EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells

Journal of Experimental & Clinical Cancer Research

Massimo Cristofanilli¹  Rugang Zhang²  Katherine R Alpaugh¹  Sandra V Fernandez¹  Hua Li²  Zhaomei Mu¹ 

[1] Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA;Wistar Institute, Philadelphia, PA 19104, USA

关键词: Tumor spheroid formation;    Cancer stem cell;    EZH2;    Inflammatory breast cancer;   

Others  :  815101
DOI  :  10.1186/1756-9966-32-70

 received in 2013-08-28, accepted in 2013-09-24,  发布年份 2013

【 摘 要 】

Introduction

Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.

Methods

SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice.

Results

The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo.

Conclusions

Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.

【 授权许可】

   
2013 Mu et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Hance KW, Anderson WF, Devesa SS, Young HA, Levine PH: Trends in inflammatory breast carcinoma incidence and survival: the surveillance, epidemiology, and end results program at the National Cancer Institute. J Natl Cancer Inst 2005, 97(13):966-975.
• [2]Dawood S, Merajver SD, Viens P, Vermeulen PB, Swain SM, Buchholz TA, Dirix LY, Levine PH, Lucci A, Krishnamurthy S, et al.: International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment. Ann Oncol 2011, 22(3):515-523.
• [3]Jaiyesimi IA, Buzdar AU, Hortobagyi G: Inflammatory breast cancer: a review. J Clin Oncol 1992, 10(6):1014-1024.
• [4]Cristofanilli M, Valero V, Buzdar AU, Kau SW, Broglio KR, Gonzalez-Angulo AM, Sneige N, Islam R, Ueno NT, Buchholz TA, et al.: Inflammatory breast cancer (IBC) and patterns of recurrence: understanding the biology of a unique disease. Cancer 2007, 110(7):1436-1444.
• [5]Alpaugh ML, Tomlinson JS, Kasraeian S, Barsky SH: Cooperative role of E-cadherin and sialyl-Lewis X/A-deficient MUC1 in the passive dissemination of tumor emboli in inflammatory breast carcinoma. Oncogene 2002, 21(22):3631-3643.
• [6]Dawood S, Cristofanilli M: Inflammatory breast cancer: what progress have we made? Oncology 2011, 25(3):264-270.
• [7]Zhang D, LaFortune TA, Krishnamurthy S, Esteva FJ, Cristofanilli M, Liu P, Lucci A, Singh B, Hung MC, Hortobagyi GN, et al.: Epidermal growth factor receptor tyrosine kinase inhibitor reverses mesenchymal to epithelial phenotype and inhibits metastasis in inflammatory breast cancer. Clin Cancer Res 2009, 15(21):6639-6648.
• [8]Cabioglu N, Gong Y, Islam R, Broglio KR, Sneige N, Sahin A, Gonzalez-Angulo AM, Morandi P, Bucana C, Hortobagyi GN, et al.: Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer. Ann Oncol 2007, 18(6):1021-1029.
• [9]Yamauchi H, Cristofanilli M, Nakamura S, Hortobagyi GN, Ueno NT: Molecular targets for treatment of inflammatory breast cancer. Nat Rev Clin Oncol 2009, 6(7):387-394.
• [10]Van Laere SJ, Van den Eynden GG, Van der Auwera I, Vandenberghe M, van Dam P, Van Marck EA, van Golen KL, Vermeulen PB, Dirix LY: Identification of cell-of-origin breast tumor subtypes in inflammatory breast cancer by gene expression profiling. Breast Cancer Res Treat 2006, 95(3):243-255.
• [11]Cao R, Zhang Y: SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 2004, 15(1):57-67.
• [12]Chang CJ, Hung MC: The role of EZH2 in tumour progression. Br J Cancer 2012, 106(2):243-247.
• [13]Sauvageau M, Sauvageau G: Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell 2010, 7(3):299-313.
• [14]Collett K, Eide GE, Arnes J, Stefansson IM, Eide J, Braaten A, Aas T, Otte AP, Akslen LA: Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer. Clin Cancer Res 2006, 12(4):1168-1174.
• [15]Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF, et al.: EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci U S A 2003, 100(20):11606-11611.
• [16]Raaphorst FM, Meijer CJ, Fieret E, Blokzijl T, Mommers E, Buerger H, Packeisen J, Sewalt RA, Otte AP, van Diest PJ: Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene. Neoplasia 2003, 5(6):481-488.
• [17]Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume O, Haukaas SA, Salvesen HB, Otte AP, Akslen LA: EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 2006, 24(2):268-273.
• [18]Ding L, Kleer CG: Enhancer of Zeste 2 as a marker of preneoplastic progression in the breast. Cancer Res 2006, 66(19):9352-9355.
• [19]Gonzalez ME, Li X, Toy K, DuPrie M, Ventura AC, Banerjee M, Ljungman M, Merajver SD, Kleer CG: Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1. Oncogene 2009, 28(6):843-853.
• [20]Pietersen AM, Horlings HM, Hauptmann M, Langerod A, Ajouaou A, Cornelissen-Steijger P, Wessels LF, Jonkers J, van de Vijver MJ, van Lohuizen M: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer. Breast Cancer Res 2008, 10(6):R109.
• [21]Gong Y, Huo L, Liu P, Sneige N, Sun X, Ueno NT, Lucci A, Buchholz TA, Valero V, Cristofanilli M: Polycomb group protein EZH2 is frequently expressed in inflammatory breast cancer and is predictive of worse clinical outcome. Cancer 2011, 117(24):5476-5484.
• [22]Fernandez SV, Robertson FM, Pei J, Aburto-Chumpitaz L, Mu Z, Chu K, Alpaugh RK, Huang Y, Cao Y, Ye Z, et al.: Inflammatory breast cancer (IBC): clues for targeted therapies. Breast Cancer Res Treat 2013, 140(1):23-33.
• [23]Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100(7):3983-3988.
• [24]Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS: Stem cells in normal breast development and breast cancer. Cell Prolif 2003, 36(Suppl 1):59-72.
• [25]Phillips TM, McBride WH, Pajonk F: The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 2006, 98(24):1777-1785.
• [26]Shafee N, Smith CR, Wei S, Kim Y, Mills GB, Hortobagyi GN, Stanbridge EJ, Lee EY: Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors. Cancer Res 2008, 68(9):3243-3250.
• [27]Korkaya H, Paulson A, Charafe-Jauffret E, Ginestier C, Brown M, Dutcher J, Clouthier SG, Wicha MS: Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling. PLoS Biol 2009, 7(6):e1000121.
• [28]Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, Hsu JM, Hortobagyi GN, Hung MC: EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-beta-catenin signaling. Cancer Cell 2011, 19(1):86-100.
• [29]Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, Mongan NP: Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells Dev 2009, 18(7):1093-1108.
• [30]Bellacosa A, Godwin AK, Peri S, Devarajan K, Caretti E, Vanderveer L, Bove B, Slater C, Zhou Y, Daly M, et al.: Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations. Cancer Prev Res 2010, 3(1):48-61.
• [31]Li H, Cai Q, Godwin AK, Zhang R: Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells. Mol Cancer Res 2010, 8(12):1610-1618.
• [32]Mu Z, Hachem P, Hensley H, Stoyanova R, Kwon HW, Hanlon AL, Agrawal S, Pollack A: Antisense MDM2 enhances the response of androgen insensitive human prostate cancer cells to androgen deprivation in vitro and in vivo. Prostate 2008, 68(6):599-609.
• [33]Van Laere SJ, Ueno NT, Finetti P, Vermeulen PB, Lucci A, Robertson FM, Marsan M, Iwamoto T, Krishnamurthy S, Masuda H, et al.: Uncovering the molecular secrets of Inflammatory Breast Cancer biology: An integrated analysis of three distinct Affymetrix gene expression data sets. Clin Cancer Res 2013, 19(17):4685-4696.
• [34]Holm K, Grabau D, Lovgren K, Aradottir S, Gruvberger-Saal S, Howlin J, Saal LH, Ethier SP, Bendahl PO, Stal O, et al.: Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes. Mol Oncol 2012, 6(5):494-506.
• [35]Alpaugh ML, Tomlinson JS, Shao ZM, Barsky SH: A novel human xenograft model of inflammatory breast cancer. Cancer Res 1999, 59(20):5079-5084.
• [36]Shirakawa K, Tsuda H, Heike Y, Kato K, Asada R, Inomata M, Sasaki H, Kasumi F, Yoshimoto M, Iwanaga T, et al.: Absence of endothelial cells, central necrosis, and fibrosis are associated with aggressive inflammatory breast cancer. Cancer Res 2001, 61(2):445-451.
• [37]Tomao F, Papa A, Rossi L, Strudel M, Vici P, Lo Russo G, Tomao S: Emerging role of cancer stem cells in the biology and treatment of ovarian cancer: basic knowledge and therapeutic possibilities for an innovative approach. J Exp Clin Cancer Res 2013, 32:48. doi: 10.1186/1756-9966 BioMed Central Full Text
• [38]Xu W, Lin H, Zhang Y, Chen X, Hua B, Hou W, Qi X, Pei Y, Zhu X, Zhao Z, Yang L: Compound Kushen Injection suppresses human breast cancer stem-like cells by down-regulating the canonical Wnt/β-catenin pathway. J Exp Clin Cancer Res 2011, 30:103. 10.1186/1756-9966 BioMed Central Full Text
• [39]Lu C, Han HD, Mangala LS, Ali-Fehmi R, Newton CS, Ozbun L, Armaiz-Pena GN, Hu W, Stone RL, Munkarah A, et al.: Regulation of tumor angiogenesis by EZH2. Cancer Cell 2010, 18(2):185-197.
• [40]Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, Zeng J, Li M, Fan H, Lin Y, et al.: Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc Natl Acad Sci U S A 2012, 109(52):21360-21365.
• [41]Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, et al.: A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 2012, 8(11):890-896.
• [42]McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Della Pietra A 3rd, Diaz E, et al.: EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 2012, 492(7427):108-112.
• [43]Amatangelo MD, Garipov A, Li H, Conejo-Garcia JR, Speicher DW, Zhang R: Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition. Cell Cycle 2013, 12(13):2113-2119.