The Expression Analysis of Intestinal Cancer Stem Cell Marker Lgr5 in Colorectal Cancer Patients and the Correlation with Histopathological Markers

  • Shirin Salehizadeh
  • Mandana Hasanzad
  • Azade Amini Kadijani
  • Abolfazl AkbariEmail author
Original Research



Cancer stem cells (CSCs) have frequently been utilized in the cell characterization and identified responsible for tumor development, metastasis, recurrence, and chemoresistance. CSC surface markers function in cancer cell signaling and are indicated as potential biomarkers for cancer diagnosis and prognosis. As well, dysregulation of cancer-related signaling pathways could promote CSC development and progression. Our aim was to evaluate the expression of colorectal CSC markers and their correlation with cancer proliferation and angiogenesis.


In this case-control study, total RNA was extracted from a total of 74 colorectal tumors and 74 adjacent normal tissue biopsies. Then, using a quantitative real-time PCR, the relative expression levels of Lgr5 and Lrig1 were measured in all malignant and healthy samples. Also, immunohistochemical (IHC) staining of tumor tissues was performed for Ki-67 (proliferation) and CD34 (angiogenesis) markers, and the immunoexpression staining scores were obtained. The diagnostic value of the genes was evaluated using receiver operating characteristic (ROC) curve. Possible correlation between CSC markers and immunohistochemical markers in CRC was analyzed by Pearson’s correlation test and linear regression.


The expression level of Lgr5 in tumor samples showed a significant increase compared with normal samples (p < 0.001) with a fold change of 2.54 (± 0.182). However, there was no significant difference in the relative expression of Lrig1 gene in tissue samples of healthy subjects and patients. The analysis of the ROC showed an AUC of 0.92 for Lgr5 and sensitivity 80% and specificity 96%. Further analysis revealed a significant correlation between mRNA levels of Lgr5 and immunoexpression of Ki-67 (r2 = 0.680, p < 0.001).


The high expression levels of Lgr5 found in tumor tissues were correlated with histological parameters, indicating a significant role in CRC development and diagnosis.


Angiogenesis Cancer stem cell Colorectal cancer Lgr5 Lrig1 Proliferation 


Funding Information

This study was supported by Iran University of Medical Sciences (Grant No. 28672).

Compliance with Ethical Standards

The study was approved by the ethics committee of Iran University of Medical Sciences (ethical code: IR.IUMS.REC 95-02-182-28672). All the individuals completed the informed consent forms.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Capp JP. Cancer stem cells: from historical roots to a new perspective. J Oncol. 2019;2019:1–10.CrossRefGoogle Scholar
  2. 2.
    Mobini GR, et al. Transforming growth factor beta-induced factor 2-linked X (TGIF2LX) regulates two morphogenesis genes, Nir1 and Nir2 in human colorectal. Acta Medica Iranica. 2016:302–7.Google Scholar
  3. 3.
    Phi LTH, Sari IN, Yang YG, Lee SH, Jun N, Kim KS, et al. Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int. 2018;2018:116.CrossRefGoogle Scholar
  4. 4.
    Karimi A, Majidzadeh-a K, Madjd Z, Akbari A, Habibi L, Akrami SM. Effect of copper sulfate on expression of endogenous L1 retrotransposons in HepG2 cells (hepatocellular carcinoma). Biol Trace Elem Res. 2015;165(2):131–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Huels DJ, Sansom OJ. Stem vs non-stem cell origin of colorectal cancer. Br J Cancer. 2015;113(1):1–5.PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683–91.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Brown DG, Rao S, Weir TL, O’Malia J, Bazan M, Brown RJ, et al. Metabolomics and metabolic pathway networks from human colorectal cancers, adjacent mucosa, and stool. Cancer Metab. 2016;4(1):11.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Puglisi MA, Tesori V, Lattanzi W, Gasbarrini GB, Gasbarrini A. Colon cancer stem cells: controversies and perspectives. World J Gastroenterol: WJG. 2013;19(20):2997–3006.PubMedCrossRefGoogle Scholar
  9. 9.
    Takebe N, Ivy SP. Controversies in cancer stem cells: targeting embryonic signaling pathways. Clin Cancer Res. 2010:1078–0432 CCR-09-2934.Google Scholar
  10. 10.
    Vidal S, et al. Targeting cancer stem cells to suppress acquired chemotherapy resistance. Oncogene. 2014;33(36):4451–63.PubMedCrossRefGoogle Scholar
  11. 11.
    Lathia JD, Mack SC, Mulkearns-Hubert EE, Valentim CLL, Rich JN. Cancer stem cells in glioblastoma. Genes Dev. 2015;29(12):1203–17.PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Vermeulen L, Snippert HJ. Stem cell dynamics in homeostasis and cancer of the intestine. Nat Rev Cancer. 2014;14(7):468–80.PubMedCrossRefGoogle Scholar
  13. 13.
    Kobayashi S, Yamada-Okabe H, Suzuki M, Natori O, Kato A, Matsubara K, et al. LGR5-positive colon cancer stem cells interconvert with drug-resistant LGR5-negative cells and are capable of tumor reconstitution. Stem Cells. 2012;30(12):2631–44.PubMedCrossRefGoogle Scholar
  14. 14.
    Ruffner H, Sprunger J, Charlat O, Leighton-Davies J, Grosshans B, Salathe A, et al. R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5. PLoS One. 2012;7(7):e40976.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Hoffmeyer K, Raggioli A, Rudloff S, Anton R, Hierholzer A, del Valle I, et al. Wnt/β-catenin signaling regulates telomerase in stem cells and cancer cells. Science. 2012;336(6088):1549–54.PubMedCrossRefGoogle Scholar
  16. 16.
    de Lau W, Peng WC, Gros P, Clevers H. The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 2014;28(4):305–16.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Krishnamurthy N, Kurzrock R. Targeting the Wnt/beta-catenin pathway in cancer: update on effectors and inhibitors. Cancer Treat Rev. 2018;62:50–60.PubMedCrossRefGoogle Scholar
  18. 18.
    Langan RC, Mullinax JE, Raiji MT, Upham T, Summers T, Stojadinovic A, et al. Colorectal cancer biomarkers and the potential role of cancer stem cells. J Cancer. 2013;4(3):241–50.PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Ljuslinder I, Golovleva I, Palmqvist R, Öberg Å, Stenling R, Jonsson Y, et al. LRIG1 expression in colorectal cancer. Acta Oncol. 2007;46(8):1118–22.PubMedCrossRefGoogle Scholar
  20. 20.
    Powell AE, Wang Y, Li Y, Poulin EJ, Means AL, Washington MK, et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell. 2012;149(1):146–58.PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Wang Y, Poulin E, Coffey R. LRIG1 is a triple threat: ERBB negative regulator, intestinal stem cell marker and tumour suppressor. Br J Cancer. 2013;108(9):1765–70.PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Reya T, et al. Stem cells, cancer, and cancer stem cells. Nature. 2001;414(6859):105.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Takahashi-Yanaga F, Kahn M. Targeting Wnt signaling: can we safely eradicate cancer stem cells? Clin Cancer Res. 2010:1078–0432 CCR-09-2943.Google Scholar
  24. 24.
    Puppa G, Sonzogni A, Colombari R, Pelosi G. TNM staging system of colorectal carcinoma: a critical appraisal of challenging issues. Archives of Pathology & Laboratory Medicine. 2010;134(6):837–52.Google Scholar
  25. 25.
    Ishiguro T, Ohata H, Sato A, Yamawaki K, Enomoto T, Okamoto K. Tumor-derived spheroids: relevance to cancer stem cells and clinical applications. Cancer Sci. 2017;108(3):283–9.PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Chalkidou A, Landau DB, Odell EW, Cornelius VR, O’Doherty MJ, Marsden PK. Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: a systematic review and meta-analysis. Eur J Cancer. 2012;48(18):3499–513.PubMedCrossRefGoogle Scholar
  27. 27.
    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18–43.PubMedGoogle Scholar
  28. 28.
    Shaheen S, Ahmed M, Lorenzi F, Nateri AS. Spheroid-formation (colonosphere) assay for in vitro assessment and expansion of stem cells in colon cancer. Stem Cell Rev Rep. 2016;12(4):492–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Eschrich S, Yang I, Bloom G, Kwong KY, Boulware D, Cantor A, et al. Molecular staging for survival prediction of colorectal cancer patients. J Clin Oncol. 2005;23(15):3526–35.PubMedCrossRefGoogle Scholar
  30. 30.
    Mínguez B, Lachenmayer A. Diagnostic and prognostic molecular markers in hepatocellular carcinoma. Dis Markers. 2011;31(3):181–90.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Hammond MEH, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48–72.PubMedGoogle Scholar
  32. 32.
    Bergamaschi A, Kim YH, Wang P, Sørlie T, Hernandez-Boussard T, Lonning PE, et al. Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer. Genes Chromosom Cancer. 2006;45(11):1033–40.PubMedCrossRefGoogle Scholar
  33. 33.
    Marisa L, de Reyniès A, Duval A, Selves J, Gaub MP, Vescovo L, et al. Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value. PLoS Med. 2013;10(5):e1001453.PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol. 2005;23(28):7212–20.PubMedCrossRefGoogle Scholar
  35. 35.
    Pelosi G, Rindi G, Travis WD, Papotti M. Ki-67 antigen in lung neuroendocrine tumors: unraveling a role in clinical practice. J Thorac Oncol. 2014;9(3):273–84.PubMedCrossRefGoogle Scholar
  36. 36.
    Moore N, Lyle S. Quiescent, slow-cycling stem cell populations in cancer: a review of the evidence and discussion of significance. J Oncol. 2011;2011:111.CrossRefGoogle Scholar
  37. 37.
    Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B, et al. A perivascular niche for brain tumor stem cells. Cancer Cell. 2007;11(1):69–82.PubMedCrossRefGoogle Scholar
  38. 38.
    Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, et al. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest. 2010;120(9):3326–39.PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Vermeulen L, de Sousa E Melo F, van der Heijden M, Cameron K, de Jong JH, Borovski T, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol. 2010;12(5):468–76.PubMedCrossRefGoogle Scholar
  40. 40.
    Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26(17):2839–45.CrossRefGoogle Scholar
  41. 41.
    Liu Y, et al. Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer. J Biol Chem. 2011;286(28):25007–15.PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    White BD, Chien AJ, Dawson DW. Dysregulation of Wnt/β-catenin signaling in gastrointestinal cancers. Gastroenterology. 2012;142(2):219–32.PubMedCrossRefGoogle Scholar
  43. 43.
    Walker F, Zhang HH, Odorizzi A, Burgess AW. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines. PLoS One. 2011;6(7):e22733.PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Wu X-S, Xi H-Q, Chen L. Lgr5 is a potential marker of colorectal carcinoma stem cells that correlates with patient survival. World J Surg Oncol. 2012;10(1):244.PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Han Y, Xue X, Jiang M, Guo X, Li P, Liu F, et al. 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(2):267–73.PubMedCrossRefGoogle Scholar
  46. 46.
    de Lau W, Barker N, Low TY, Koo BK, Li VSW, Teunissen H, et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 2011;476(7360):293–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Jiang 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(1):12.PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Dame MK, et al. Identification, isolation, and characterization of human LGR5-positive colon adenoma cells. Development. 2018;145.Google Scholar
  49. 49.
    He S, Zhou H, Zhu X, Hu S, Fei M, Wan D, et al. Expression of Lgr5, a marker of intestinal stem cells, in colorectal cancer and its clinicopathological significance. Biomed Pharmacother. 2014;68(5):507–13.PubMedCrossRefGoogle Scholar
  50. 50.
    Takaishi S, Okumura T, Wang TC. Gastric cancer stem cells. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26(17):2876–82.CrossRefGoogle Scholar
  51. 51.
    Lang SA, Gaumann A, Koehl GE, Seidel U, Bataille F, Klein D, et al. Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model. Int J Cancer. 2007;120(8):1803–10.PubMedCrossRefGoogle Scholar
  52. 52.
    Hermann PC, et al. Cancer stem cells in solid tumors. In: Seminars in cancer biology. Amsterdam: Elsevier; 2010.Google Scholar
  53. 53.
    Takeda K, Kinoshita I, Shimizu Y, Matsuno Y, Shichinohe T, Dosaka-Akita H. Expression of LGR5, an intestinal stem cell marker, during each stage of colorectal tumorigenesis. Anticancer Res. 2011;31(1):263–70.PubMedGoogle Scholar
  54. 54.
    Scheel C, Weinberg RA. Phenotypic plasticity and epithelial-mesenchymal transitions in cancer and normal stem cells? Int J Cancer. 2011;129(10):2310–4.PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Ye X, Weinberg RA. Epithelial–mesenchymal plasticity: a central regulator of cancer progression. Trends Cell Biol. 2015;25(11):675–86.PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Osawa H, Takahashi H, Nishimura J, Ohta K, Haraguchi N, Hata T, et al. Full-length LGR5-positive cells have chemoresistant characteristics in colorectal cancer. Br J Cancer. 2016;114(11):1251–60.PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    Lindquist D, Kvarnbrink S, Henriksson R, Hedman H. LRIG and cancer prognosis. Acta Oncol. 2014;53(9):1135–42.PubMedPubMedCentralCrossRefGoogle Scholar
  58. 58.
    Krig SR, Frietze S, Simion C, Miller JK, Fry WHD, Rafidi H, et al. Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer. Mol Cancer Res. 2011;9:1406–17.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Shirin Salehizadeh
    • 1
  • Mandana Hasanzad
    • 2
    • 3
  • Azade Amini Kadijani
    • 4
  • Abolfazl Akbari
    • 5
    • 6
    Email author
  1. 1.Department of Genetics, Tehran Medical Sciences BranchIslamic Azad UniversityTehranIran
  2. 2.Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical SciencesIslamic Azad UniversityTehranIran
  3. 3.Medical Genomics Research Center, Tehran Medical SciencesIslamic Azad UniversityTehranIran
  4. 4.Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver DiseasesShahid Beheshti University of Medical SciencesTehranIran
  5. 5.Colorectal Research CenterIran University of Medical SciencesTehranIran
  6. 6.Colorectal Research Center, Rasoul-e-Akram HospitalTehranIran

Personalised recommendations