Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition

  • Authors:
    • Miho Okano
    • Masamitsu Konno
    • Yoshihiro Kano
    • Hirotoshi Kim
    • Koichi Kawamoto
    • Masahisa Ohkuma
    • Naotsugu Haraguchi
    • Takehiko Yokobori
    • Koshi Mimori
    • Hirofumi Yamamoto
    • Mitsugu Sekimoto
    • Yuichiro Doki
    • Masaki Mori
    • Hideshi Ishii
  • View Affiliations

  • Published online on: May 23, 2014     https://doi.org/10.3892/ijo.2014.2462
  • Pages: 575-580
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Conventional cancer chemotherapy preferentially destroys non-stem cancer cells within a tumor, and a subpopulation of cancer stem cells (CSCs) is more resistant and survives, leading to relapses and metastasis. Howeve, recent studies suggest that CD24 and susceptibility to epithelial-mesenchymal transition (EMT) can serve as markers of CSCs. We report that CD24+ cells are susceptible to induction of EMT, a phenotype important for cancer metastasis. We studied the responsiveness of CSC markers to TGF-?, an effective EMT inducer. The data on CD24 demonstrated that CD24+ cells are susceptible to EMT, a phenotype important for cancer metastasis in two colorectal cancer cell lines, the CaR-1 and CCK81. CD24+ cells expressed Notch 1 in response to exposure to TGF-? in culture and showed higher tumorigenic activity compared to controls. This evidence shows that CD24+ cells are susceptible to EMT induction and to cancer progression and is indicative of the candidacy of CD24 as a therapeutic target in CSC.

Introduction

Conventional cancer treatments preferentially destroy non-stem cancer cells within tumors, whereas cancer stem cells (CSCs) are more resistant and survive, which can subsequently cause a relapse, and in some cases, life-threatening metastasis (1,2). Identification of a regulatory mechanism, such as a functional cell surface marker, would be useful for distinguishing CSCs from non-stem cancer cells, which would allow for reduction of the dosage of chemo- and radiotherapy and maximize tumor targeting (3). Previously, we identified CD13/aminopeptidase as a cell surface marker, which is preferentially expressed in CSCs of gastrointestinal organs. CD13/aminopeptidase has a functional role in the reduction of reactive oxygen species (ROS) in tumor cells by modulating glutathione synthesis, thereby contributing to the survival of CSCs after chemo- and radiotherapy (4). CD13 is also upregulated during the epithelial-mesenchymal transition (EMT), a phenotype important for cancer metastasis (5). Recent studies suggest that susceptibility to EMT can serve as a marker of CSCs (6). Because CSCs are a heterogeneous cell population (7), further research is necessary for identification of CSC markers.

Since the identification of rare CSCs in leukemia (810), molecular markers for detection of CSCs have been reported in solid tumors of the head and neck (11), gastrointestinal system (12), colon (13,14), breast (15) and brain (16,17). CD44 (hyaluronic acid receptor) is one of the most commonly studied surface markers, which is expressed by almost all cancer stem cells, and CD24 (heat-stable antigen) is another surface marker expressed in many tumor types (18). Although CD44+CD24 cell populations have been identified as CSCs in breast cancer (19), expression of CD133, CD166, CD44, CD29, CD24, Lgr5, and nuclear β-catenin has been suggested to mark the CSC population in the colon (20).

Subsequent studies showed that although it is unclear whether CD133 is a marker of colon CSCs, other cell surface markers, such as epithelial-specific antigen, CD44, CD166, Musashi-1, CD29, CD24, leucine-rich repeat-containing G protein-coupled receptor 5, and aldehyde dehydrogenase 1, have been shown to be promising candidates (21). A recent study on mice narrowed down the targets and demonstrated that CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells; their data suggest that the presence of CD24 expression in normal colonic epithelium may have important implications in the use of colorectal cancer therapies targeting CD24 (22). Here we studied the responsiveness to TGF-β of CSCs carrying various markers (TGF-β is an effective EMT inducer). The data on CD24 demonstrated that CD24+ cells are susceptible to EMT induction and are associated with tumorigenesis in mice.

Materials and methods

Cell culture

Human CRC cell lines were obtained from American Type Culture Collection (ATCC) and cultured in minimum essential medium (MEM; Invitrogen, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2. For flow cytometry and cell sorting, an allophycocyanin (APC)-conjugated anti-human CD44 antibody, a fluorescein isothiocyanate (FITC)-conjugated anti-human CD24 antibody, and a phycoerythrin (PE)-conjugated anti-human N-CAD antibody (BD Bioscience, San Jose, CA, USA) were used for characterization of cancer cells. Labeled cells were analyzed on a BD FACS Aria II Cell Sorter System (Becton-Dickinson, Franklin Lakes, NJ, USA), followed by data analysis using the Diva program (Becton-Dickinson), as described previously (4,5).

The expression study

Total RNA was extracted from cells, reverse-transcribed to cDNA, and subjected to PCR analysis using specific primers as described previously (4,5).

Animal experiments

Cells were injected subcutaneously into NOD/SCID mice as described previously (4,5). These mice were monitored for up to 10 weeks and sacrificed when the tumors reached a maximum diameter of 15 mm. All animal studies were approved by the Animal Experiments Committee of Osaka University.

Statistical analysis

For continuous variables used in an in vitro analysis, the data were calculated as mean ± SD and were analyzed using the Wilcoxon rank test. The relationship between mRNA expression and clinicopathological factors was analyzed using the χ2 test and Student’s t-test. Kaplan-Meier survival curves were plotted and compared using the generalized log-rank test. Univariate and multivariate analyses for identification of factors prognostic of overall survival were performed using the Cox proportional hazards regression model. All calculations were performed using the JMP software (SAS Institute, Cary, NC, USA). Differences with a p-value of <0.05 were considered statistically significant.

Results

TGF-β stimulates EMT

To study the EMT mechanism, we cultured the colorectal cancer cell lines CaR-1 and CCK81 in the medium containing TGF-β. The data from quantitative PCR indicated that expression of EMT markers such as N-cadherin, vimentin, and fibronectin was increased in a dose-dependent manner with TGF-β concentration in the culture medium (Fig. 1A). Immunohistochemical analysis indicated that expression of these genes was increased after exposure to TGF-β (Fig. 1B). The data show that EMT was induced in the cell lines we examined under these conditions.

TGF-β increases CD24 expression in colorectal cancer cells

To study the effect of TGF-β, we investigated the expression of CSC markers CD44, CD24, and N-cadherin. The data indicated that the expression of these markers was increased after TGF-β exposure; the effect was strong in CD24 compared with the other two markers (Fig. 2). Accordingly, in subsequent experiments, we focused on CD24.

CD24 is enriched in the EMT cells

We were interested in whether CD24+ cells are susceptible to TGF-β-induced EMT. To this end, we subjected the colorectal cancer cell lines CaR-1 and CCK81, to fluorescence-activated cell sorting (FACS) and analyzed the results. The data showed that EMT-primed cells, which were marked by the expression of N-cadherin, were enriched in CD24+ cells (Fig. 3). The data were consistent between the two cell lines, suggesting that CD24 is a marker of EMT.

CD24 stem markers

We wanted to identify the molecules expressed in CD24+ cells, which could be associated with cancer stemness. We analyzed the expression of several markers, including c-kit, Bmi1, SCF, and Notch 1 (Fig. 4). Then we assessed the effect of adding TGF-β (EMT inducer) to cell culture medium on each separate cell population by FACS sorting. The data on Notch 1 expression indicated that CD24+ cells are likely to be CSCs. CD24+ cells responded to TGF-β, and this effect was more appreciable in N-cadherin+ cells (Fig. 5), suggesting that CD24+ cells are prone to EMT, and CD24+ N-cadherin+ cells are more sensitive to TGF-β than are CD24+N-cadherin cells. The data showed that the expression of Notch 1 correlated with expression of CD24, suggesting that there was a link between the CD24+ CSCs and the Notch 1 pathway.

CD24+ cells show high tumorigenic activity

To assess the tumorigenic potential of CD24+ cells, we injected the cancer cells into immunocompetent NOD/SCID mice subcutaneously. The results showed that CD24+ cancer cells had a higher tumorigenic potential compared with CD24 cells with respect to both tumor frequency and tumor size (Fig. 6). These data suggest that in our experimental model, CD24+ cells drive tumorigenicity, which is one of the characteristics of CSCs.

Discussion

Previous studies pointed to the candidacy of CD24 as a CSC marker in colorectal cancer (20.21). The present study shows that CD24+ colonic cancer cells increase in number after exposure to TGF-β in culture, compared with N-CAD+ and CD44+ cells, suggesting that CD24+ cells are susceptible to EMT, a cellular trait important for cancer metastasis. The data were consistent between the two cell lines that were studied, CaR-1 and CCK81. The cell sorting experiment indicated that CD24 is a more useful marker than CD44, for separation of EMT-prone cells, according to assessment of the expression of N-CAD, a marker of EMT. The present study indicates that CD24 is associated with EMT, and the association is more pronounced compared with other possible markers, such as CD44. The findings are compatible with the data from other cell lines (23).

Because CD24+ cancer cells formed larger tumors in immunocompetent NOD/SCID mice, we determined if any stemness markers were expressed preferentially in CD24+ cells. The FACS experiment indicated that exposure to TGF-β in culture resulted in increased Notch 1 expression. Recent studies have indicated that Notch signaling has a critical role at the intersection of EMT and cancer stemness and that Notch inhibition is an attractive strategy for the treatment of several cancers, at least in part because of its ability to reverse or prevent EMT (24).

A previous study indicated that TGF-β is a possible niche signal in the bone marrow to induce hibernation of hematopoietic stem cells (25), a dormant phenotype of cancer cells, showing resistance to chemotherapy. The hibernation state is associated with inhibition of lipid raft clustering; this change results in inhibition of signaling of growth factors or cytokines through cell surface receptors (25). The study of Listeria monocytogens indicated that CD14 and CD24, which normally exhibit uniform distribution on cells undergo clustering upon treatment with the stimulation (26); the phenomenon is suggestive of lipid raft clustering and signaling through CD24. A recent study of protein clustering showed enrichment of CD24 in lipid rafts and a more random distribution of CD44 in the plasma membrane (27). Taken together, the data are indicative of the significance of CD24 as a functional marker of CSCs and suggest that this protein is a possible therapeutic target in colorectal cancer.

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology; a Grant-in-Aid from the Third Comprehensive 10-year Strategy for Cancer Control, Ministry of Health, Labor and Welfare; a grant from the Kobayashi Cancer Research Foundation; a grant from the Princess Takamatsu Cancer Research Fund; a grant from the Senshin Medical Research Foundation; a grant from the National Institute of Biomedical Innovation, Japan.

References

1 

Reya T, Morrison SJ, Clarke MF and Weissman IL: Stem cells, cancer, and cancer stem cells. Nature. 414:105–111. 2001. View Article : Google Scholar : PubMed/NCBI

2 

Dewi DL, Ishii H, Kano Y, Nishikawa S, Haraguchi N, Sakai D, Satoh T, Doki Y and Mori M: Cancer stem cell theory in gastrointestinal malignancies: recent progress and upcoming challenges. J Gastroenterol. 46:1145–1157. 2011. View Article : Google Scholar : PubMed/NCBI

3 

Ishii H, Iwatsuki M, Ieta K, Ohta D, Haraguchi N, Mimori K and Mori M: Cancer stem cells and chemoradiation resistance. Cancer Sci. 99:1871–1877. 2008. View Article : Google Scholar

4 

Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H, Barnard GF, Doki Y and Mori M: CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest. 120:3326–3339. 2010. View Article : Google Scholar : PubMed/NCBI

5 

Kim HM, Haraguchi N, Ishii H, Ohkuma M, Okano M, Mimori K, Eguchi H, Yamamoto H, Nagano H, Sekimoto M, Doki Y and Mori M: Increased CD13 expression reduces reactive oxygen species, promoting survival of liver cancer stem cells via an epithelial-mesenchymal transition-like phenomenon. Ann Surg Oncol. (Suppl 3): S539–S548. 2012. View Article : Google Scholar : PubMed/NCBI

6 

Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J and Weinberg RA: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 133:704–715. 2008. View Article : Google Scholar : PubMed/NCBI

7 

Meacham CE and Morrison SJ: Tumour heterogeneity and cancer cell plasticity. Nature. 501:328–337. 2013. View Article : Google Scholar : PubMed/NCBI

8 

Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK, Andreeff M and Goodell MA: A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood. 98:1166–1173. 2001. View Article : Google Scholar : PubMed/NCBI

9 

Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA and Dick JE: A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 367:645–648. 1994. View Article : Google Scholar : PubMed/NCBI

10 

Bonnet D and Dick JE: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 3:730–737. 1997. View Article : Google Scholar : PubMed/NCBI

11 

Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF and Ailles LE: Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA. 104:973–978. 2007. View Article : Google Scholar : PubMed/NCBI

12 

Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF and Mori M: Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells. 24:506–513. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C and De Maria R: Identification and expansion of human colon-cancer-initiating cells. Nature. 445:111–115. 2007. View Article : Google Scholar : PubMed/NCBI

14 

O’Brien CA, Pollett A, Gallinger S and Dick JE: A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 445:106–110. 2007.PubMed/NCBI

15 

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ and Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 100:3983–3988. 2003. View Article : Google Scholar : PubMed/NCBI

16 

Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G, Brem H, Olivi A, Dimeco F and Vescovi AL: Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature. 444:761–765. 2006. View Article : Google Scholar : PubMed/NCBI

17 

Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD and Rich JN: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 444:756–760. 2006. View Article : Google Scholar : PubMed/NCBI

18 

Jaggupilli A and Elkord E: Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity. Clin Dev Immunol. 2012:7080362012. View Article : Google Scholar : PubMed/NCBI

19 

Ponti D, Zaffaroni N, Capelli C and Daidone MG: Breast cancer stem cells: an overview. Eur J Cancer. 42:1219–12124. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Perez Alea M, Richel DJ, Stassi G and Medema JP: Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci USA. 105:13427–13432. 2008. View Article : Google Scholar : PubMed/NCBI

21 

Todaro M, Francipane MG, Medema JP and Stassi G: Colon cancer stem cells: promise of targeted therapy. Gastroenterology. 138:2151–2162. 2010. View Article : Google Scholar : PubMed/NCBI

22 

King JB, von Furstenberg RJ, Smith BJ, McNaughton KK, Galanko JA and Henning SJ: CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice. Am J Physiol Gastrointest Liver Physiol. 303:G443–G452. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Ke J, Wu X, Wu X, He X, Lian L, Zou Y, He X, Wang H, Luo Y, Wang L and Lan P: A subpopulation of CD24+ cells in colon cancer cell lines possess stem cell characteristics. Neoplasma. 59:282–288. 2012.

24 

Espinoza I and Miele L: Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells. Cancer Lett. 341:41–45. 2013. View Article : Google Scholar : PubMed/NCBI

25 

Yamazaki S, Iwama A, Takayanagi S, Eto K, Ema H and Nakauchi H: TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation. Blood. 113:1250–1256. 2009. View Article : Google Scholar : PubMed/NCBI

26 

Gekara NO and Weiss S: Lipid rafts clustering and signalling by listeriolysin O. Biochem Soc Trans. 32:712–714. 2004. View Article : Google Scholar : PubMed/NCBI

27 

Yu X, Wang J, Feizpour A and Reinhard BM: Illuminating the lateral organization of cell-surface CD24 and CD44 through plasmon coupling between Au nanoparticle immunolabels. Anal Chem. 85:1290–1294. 2013. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August-2014
Volume 45 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Okano M, Konno M, Kano Y, Kim H, Kawamoto K, Ohkuma M, Haraguchi N, Yokobori T, Mimori K, Yamamoto H, Yamamoto H, et al: Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition. Int J Oncol 45: 575-580, 2014
APA
Okano, M., Konno, M., Kano, Y., Kim, H., Kawamoto, K., Ohkuma, M. ... Ishii, H. (2014). Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition. International Journal of Oncology, 45, 575-580. https://doi.org/10.3892/ijo.2014.2462
MLA
Okano, M., Konno, M., Kano, Y., Kim, H., Kawamoto, K., Ohkuma, M., Haraguchi, N., Yokobori, T., Mimori, K., Yamamoto, H., Sekimoto, M., Doki, Y., Mori, M., Ishii, H."Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition". International Journal of Oncology 45.2 (2014): 575-580.
Chicago
Okano, M., Konno, M., Kano, Y., Kim, H., Kawamoto, K., Ohkuma, M., Haraguchi, N., Yokobori, T., Mimori, K., Yamamoto, H., Sekimoto, M., Doki, Y., Mori, M., Ishii, H."Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition". International Journal of Oncology 45, no. 2 (2014): 575-580. https://doi.org/10.3892/ijo.2014.2462