Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine

BMC Cancer

Anke Van den broeck¹  Lies Gremeaux¹  Baki Topal²  Hugo Vankelecom¹ 

[1] Laboratory of Tissue Plasticity, Research Unit of Embryo and Stem Cells, Department of Development & Regeneration, University of Leuven (KU Leuven), Leuven, Belgium

[2] Department of Abdominal Surgery, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium

关键词: Side population;    Chemoresistance;    Pancreatic cancer;   

Others  :  1080254
DOI  :  10.1186/1471-2407-12-354

 received in 2012-03-10, accepted in 2012-07-30,  发布年份 2012

【 摘 要 】

Background

Therapy resistance remains one of the major challenges to improve the prognosis of patients with pancreatic cancer. Chemoresistant cells, which potentially also display cancer stem cell (CSC) characteristics, can be isolated using the side population (SP) technique. Our aim was to search for a SP in human pancreatic ductal adenocarcinoma (PDAC) and to examine its chemoresistance and CSC(−like) phenotype.

Methods

Human PDAC samples were expanded in immunodeficient mice and first-generation xenografts analyzed for the presence of a Hoechst dye-effluxing SP using flow cytometry (FACS). To investigate chemoresistance of the SP, mice bearing PDAC xenografts were treated with gemcitabine and SP proportion determined. In addition, the SP and the main tumour cell population (MP) were sorted by FACS for RNA extraction to profile gene expression, and for culturing under sphere-forming conditions.

Results

A SP was identified in all PDAC samples, analyzed. This SP was more resistant to gemcitabine than the other tumour cells as examined in vivo. Whole-genome expression profiling of the SP revealed upregulation of genes related to therapy resistance, apoptotic regulation and epithelial-mesenchymal transition. In addition, the SP displayed higher tumourigenic (CSC) activity than the MP as analyzed in vitro by sphere-forming capacity.

Conclusion

We identified a SP in human PDAC and uncovered a chemoresistant and CSC-associated phenotype. This SP may represent a new therapeutic target in pancreatic cancer.

Trial registration

Clinicaltrials.gov NCT00936104

【 授权许可】

   
2012 Van den broeck et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics, 2008. CA Cancer J Clin 2008, 58(2):71-96.
• [2]Van den Broeck A, Sergeant G, Ectors N, Van Steenbergen W, Aerts R, Topal B: Patterns of recurrence after curative resection of pancreatic ductal adenocarcinoma. Eur J Surg Oncol 2009, 35(6):600-604.
• [3]Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D, Frese KK, Denicola G, Feig C, Combs C, Winter SP, Ireland-Zecchini H, Reichelt S, Howat WJ, Chang A, Dhara M, Wang L, Rückert F, Grützmann R, Pilarsky C, Izeradjene K, Hingorani SR, Huang P, Davies SE, Plunkett W, Egorin M, et al.: Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009, 324(5933):1457-1461.
• [4]Olson P, Hanahan D: Cancer. Breaching the cancer fortress. Science 2009, 324(5933):1400-1401.
• [5]Goodell MA: Stem cell identification and sorting using the Hoechst 33342 side population (SP). Curr Protoc Cytom 2005, suppl 9:18.1-18.11.
• [6]Yao J, Cai HH, Wei JS, An Y, Ji ZL, Lu ZP, Wu JL, Chen P, Jiang KR, Dai CC, Qian ZY, Xu ZK, Miao Y: Side population in the pancreatic cancer cell lines SW1990 and CFPAC-1 is enriched with cancer stem-like cells. Oncol Rep 2010, 23(5):1375-1382.
• [7]Zhang SN, Huang FT, Huang YJ, Zhong W, Yu Z: Characterization of a cancer stem cell-like side population derived from human pancreatic adenocarcinoma cells. Tumori 2010, 96(6):985-992.
• [8]Zhou J, Wang CY, Liu T, Wu B, Zhou F, Xiong JX, Wu HS, Tao J, Zhao G, Yang M, Gou SM: Persistence of side population cells with high drug efflux capacity in pancreatic cancer. World J Gastroenterol 2008, 14(6):925-930.
• [9]Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF, Mori M: Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 2006, 24(3):506-513.
• [10]Wu C, Alman BA: Side population cells in human cancers. Cancer Lett 2008, 268(1):1-9.
• [11]Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM: Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006, 66(19):9339-9344.
• [12]Sergeant G, Vankelecom H, Gremeaux L, Topal B: Role of cancer stem cells in pancreatic ductal adenocarcinoma. Nat Rev Clin Oncol 2009, 6(10):580-586.
• [13]Boman BM, Wicha MS: Cancer stem cells: a step toward the cure. J Clin Oncol 2008, 26(17):2795-2799.
• [14]Eaves CJ: Cancer stem cells: Here, there, everywhere? Nature 2008, 456(7222):581-582.
• [15]Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, Metzeler KH, Poeppl A, Ling V, Beyene J, Canty AJ, Danska JS, Bohlander SK, Buske C, Minden MD, Golub TR, Jurisica I, Ebert BL, Dick JE: Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011, 17(9):1086-1093.
• [16]Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM: Identification of pancreatic cancer stem cells. Cancer Res 2007, 67(3):1030-1037.
• [17]Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, Bruns CJ, Heeschen C: Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007, 1(3):313-323.
• [18]Tomayko MM, Reynolds CP: Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989, 24(3):148-154.
• [19]Baugh LR, Hill AA, Brown EL, Hunter CP: Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 2001, 29(5):E29.
• [20]Chen J, Gremeaux L, Fu Q, Liekens D, Van Laere S, Vankelecom H: Pituitary progenitor cells tracked down by side population dissection. Stem Cells 2009, 27(5):1182-1195.
• [21]Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004, 3(1):1544-6115.
• [22]Gou S, Liu T, Wang C, Yin T, Li K, Yang M, Zhou J: Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties. Pancreas 2007, 34(4):429-435.
• [23]Bleau AM, Hambardzumyan D, Ozawa T, Fomchenko EI, Huse JT, Brennan CW, Holland EC: PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 2009, 4(3):226-235.
• [24]Kabashima A, Higuchi H, Takaishi H, Matsuzaki Y, Suzuki S, Izumiya M, Iizuka H, Sakai G, Hozawa S, Azuma T, Hibi T: Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer 2009, 124(12):2771-2779.
• [25]Wang YH, Li F, Luo B, Wang XH, Sun HC, Liu S, Cui YQ, Xu XX: A side population of cells from a human pancreatic carcinoma cell line harbors cancer stem cell characteristics. Neoplasma 2009, 56(5):371-378.
• [26]Dittmer J: The biology of the Ets1 proto-oncogene. Mol Cancer 2003, 2:29. BioMed Central Full Text
• [27]Khanna A, Mahalingam K, Chakrabarti D, Periyasamy G: Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells. Cell Mol Biol Lett 2011, 16(1):101-113.
• [28]Zhang M, Ma Q, Hu H, Zhang D, Li J, Ma G, Bhat K, Wu E: Stem cell factor/c-kit signaling enhances invasion of pancreatic cancer cells via HIF-1alpha under normoxic condition. Cancer Lett 2011, 303(2):108-117.
• [29]Yasuda A, Sawai H, Takahashi H, Ochi N, Matsuo Y, Funahashi H, Sato M, Okada Y, Takeyama H, Manabe T: The stem cell factor/c-kit receptor pathway enhances proliferation and invasion of pancreatic cancer cells. Mol Cancer 2006, 5:46. BioMed Central Full Text
• [30]Cobaleda C, Perez-Caro M, Vicente-Duenas C, Sanchez-Garcia I: Function of the zinc-finger transcription factor SNAI2 in cancer and development. Annu Rev Genet 2007, 41:41-61.
• [31]Nishioka R, Itoh S, Gui T, Gai Z, Oikawa K, Kawai M, Tani M, Yamaue H, Muragaki Y: SNAIL induces epithelial-to-mesenchymal transition in a human pancreatic cancer cell line (BxPC3) and promotes distant metastasis and invasiveness in vivo. Exp Mol Pathol 2010, 89(2):149-157.
• [32]Hotz B, Arndt M, Dullat S, Bhargava S, Buhr HJ, Hotz HG: Epithelial to mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer. Clin Cancer Res 2007, 13(16):4769-4776.
• [33]Zhang K, Chen D, Jiao X, Zhang S, Liu X, Cao J, Wu L, Wang D: Slug enhances invasion ability of pancreatic cancer cells through upregulation of matrix metalloproteinase-9 and actin cytoskeleton remodeling. Lab Invest 2011, 91(3):426-438.
• [34]Jesse S, Koenig A, Ellenrieder V, Menke A: Lef-1 isoforms regulate different target genes and reduce cellular adhesion. Int J Cancer 2010, 126(5):1109-1120.
• [35]Ruckert F, Samm N, Lehner AK, Saeger HD, Grutzmann R, Pilarsky C: Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis. BMC Cancer 2010, 10:379. BioMed Central Full Text
• [36]Bold RJ, Virudachalam S, McConkey DJ: BCL2 expression correlates with metastatic potential in pancreatic cancer cell lines. Cancer 2001, 92(5):1122-1129.
• [37]Ohta T, Elnemr A, Kitagawa H, Kayahara M, Takamura H, Fujimura T, Nishimura G, Shimizu K, Yi SQ, Miwa K: Fas ligand expression in human pancreatic cancer. Oncol Rep 2004, 12(4):749-754.
• [38]Chen M, Xue X, Wang F, An Y, Tang D, Xu Y, Wang H, Yuan Z, Gao W, Wei J, Zhang J, Miao Y: Expression and promoter methylation analysis of ATP-binding cassette. Oncol Rep 2012, 27(1):265-269.
• [39]Chen KG, Valencia JC, Gillet JP, Hearing VJ, Gottesman MM: Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma. Pigment Cell Melanoma Res 2009, 22(6):740-749.
• [40]Barrett LE, Granot Z, Coker C, Iavarone A, Hambardzumyan D, Holland EC, Nam HS, Benezra R: Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma. Cancer Cell 2012, 21(1):11-24.
• [41]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: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133(4):704-715.
• [42]Ma Y, Liang D, Liu J, Axcrona K, Kvalheim G, Giercksky KE, Nesland JM, Suo Z: Synergistic effect of SCF and G-CSF on stem-like properties in prostate cancer cell lines. Tumour Biol 2012, 33(4):967-978.
• [43]Levina V, Marrangoni A, Wang T, Parikh S, Su Y, Herberman R, Lokshin A, Gorelik E: Elimination of human lung cancer stem cells through targeting of the stem cell factor-c-kit autocrine signaling loop. Cancer Res 2010, 70(1):338-346.
• [44]Simeone DM: Pancreatic cancer stem cells: implications for the treatment of pancreatic cancer. Clin Cancer Res 2008, 14(18):5646-5648.