfection with 5 μg of cDNA (or control vector) or LGR5 siRNA using the Neon transfection system (Thermo Fisher Scientific). One or two days after transfection, cells were subjected to real-time PCR, Western blot, and functional assays. All experiments were performed at least two to three times independently. As a proliferation assay, 24 hours after transfection in 6-well plates, cells were harvested and seeded at 5 × 103 cells/well on 96-well plates and incubated at 37°C. After the addition of 10 μL of Cell Counting Kit-8 reagent (Dojindo, Kumamoto, Japan) into each well and incubation for 1 hour, absorbance was measured at 450 nm using a spectrophotometer (Tecan, Mannedorf, Switzerland). As a colony-formation assay, 5000 to 10,000 cells were counted using Luna-II (Logos Biosystems, Gyeonggi-do, Republic of Korea) and seeded in a 60-mm culture dish and incubated for 10 to 20 days until distinguishable colony sizes appeared. Colonies were fixed with 70% methanol solution and stained with a 0.01% to 0.1% crystal violet solution. All experiments were performed at least two to three times independently. For a wound-healing assay, cells were cultured in a SPLScar Block (SPL Life Sciences, Seongnam, Republic of Korea) in a 6-well plate until they were confluent after transfection with the control vector or LGR5. The block was composed of 500 μm–thick walls to artificially generate cell-free gaps. After removal of the block from the plate, culture medium was added and cellular migration was photographed at 0 and 48 hours. For a migration assay, cells were starved in serum-free RPMI medium for 3 hours. After starvation, cells were harvested and resuspended by trypsin treatment. Twenty-four–well culture plates were divided into upper and lower wells by a transwell insert (pore size, 8 μm; BD Biosciences). The upper surface of the transwell was loaded with 2 × 105 cells in 300 μL of serum-free RPMI medium, and the lower wells contained 500 μL of RPMI with 10% fetal bovine serum. After 24 hours of incubation, nonmigrated cells were removed from the top of each insert with a cotton swab. Migrated cells on the bottom surface were fixed with methanol for 10 minutes and counted after staining with crystal violet for 1 hour. All experiments were performed at least two to three times independently. Statistical analyses were performed using SPSS statistical software version 18.0 (SPSS, Chicago, IL) and Prism software version 5.0 (GraphPad Software, San Diego, CA). The correlation between LGR5 positivity and clinicopathologic parameters was tested using the Pearson χ2 test or the Fisher exact test. Between-group comparisons o
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