Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma


It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.


Vyšlo v časopise: Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma. PLoS Pathog 6(6): e32767. doi:10.1371/journal.ppat.1000940
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000940

Souhrn

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.


Zdroje

1. SpanoJP

BussonP

AtlanD

BourhisJ

PignonJP

2003 Nasopharyngeal carcinomas: an update. Eur J Cancer 39 2121 2135

2. OngYK

HengDM

ChungB

LeongSS

WeeJ

2003 Design of a prognostic index score for metastatic nasopharyngeal carcinoma. Eur J Cancer 39 1535 1541

3. LeeAW

PoonYF

FooW

LawSC

CheungFK

1992 Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985: overall survival and patterns of failure. Int J Radiat Oncol Biol Phys 23 261 270

4. ThieryJP

SleemanJP

2006 Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7 131 142

5. ManiSA

GuoW

LiaoMJ

EatonEN

AyyananA

2008 The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133 704 715

6. RobinsonSN

SeinaSM

GohrJC

KuszynskiCA

SharpJG

2005 Evidence for a qualitative hierarchy within the Hoechst-33342 ‘side population’ (SP) of murine bone marrow cells. Bone Marrow Transplant 35 807 818

7. ZhouS

SchuetzJD

BuntingKD

ColapietroAM

SampathJ

2001 The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7 1028 1034

8. ZhouS

MorrisJJ

BarnesY

LanL

SchuetzJD

2002 Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 99 12339 12344

9. WangJ

GuoLP

ChenLZ

ZengYX

LuSH

2007 Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res 67 3716 3724

10. YoungLS

RickinsonAB

2004 Epstein-Barr virus: 40 years on. Nat Rev Cancer 4 757 768

11. YoungLS

MurrayPG

2003 Epstein-Barr virus and oncogenesis: from latent genes to tumours. Oncogene 22 5108 5121

12. LoKW

ToKF

HuangDP

2004 Focus on nasopharyngeal carcinoma. Cancer Cell 5 423 428

13. Andersson-AnvretM

ForsbyN

KleinG

HenleW

BiorklundA

1979 Relationship between the Epstein-Barr virus genome and nasopharyngeal carcinoma in Caucasian patients. Int J Cancer 23 762 767

14. YatesJL

WarrenN

SugdenB

1985 Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313 812 815

15. Thorley-LawsonDA

2001 Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol 1 75 82

16. HungSC

KangMS

KieffE

2001 Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1. Proc Natl Acad Sci U S A 98 1865 1870

17. NiedobitekG

YoungLS

SamCK

BrooksL

PrasadU

1992 Expression of Epstein-Barr virus genes and of lymphocyte activation molecules in undifferentiated nasopharyngeal carcinomas. Am J Pathol 140 879 887

18. YoungLS

DawsonCW

ClarkD

RupaniH

BussonP

1988 Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol 69 (Pt5) 1051 1065

19. HeussingerN

ButtnerM

OttG

BrachtelE

PilchBZ

2004 Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in EBV-associated nasopharyngeal carcinoma. J Pathol 203 696 699

20. BrooksL

YaoQY

RickinsonAB

YoungLS

1992 Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 66 2689 2697

21. BussonP

McCoyR

SadlerR

GilliganK

TurszT

1992 Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma. J Virol 66 3257 3262

22. PegtelDM

SubramanianA

SheenTS

TsaiCH

GolubTR

2005 Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis. J Virol 79 15430 15442

23. CaldwellRG

WilsonJB

AndersonSJ

LongneckerR

1998 Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9 405 411

24. ChenSY

LuJ

ShihYC

TsaiCH

2002 Epstein-Barr virus latent membrane protein 2A regulates c-Jun protein through extracellular signal-regulated kinase. J Virol 76 9556 9561

25. FukudaM

LongneckerR

2007 Epstein-Barr virus latent membrane protein 2A mediates transformation through constitutive activation of the Ras/PI3-K/Akt Pathway. J Virol 81 9299 9306

26. ScholleF

BendtKM

Raab-TraubN

2000 Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt. J Virol 74 10681 10689

27. StewartS

DawsonCW

TakadaK

CurnowJ

MoodyCA

2004 Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway. Proc Natl Acad Sci U S A 101 15730 15735

28. SwartR

RufIK

SampleJ

LongneckerR

2000 Latent membrane protein 2A-mediated effects on the phosphatidylinositol 3-Kinase/Akt pathway. J Virol 74 10838 10845

29. MorrisonJA

KlingelhutzAJ

Raab-TraubN

2003 Epstein-Barr virus latent membrane protein 2A activates beta-catenin signaling in epithelial cells. J Virol 77 12276 12284

30. LuJ

LinWH

ChenSY

LongneckerR

TsaiSC

2006 Syk tyrosine kinase mediates Epstein-Barr virus latent membrane protein 2A-induced cell migration in epithelial cells. J Biol Chem 281 8806 8814

31. HinoR

UozakiH

MurakamiN

UshikuT

ShinozakiA

2009 Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma. Cancer Res 69 2766 2774

32. LiuM

JuX

WillmarthNE

CasimiroMC

OjeifoJ

2009 Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo. Am J Pathol 174 1910 1920

33. PalingNR

WheadonH

BoneHK

WelhamMJ

2004 Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem 279 48063 48070

34. JirmanovaL

AfanassieffM

Gobert-GosseS

MarkossianS

SavatierP

2002 Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells. Oncogene 21 5515 5528

35. AllenMD

YoungLS

DawsonCW

2005 The Epstein-Barr virus-encoded LMP2A and LMP2B proteins promote epithelial cell spreading and motility. J Virol 79 1789 1802

36. DontuG

AbdallahWM

FoleyJM

JacksonKW

ClarkeMF

2003 In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17 1253 1270

37. BrazilDP

HemmingsBA

2001 Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci 26 657 664

38. SpeckP

KlineKA

ChereshP

LongneckerR

1999 Epstein-Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus. J Gen Virol 80 (Pt8) 2193 2203

39. LongneckerR

MillerCL

MiaoXQ

MarchiniA

KieffE

1992 The only domain which distinguishes Epstein-Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential. J Virol 66 6461 6469

40. LongneckerR

MillerCL

MiaoXQ

TomkinsonB

KieffE

1993 The last seven transmembrane and carboxy-terminal cytoplasmic domains of Epstein-Barr virus latent membrane protein 2 (LMP2) are dispensable for lymphocyte infection and growth transformation in vitro. J Virol 67 2006 2013

41. MillerCL

BurkhardtAL

LeeJH

StealeyB

LongneckerR

1995 Integral membrane protein 2 of Epstein-Barr virus regulates reactivation from latency through dominant negative effects on protein-tyrosine kinases. Immunity 2 155 166

42. FukudaM

LongneckerR

2005 Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation. J Virol 79 8655 8660

43. PortisT

LongneckerR

2003 Epstein-Barr virus LMP2A interferes with global transcription factor regulation when expressed during B-lymphocyte development. J Virol 77 105 114

44. MillerCL

LeeJH

KieffE

LongneckerR

1994 An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking. Proc Natl Acad Sci U S A 91 772 776

45. PortisT

DyckP

LongneckerR

2003 Epstein-Barr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma. Blood 102 4166 4178

46. PortisT

LongneckerR

2004 Epstein-Barr virus (EBV) LMP2A mediates B-lymphocyte survival through constitutive activation of the Ras/PI3K/Akt pathway. Oncogene 23 8619 8628

47. HammerschmidtW

SugdenB

2004 Epstein-Barr virus sustains Burkitt's lymphomas and Hodgkin's disease. Trends Mol Med 10 331 336

48. ThieryJP

2003 Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15 740 746

49. BrabletzT

JungA

SpadernaS

HlubekF

KirchnerT

2005 Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression. Nat Rev Cancer 5 744 749

50. Al-HajjM

BeckerMW

WichaM

WeissmanI

ClarkeMF

2004 Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 14 43 47

51. DickJE

2003 Breast cancer stem cells revealed. Proc Natl Acad Sci U S A 100 3547 3549

52. WangJC

DickJE

2005 Cancer stem cells: lessons from leukemia. Trends Cell Biol 15 494 501

53. ReyaT

MorrisonSJ

ClarkeMF

WeissmanIL

2001 Stem cells, cancer, and cancer stem cells. Nature 414 105 111

54. BoyerLA

LeeTI

ColeMF

JohnstoneSE

LevineSS

2005 Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122 947 956

55. RoddaDJ

ChewJL

LimLH

LohYH

WangB

2005 Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280 24731 24737

56. ChewJL

LohYH

ZhangW

ChenX

TamWL

2005 Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 25 6031 6046

57. LohYH

WuQ

ChewJL

VegaVB

ZhangW

2006 The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38 431 440

58. ChuaHL

Bhat-NakshatriP

ClareSE

MorimiyaA

BadveS

2007 NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 26 711 724

59. MinC

EddySF

SherrDH

SonensheinGE

2008 NF-kappaB and epithelial to mesenchymal transition of cancer. J Cell Biochem 104 733 744

60. KongD

WangZ

SarkarSH

LiY

BanerjeeS

2008 Platelet-derived growth factor-D overexpression contributes to epithelial-mesenchymal transition of PC3 prostate cancer cells. Stem Cells 26 1425 1435

61. LeeMY

LimHW

LeeSH

HanHJ

2009 Smad, PI3K/Akt, and Wnt-Dependent Signaling Pathways Are Involved in BMP-4-Induced ESC Self-Renewal. Stem Cells 27 1858 1868

62. SongLB

LiJ

LiaoWT

FengY

YuCP

2009 The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells. J Clin Invest 119 3626 3636

63. HorikawaT

YangJ

KondoS

YoshizakiT

JoabI

2007 Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma. Cancer Res 67 1970 1978

64. FarwellDG

SheraKA

KoopJI

BonnetGA

MatthewsCP

2000 Genetic and epigenetic changes in human epithelial cells immortalized by telomerase. Am J Pathol 156 1537 1547

65. ShackelfordJ

MaierC

PaganoJS

2003 Epstein-Barr virus activates beta-catenin in type III latently infected B lymphocyte lines: association with deubiquitinating enzymes. Proc Natl Acad Sci U S A 100 15572 15576

66. DimriGP

ItahanaK

AcostaM

CampisiJ

2000 Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor. Mol Cell Biol 20 273 285

67. SongLB

ZengMS

LiaoWT

ZhangL

MoHY

2006 Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells. Cancer Res 66 6225 6232

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