Archives of Dermatological Research

, Volume 305, Issue 1, pp 35–47 | Cite as

Cancer stem-like cells enriched with CD29 and CD44 markers exhibit molecular characteristics with epithelial–mesenchymal transition in squamous cell carcinoma

  • Songmei Geng
  • Yuanyuan Guo
  • Qianqian Wang
  • Lan Li
  • Jianli Wang
Original Paper

Abstract

Increasing evidences have indicated that only a phenotypic subset of cancer cells, termed as the cancer stem cells (CSCs), is capable of initiating tumor growth and provide a reservoir of cells that cause tumor recurrence after therapy. Epithelial–mesenchymal transition (EMT), a cell type change from an epithelial cobblestone phenotype to an elongated fibroblastic phenotype, plays a critical role not only in tumor metastasis but also in tumor recurrence and contributes to drug resistance. Accumulating evidence has shown that cells with an EMT phenotype are rich sources for CSCs, suggesting a biological link between EMT and CSCs; thus study on the link will help understand the cellular and molecular mechanisms of tumor metastasis and drug resistance. CD29 is involved in EMT through cross-talk with cadherins and CD44 has been reported as a successful used marker for CSCs. Here, we try to address whether combination of CD29 and CD44 could be used to identify cancer stem-like cells undergoing EMT in squamous cell carcinoma (SCC) and compare the molecular differences between CD29high/CD44high and CD29low/CD44low cells in SCC. Expression pattern of CD29 and CD44 was analyzed in tissues of skin SCC and cultured A431 cells by immunostaining. Subtype cells of CD29high/CD44high and CD29low/CD44low A431 were sorted by fluorescence-activated cell sorting and proliferating abilities were assayed by cell counting, colony forming and tumorigenicity in NOD/SCID mice. Finally, to probe more deeply into the molecular differences between CD29high/CD44high and CD29low/CD44low A431 cells, gene microarray analysis was applied to compare gene expression profiling. Staining of CD29 and CD44 showed similar heterogeneous expression pattern with positive cells located in the invasion front of SCC tissue as well as in cultured A431 cells. Sorted CD29high/CD44high A431 cells had higher proliferating ability in vitro and in NOD/SCID mice as compared with CD29low/CD44low cells. Gene profiling identified differentiated gene expressions between CD29high/CD44high and CD29low/CD44low A431 cells. These genes are involved in cell cycle, cell malignant transformation, metastasis, drug resistance and EMT, implying that CD29high/CD44high cells have properties of CSCs and EMT. Our present results demonstrated heterogeneous gene expression patterns and different biological behavior in SCC. Combination of CD29 and CD44 can be used as markers to enrich CSCs in human SCC. Moreover, CD29high/CD44high cells exhibit molecular characteristics of EMT, suggesting that CSC-associated pathways were involved in EMT. Studies on correlation of CSCs and the cells undergoing EMT may explain some aspects of tumor progression and drug resistance.

Keywords

Squamous cell carcinoma Cancer stem cells CD29 CD44 EMT 

References

  1. 1.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988PubMedCrossRefGoogle Scholar
  2. 2.
    Allen WL, McLean EG, Boyer J, McCulla A, Wilson PM, Coyle V, Longley DB, Casero RA Jr, Johnston PG (2007) The role of spermidine/spermine N1-acetyltransferase in determining response to chemotherapeutic agents in colorectal cancer cells. Mol Cancer Ther 6(1):128–137PubMedCrossRefGoogle Scholar
  3. 3.
    Alvarez H, Montgomery EA, Karikari C, Canto M, Dunbar KB, Wang JS, Feldmann G, Hong SM, Haffner MC, Meeker AK, Holland SJ, Yu J, Heckrodt TJ, Zhang J, Ding P, Goff D, Singh R, Roa JC, Marimuthu A, Riggins GJ, Eshleman JR, Nelkin BD, Pandey A, Maitra A (2010) The Axl receptor tyrosine kinase is an adverse prognostic factor and a therapeutic target in esophageal adenocarcinoma. Cancer Biol Ther 10(10):18–1009Google Scholar
  4. 4.
    Argast GM, Croy CH, Couts KL, Zhang Z, Litman E, Chan DC, Ahn NG (2009) Plexin B1 is repressed by oncogenic B-Raf signaling and functions as a tumor suppressor in melanoma cells. Oncogene 28(30):2697–2709PubMedCrossRefGoogle Scholar
  5. 5.
    Atsumi N, Ishii G, Kojima M, Sanada M, Fujii S, Ochiai A (2008) Podoplanin, a novel marker of tumor-initiating cells in human squamous cell carcinoma A431. Biochem Biophys Res Commun 373(1):36–41PubMedCrossRefGoogle Scholar
  6. 6.
    Biddle A, Liang X, Gammon L, Fazil B, Harper LJ, Emich H, Costea DE, Mackenzie IC (2011) Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative. Cancer Res 71(15):5317–5326PubMedCrossRefGoogle Scholar
  7. 7.
    Blick T, Hugo H, Widodo E, Waltham M, Pinto C, Mani SA, Weinberg RA, Neve RM, Lenburg ME, Thompson EW (2010) Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. J Mammary Gland Biol Neoplasia 15(2):235–252PubMedCrossRefGoogle Scholar
  8. 8.
    Bortolomai I, Canevari S, Facetti I, De Cecco L, Castellano G, Zacchetti A, Alison MR, Miotti S (2010) Tumor initiating cells: development and critical characterization of a model derived from the A431 carcinoma cell line forming spheres in suspension. Cell Cycle 9(6):1194–1206PubMedCrossRefGoogle Scholar
  9. 9.
    Brakebusch C, Fässler R (2005) β1 integrin function in vivo: adhesion, migration and more. Cancer Metastasis Rev 24(3):403–411PubMedCrossRefGoogle Scholar
  10. 10.
    Chovanec M, Smetana K Jr, Betka J, Plzák J, Brabec J, Moya-Alvarez V, André S, Kodet R, Gabius HJ (2005) Correlation of expression of nuclear proteins pKi67 and p63 with lectin histochemical features in head and neck squamous cell cancer. Int J Oncol 27(2):409–415PubMedGoogle Scholar
  11. 11.
    Christiansen JJ, Rajasekaran AK (2006) Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res 66(17):8319–8326PubMedCrossRefGoogle Scholar
  12. 12.
    Chu P, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E, Peach RJ (2009) Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer 124(6):1312–1321PubMedCrossRefGoogle Scholar
  13. 13.
    Cui H, Darmanin S, Natsuisaka M, Kondo T, Asaka M, Shindoh M, Higashino F, Hamuro J, Okada F, Kobayashi M, Nakagawa K, Koide H, Kobayashi M (2007) Enhanced expression of asparagine synthetase under glucose-deprived conditions protects pancreatic cancer cells from apoptosis induced by glucose deprivation and cisplatin. Cancer Res 67(7):3345–3355PubMedCrossRefGoogle Scholar
  14. 14.
    Dokmanovic M, Chang BD, Fang J, Roninson IB (2002) Retinoid-induced growth arrest of breast carcinoma cells involves co-activation of multiple growth-inhibitory genes. Cancer Biol Ther 1(1):24–27PubMedGoogle Scholar
  15. 15.
    Finlan LE, Hupp TR (2006) Epidermal stem cells and cancer stem cells: insights into cancer and potential therapeutic strategies. Eur J Cancer 42(9):1283–1292PubMedCrossRefGoogle Scholar
  16. 16.
    Geng S, Wang Q, Wang J, Hu Z, Liu C, Qiu J, Zeng W (2011) Isolation and identification of a distinct side population cancer cells in the human epidermal squamous cancer cell line A431. Arch Dermatol Res 303(3):181–189PubMedCrossRefGoogle Scholar
  17. 17.
    Gerdes MJ, Yuspa SH (2005) The contribution of epidermal stem cells to skin cancer. Stem Cell Rev 1(3):225–231PubMedCrossRefGoogle Scholar
  18. 18.
    Gómez Román JJ, Garay GO, Saenz P, Escuredo K, Sanz Ibayondo C, Gutkind S, Junquera C, Simón L, Martínez A, Fernández Luna JL, Val-Bernal JF (2008) Plexin B1 is downregulated in renal cell carcinomas and modulates cell growth. Transl Res 151(3):134–140PubMedCrossRefGoogle Scholar
  19. 19.
    Green J, Ikram M, Vyas J, Patel N, Proby CM, Ghali L, Leigh IM, O’Toole EA, Storey A (2006) Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours. Br J Cancer 94(10):1446–1451PubMedCrossRefGoogle Scholar
  20. 20.
    Hölzel M, Huang S, Koster J, Ora I, Lakeman A, Caron H, Nijkamp W, Xie J, Callens T, Asgharzadeh S, Seeger RC, Messiaen L, Versteeg R, Bernards R (2010) NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell 142(2):218–229PubMedCrossRefGoogle Scholar
  21. 21.
    Horst D, Kriegl L, Engel J, Kirchner T, Jung A (2009) Prognostic significance of the cancer stem cell markers CD133, CD44, and CD166 in colorectal cancer. Cancer Invest 27(8):844–850PubMedCrossRefGoogle Scholar
  22. 22.
    Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, Thompson EW (2007) Epithelial-mesenchymal and mesenchymal-epithelial transitions in carcinoma progression. J Cell Physiol 213(2):374–383PubMedCrossRefGoogle Scholar
  23. 23.
    Jensen KB, Jones J, Watt FM (2008) A stem cell gene expression profile of human squamous cell carcinomas. Cancer Lett 272(1):23–31PubMedCrossRefGoogle Scholar
  24. 24.
    Jones PH, Watt FM (1993) Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73(4):713–724PubMedCrossRefGoogle Scholar
  25. 25.
    Kamstrup MR, Gniadecki R, Skovgaard GL (2007) Putative cancer stem cells in cutaneous malignancies. Exp Dermatol 16(4):297–301PubMedCrossRefGoogle Scholar
  26. 26.
    Kanehira M, Harada Y, Takata R, Shuin T, Miki T, Fujioka T, Nakamura Y, Katagiri T (2007) Involvement of upregulation of DEPDC1 (DEP domain containing 1) in bladder carcinogenesis. Oncogene 26(44):6448–6455PubMedCrossRefGoogle Scholar
  27. 27.
    Keating AK, Kim GK, Jones AE, Donson AM, Ware K, Mulcahy JM, Salzberg DB, Foreman NK, Liang X, Thorburn A, Graham DK (2010) Inhibition of Mer and Axl receptor tyrosine kinases in astrocytoma cells leads to increased apoptosis and improved chemosensitivity. Mol Cancer Ther 9(5):1298–1307PubMedCrossRefGoogle Scholar
  28. 28.
    Koo BH, Hurskainen T, Mielke K, Aung PP, Casey G, Autio-Harmainen H, Apte SS (2007) ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer. Int J Cancer 121(8):1710–1716PubMedCrossRefGoogle Scholar
  29. 29.
    Liu B, Sun H, Wang W, Li W, Yan YF, Chen SM, Yang YP, Xu CX, Xin JX, Liu XX (2009) Adenovirus vector-mediated upregulation of spermidine/spermine N1-acetyltransferase impairs human gastric cancer growth in vitro and in vivo. Cancer Sci 100(11):2126–2132PubMedCrossRefGoogle Scholar
  30. 30.
    Locke M, Heywood M, Fawell S, Mackenzie IC (2005) Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. Cancer Res 65(19):8944–8950PubMedCrossRefGoogle Scholar
  31. 31.
    Mackenzie IC (2004) Growth of malignant oral epithelial stem cells after seeding into organotypical cultures of normal mucosa. J Oral Pathol Med 33(2):71–78PubMedCrossRefGoogle Scholar
  32. 32.
    Mackenzie IC (2004) Retention of stem cell patterns in malignant cell lines. Cell Prolif 38(6):347–355CrossRefGoogle Scholar
  33. 33.
    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, Weinberg RA (2008) The epithelial mesenchymal transition generates cells with properties of stem cells. Cell 133(4):704–715PubMedCrossRefGoogle Scholar
  34. 34.
    Meester-Smoor MA, Janssen MJ, Grosveld GC, de Klein A, van IJcken WF, Douben H, Zwarthoff EC (2008) MN1 affects expression of genes involved in hematopoiesis and can enhance as well as inhibit RAR/RXR-induced gene expression. Carcinogenesis 29(10):2025–2034PubMedCrossRefGoogle Scholar
  35. 35.
    Patrawala L, Calhoun T, Schneider-Broussard R, Li H, Bhatia B, Tang S, Reilly JG, Chandra D, Zhou J, Claypool K, Coghlan L, Tang DG (2006) Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25(12):1696–1708PubMedCrossRefGoogle Scholar
  36. 36.
    Pries R, Witrkopf N, Trenkle T, Nitsch SM, Wollenberg B (2008) Potential stem cell marker CD44 is constitutively expressed in permanent cell lines of head and neck cancer. In Vivo (Athens, Greece) 22(1):89–92Google Scholar
  37. 37.
    Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 104(3):973–978PubMedCrossRefGoogle Scholar
  38. 38.
    Rankin EB, Fuh KC, Taylor TE, Krieg AJ, Musser M, Yuan J, Wei K, Kuo CJ, Longacre TA, Giaccia AJ (2010) AXL is an essential factor and therapeutic target for metastatic ovarian cancer. Cancer Res 70(19):7570–7579PubMedCrossRefGoogle Scholar
  39. 39.
    Rody A, Holtrich U, Gaetje R, Gehrmann M, Engels K, von Minckwitz G, Loibl S, Diallo-Danebrock R, Ruckhäberle E, Metzler D, Ahr A, Solbach C, Karn T, Kaufmann M (2007) Poor outcome in estrogen receptor-positive breast cancers predicted by loss of plexin B1. Clin Cancer Res 13(4):1115–1122PubMedCrossRefGoogle Scholar
  40. 40.
    Rody A, Karn T, Holtrich U, Kaufmann M (2008) “Stem cell like” breast cancers-a model for the identification of new prognostic/predictive markers in endocrine responsive breast cancer exemplified by Plexin B1. Eur J Obstet Gynecol Reprod Biol 139(1):11–15PubMedCrossRefGoogle Scholar
  41. 41.
    Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29(34):4741–4751PubMedCrossRefGoogle Scholar
  42. 42.
    Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks P (2004) Identification of human brain tumour initiating cells. Nature 432(7015):396–401PubMedCrossRefGoogle Scholar
  43. 43.
    Sugiura T, Miyamoto K (2008) Characterization of TRIM31, upregulated in gastric adenocarcinoma, as a novel RBCC protein. J Cell Biochem 105(4):1081–1091PubMedCrossRefGoogle Scholar
  44. 44.
    Takaishi S, Okumura T, Tu S, Wang SS, Shibata W, Vigneshwaran R, Gordon SA, Shimada Y, Wang TC (2009) Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells 27(5):1006–1020PubMedCrossRefGoogle Scholar
  45. 45.
    Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol 7(2):131–142PubMedCrossRefGoogle Scholar
  46. 46.
    Tian K, Jurukovski V, Wang XP, Kaplan MH, Xu H (2005) Epigenetic regulation of WTH3 in primary and cultured drug-resistant breast cancer cells. Cancer Res 65(21):10024–10031PubMedCrossRefGoogle Scholar
  47. 47.
    Tian K, Wang Y, Huang Y, Sun B, Li Y, Xu H (2008) Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression. BMC Cancer 8:327PubMedCrossRefGoogle Scholar
  48. 48.
    Tusher V, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to transcriptional responses to ionizing radiation. Proc Natl Acad Sci USA 98(9):5116–5121PubMedCrossRefGoogle Scholar
  49. 49.
    von Schlippe M, Marshall JF, Perry P, Stone M, Zhu AJ, Hart IR (2000) Functional interaction between E-cadherin and alphav-containing integrins in carcinoma cells. J Cell Sci 113(Pt 3):425–437Google Scholar
  50. 50.
    Wang A, Zeng R, Huang H (2008) Retinoic acid and sodium butyrate as cell cycle regulators in the treatment of oral squamous carcinoma cells. Oncol Res 17(4):175–182PubMedCrossRefGoogle Scholar
  51. 51.
    Wang L, Zhou X, Zhou T, Ma D, Chen S, Zhi X, Yin L, Shao Z, Ou Z, Zhou P (2008) Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells. J Cancer Res Clin Oncol 134(3):365–372PubMedCrossRefGoogle Scholar
  52. 52.
    Ye X, Li Y, Stawicki S, Couto S, Eastham-Anderson J, Kallop D, Weimer R, Wu Y, Pei L (2010) An anti-Axl monoclonal antibody attenuates xenograft tumor growth and enhances the effect of multiple anticancer therapies. Oncogene 29(38):5254–5264PubMedCrossRefGoogle Scholar
  53. 53.
    Zavadil J (2010) A spotlight on regulatory networks connecting EMT and cancer stem cells. Cell Cycle 9(15):2927PubMedCrossRefGoogle Scholar
  54. 54.
    Zhang P, Zhang Y, Mao L, Zhang Z, Chen W (2009) Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes. Cancer Lett 277(2):227–234PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Songmei Geng
    • 1
  • Yuanyuan Guo
    • 1
  • Qianqian Wang
    • 1
  • Lan Li
    • 1
  • Jianli Wang
    • 2
  1. 1.Department of Dermatology, Northwest HospitalXi’an Jiaotong UniversityXi’anChina
  2. 2.Department of Hematology, Northwest HospitalXi’an Jiaotong UniversityXi’anChina

Personalised recommendations