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Krüppel-like factor 4 (KLF4) suppresses neuroblastoma cell growth and determines non-tumorigenic lineage differentiation

Oncogene volume 32, pages 4086–4099 (2013)Cite this article

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Abstract

Neuroblastoma (NB) is an embryonal tumor and possesses a unique propensity to exhibit either a spontaneous regression or an unrestrained growth. However, the underlying mechanism for this paradoxical clinical outcome remains largely unclear. Quantitative RT–PCR analysis on 102 primary NB tumors revealed that lower Krüppel-like factor 4 (KLF4) expression is frequently found in the unfavorable NB (Mann–Whitney test, P=0.027). In particular with the high-risk factors such as age of patient >1 year, MYCN amplification and low TRKA expression, the decreased expression of KLF4 was significantly associated with an unfavorable NB outcome. Despite knockdown of KLF4 alone is not sufficient to increase tumorigenicity of NB cells in vivo, stable expression of KLF4 short hairpin RNA in Be(2)-C cells significantly promoted growth of NB cells and inhibited cell differentiation toward fibromuscular lineage. In concordant with these observations, overexpression of KLF4 in SH-SY-5Y cells profoundly suppressed cell proliferation by direct upregulation of cell-cycle inhibitor protein p21WAF1/CIP1, and knocking down p21WAF1/CIP1 could partially rescue the suppressive effect of KLF4. Importantly, KLF4 overexpressing cells have lost their neuroblastic phenotypes, they were epithelial-like, strongly substrate-adherent, expressing smooth muscle marker and became non-tumorigenic, suggesting that KLF4 expression is crucial for lineage determination of NB cells, probably, favoring spontaneous tumor regression. Subsequent global gene expression profiling further revealed that transforming growth factor beta (TGFβ) and cell-cycle pathways are highly dysregulated upon KLF4 overexpression, and myogenic modulators, MEF2A and MYOD1 were found significantly upregulated. Taken together, we have demonstrated that KLF4 contributes to the favorable disease outcome by directly mediating the growth and lineage determination of NB cells.

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References

  1. Breslow N, McCann B . Statistical estimation of prognosis for children with neuroblastoma. Cancer Res 1971; 31: 2098–2103.

    CAS  PubMed  Google Scholar 

  2. Silber JH, Evans AE, Fridman M . Models to predict outcome from childhood neuroblastoma: the role of serum ferritin and tumor histology. Cancer Res 1991; 51: 1426–1433.

    CAS  PubMed  Google Scholar 

  3. Brodeur GM, Pritchard J, Berthold F, Carlsen NL, Castel V, Castelberry RP et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993; 11: 1466–1477.

    Article  CAS  PubMed  Google Scholar 

  4. Hann HW, Evans AE, Siegel SE, Wong KY, Sather H, Dalton A et al. Prognostic importance of serum ferritin in patients with Stages III and IV neuroblastoma: the Childrens Cancer Study Group experience. Cancer Res 1985; 45: 2843–2848.

    CAS  PubMed  Google Scholar 

  5. Look AT, Hayes FA, Shuster JJ, Douglass EC, Castleberry RP, Bowman LC et al. Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study. J Clin Oncol 1991; 9: 581–591.

    Article  CAS  PubMed  Google Scholar 

  6. Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 1985; 313: 1111–1116.

    Article  CAS  PubMed  Google Scholar 

  7. Shimada H, Chatten J, Newton WA, Sachs N, Hamoudi AB, Chiba T et al. Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J Natl Cancer Inst 1984; 73: 405–416.

    Article  CAS  PubMed  Google Scholar 

  8. Shuster JJ, McWilliams NB, Castleberry R, Nitschke R, Smith EI, Altshuler G et al. Serum lactate dehydrogenase in childhood neuroblastoma. A Pediatric Oncology Group recursive partitioning study. Am J Clin Oncol 1992; 15: 295–303.

    Article  CAS  PubMed  Google Scholar 

  9. Nakagawara A, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor AB, Brodeur GM . Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N Engl J Med 1993; 328: 847–854.

    Article  CAS  PubMed  Google Scholar 

  10. Tang XX, Zhao H, Robinson ME, Cohen B, Cnaan A, London W et al. Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma. Proc Natl Acad Sci USA 2000; 97: 10936–10941.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Peterson S, Bogenmann E . The RET and TRKA pathways collaborate to regulate neuroblastoma differentiation. Oncogene 2004; 23: 213–225.

    Article  CAS  PubMed  Google Scholar 

  12. Brodeur GM, Minturn JE, Ho R, Simpson AM, Iyer R, Varela CR et al. Trk receptor expression and inhibition in neuroblastomas. Clin Cancer Res 2009; 15: 3244–3250.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Iraci N, Diolaiti D, Papa A, Porro A, Valli E, Gherardi S et al. A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TRKA and p75NTR promoters and affects neuroblastoma malignancy by inhibiting the cell response to NGF. Cancer Res 2011; 71: 404–412.

    Article  CAS  PubMed  Google Scholar 

  14. Janoueix-Lerosey I, Schleiermacher G, Michels E, Mosseri V, Ribeiro A, Lequin D et al. Overall genomic pattern is a predictor of outcome in neuroblastoma. J Clin Oncol 2009; 27: 1026–1033.

    Article  PubMed  Google Scholar 

  15. Lastowska M, Cullinane C, Variend S, Cotterill S, Bown N, O'Neill S et al. Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors. J Clin Oncol 2001; 19: 3080–3090.

    Article  CAS  PubMed  Google Scholar 

  16. Michels E, Vandesompele J, De Preter K, Hoebeeck J, Vermeulen J, Schramm A et al. ArrayCGH-based classification of neuroblastoma into genomic subgroups. Genes Chromosomes Cancer 2007; 46: 1098–1108.

    Article  CAS  PubMed  Google Scholar 

  17. Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K et al. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer 2007; 46: 936–949.

    Article  CAS  PubMed  Google Scholar 

  18. Ohira M, Oba S, Nakamura Y, Isogai E, Kaneko S, Nakagawa A et al. Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas. Cancer Cell 2005; 7: 337–350.

    Article  CAS  PubMed  Google Scholar 

  19. Tomioka N, Oba S, Ohira M, Misra A, Fridlyand J, Ishii S et al. Novel risk stratification of patients with neuroblastoma by genomic signature, which is independent of molecular signature. Oncogene 2008; 27: 441–449.

    Article  CAS  PubMed  Google Scholar 

  20. Vandesompele J, Baudis M, De Preter K, Van Roy N, Ambros P, Bown N et al. Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma. J Clin Oncol 2005; 23: 2280–2299.

    Article  CAS  PubMed  Google Scholar 

  21. Janoueix-Lerosey I, Schleiermacher G, Delattre O . Molecular pathogenesis of peripheral neuroblastic tumors. Oncogene 2010; 29: 1566–1579.

    Article  CAS  PubMed  Google Scholar 

  22. Biagiotti T, D'Amico M, Marzi I, Di Gennaro P, Arcangeli A, Wanke E et al. Cell renewing in neuroblastoma: electrophysiological and immunocytochemical characterization of stem cells and derivatives. Stem Cells 2006; 24: 443–453.

    Article  PubMed  Google Scholar 

  23. Marzi I, D’Amico M, Biagiotti T, Giunti S, Carbone MV, Fredducci D et al. Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment. Cancer Res 2007; 67: 2402–2407.

    Article  CAS  PubMed  Google Scholar 

  24. Rowland BD . Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 2006; 6: 11–23.

    Article  CAS  PubMed  Google Scholar 

  25. Dang DT, Chen X, Feng J, Torbenson M, Dang LH, Yang VW . Overexpression of Kruppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity. Oncogene 2003; 22: 3424–3430.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res 2005; 65: 2746–2754.

    Article  CAS  PubMed  Google Scholar 

  27. Foster KW, Ren S, Louro ID, Lobo-Ruppert SM, McKie-Bell P, Grizzle W et al. Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ 1999; 10: 423–434.

    CAS  PubMed  Google Scholar 

  28. Foster KW, Liu Z, Nail CD, Li X, Fitzgerald TJ, Bailey SK et al. Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia. Oncogene 2005; 24: 1491–1500.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Foster KW, Frost AR, McKie-Bell P, Lin CY, Engler JA, Grizzle WE et al. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res 2000; 60: 6488–6495.

    CAS  PubMed  Google Scholar 

  30. Yu F, Li J, Chen H, Fu J, Ray S, Huang S et al. Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene 2011; 30: 2161–2172.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Akaogi K, Nakajima Y, Ito I, Kawasaki S, Oie SH, Murayama A et al. KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ERalpha. Oncogene 2009; 28: 2894–2902.

    Article  CAS  PubMed  Google Scholar 

  32. Yori JL, Seachrist DD, Johnson E, Lozada KL, Abdul-Karim FW, Chodosh LA et al. Kruppel-like factor 4 inhibits tumorigenic progression and metastasis in a mouse model of breast cancer. Neoplasia 2011; 13: 601–610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jaubert J, Cheng J, Segre JA . Ectopic expression of kruppel like factor 4 (Klf4) accelerates formation of the epidermal permeability barrier. Development 2003; 130: 2767–2777.

    Article  CAS  PubMed  Google Scholar 

  34. Katz JP, Perreault N, Goldstein BG, Lee CS, Labosky PA, Yang VW et al. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 2002; 129: 2619–2628.

    CAS  PubMed  Google Scholar 

  35. Segre JA, Bauer C, Fuchs E . Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 1999; 22: 356–360.

    Article  CAS  PubMed  Google Scholar 

  36. Feinberg MW, Wara AK, Cao Z, Lebedeva MA, Rosenbauer F, Iwasaki H et al. The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation. EMBO J 2007; 26: 4138–4148.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN et al. miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 2009; 460: 705–710.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Yori JL, Johnson E, Zhou G, Jain MK, Keri RA . Kruppel-like factor 4 inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin gene expression. J Biol Chem 2010; 285: 16854–16863.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen J, Liu J, Yang J, Chen Y, Ni S, Song H et al. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone. Cell Res 2011; 21: 205–212.

    Article  CAS  PubMed  Google Scholar 

  40. Li R, Liang J, Ni S, Zhou T, Qing X, Li H et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 2010; 7: 51–63.

    Article  CAS  PubMed  Google Scholar 

  41. Okita K, Ichisaka T, Yamanaka S . Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448: 313–317.

    Article  CAS  PubMed  Google Scholar 

  42. Ross RA, Spengler BA, Domenech C, Porubcin M, Rettig WJ, Biedler JL . Human neuroblastoma I-type cells are malignant neural crest stem cells. Cell Growth Differ 1995; 6: 449–456.

    CAS  PubMed  Google Scholar 

  43. Walton JD, Kattan DR, Thomas SK, Spengler BA, Guo HF, Biedler JL et al. Characteristics of stem cells from human neuroblastoma cell lines and in tumors. Neoplasia 2004; 6: 838–845.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Acosta S, Lavarino C, Paris R, Garcia I, de Torres C, Rodriguez E et al. Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells. BMC Dev Biol 2009; 9: 12.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Mahller YY, Williams JP, Baird WH, Mitton B, Grossheim J, Saeki Y et al. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus. PLoS One 2009; 4: e4235.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Lutz W, Fulda S, Jeremias I, Debatin KM, Schwab M . MycN IFNgamma cooperate in apoptosis of human neuroblastoma cells. Oncogene 1998; 17: 339–346.

    Article  CAS  PubMed  Google Scholar 

  47. Chen S, Crawford M, Day RM, Briones VR, Leader JE, Jose PA et al. RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation. J Biol Chem 2006; 281: 1765–1770.

    Article  CAS  PubMed  Google Scholar 

  48. McKinsey TA, Olson EN . Toward transcriptional therapies for the failing heart: chemical screens to modulate genes. J Clin Invest 2005; 115: 538–546.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhang CL, McKinsey TA, Chang S, Antos CL, Hill JA, Olson EN . Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell 2002; 110: 479–488.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Iehara T, Hosoi H, Akazawa K, Matsumoto Y, Yamamoto K, Suita S et al. MYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening. Br J Cancer 2006; 94: 1510–1515.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kaneko M, Tsuchida Y, Mugishima H, Ohnuma N, Yamamoto K, Kawa K et al. Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification. J Pediatr Hematol Oncol 2002; 24: 613–621.

    Article  PubMed  Google Scholar 

  52. Islam A, Kageyama H, Takada N, Kawamoto T, Takayasu H, Isogai E et al. High expression of Survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma. Oncogene 2000; 19: 617–623.

    Article  CAS  PubMed  Google Scholar 

  53. Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS . Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res 1978; 38: 3751–3757.

    CAS  PubMed  Google Scholar 

  54. Tweddle DA, Malcolm AJ, Cole M, Pearson AD, Lunec J . p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells. Am J Pathol 2001; 158: 2067–2077.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Keshelava N, Zuo JJ, Chen P, Waidyaratne SN, Luna MC, Gomer CJ et al. Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines. Cancer Res 2001; 61: 6185–6193.

    CAS  PubMed  Google Scholar 

  56. Tweddle DA, Malcolm AJ, Bown N, Pearson AD, Lunec J . Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line. Cancer Res 2001; 61: 8–13.

    CAS  PubMed  Google Scholar 

  57. Beppu K, Nakamura K, Linehan WM, Rapisarda A, Thiele CJ . Topotecan blocks hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression induced by insulin-like growth factor-I in neuroblastoma cells. Cancer Res 2005; 65: 4775–4781.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was funded by seed funding grant for basic research from the University of Hong Kong, General Research Grant HKU773909 from the Hong Kong Research Grants Council and research grant from Hong Kong Children Cancer Foundation to ESWN.

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  1. C K Y Shum and S T Lau: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, University of Hong Kong, Pokfulam, Hong Kong, SAR, China

    C K Y Shum, S T Lau, L L S Tsoi, P K H Tam & E S W Ngan

  2. Department of Pathology, University of Hong Kong, Pokfulam, Hong Kong, SAR, China

    L K Chan & J W P Yam

  3. Chiba Cancer Center, Research Institute, Chiba, Japan

    M Ohira & A Nakagawara

  4. Centre for Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong, SAR, China

    P K H Tam & E S W Ngan

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Shum, C., Lau, S., Tsoi, L. et al. Krüppel-like factor 4 (KLF4) suppresses neuroblastoma cell growth and determines non-tumorigenic lineage differentiation. Oncogene 32, 4086–4099 (2013). https://doi.org/10.1038/onc.2012.437

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