Background Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure. Methods/Findings To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant. Conclusions These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.
References
[1]
Lobo NA, Shimono Y, Qian D, Clarke MF (2007) The Biology of Cancer Stem Cells. Annu Rev Cell Dev Biol.
[2]
Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367: 645–648.
[3]
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100: 3983–3988.
[4]
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, et al. (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63: 5821–5828.
[5]
Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65: 10946–10951.
[6]
O'Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445: 106–110.
[7]
Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, et al. (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24: 506–513.
[8]
Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, et al. (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65: 9328–9337.
[9]
Zhang S, Balch C, Chan MW, Lai HC, Matei D, et al. (2008) Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 68: 4311–4320.
[10]
Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, et al. (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104: 973–978.
[11]
Shoemaker RH (2006) The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 6: 813–823.
[12]
Chabner BA, Roberts TG Jr (2005) Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 5: 65–72.
[13]
Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, et al. (2001) A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 98: 1166–1173.
[14]
Eramo A, Ricci-Vitiani L, Zeuner A, Pallini R, Lotti F, et al. (2006) Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ 13: 1238–1241.
[15]
Ghods AJ, Irvin D, Liu G, Yuan X, Abdulkadir IR, et al. (2007) Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells. Stem Cells 25: 1645–1653.
[16]
Kang MK, Kang SK (2007) Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma. Stem Cells Dev 16: 837–847.
[17]
Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY (2008) CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27: 1749–1758.
[18]
Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, et al. (2008) Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100: 672–679.
[19]
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444: 756–760.
[20]
Baumann M, Krause M, Hill R (2008) Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8: 545–554.
[21]
Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, et al. (2007) WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A 104: 618–623.
[22]
Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, et al. (1992) Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 89: 10578–10582.
[23]
Kondratyev M, Kreso A, Hallett RM, Girgis-Gabardo A, Barcelon ME, et al. (2011) Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer. Oncogene 31: 93–103.
[24]
Brennan KR, Brown AM (2004) Wnt proteins in mammary development and cancer. J Mammary Gland Biol Neoplasia 9: 119–131.
[25]
Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480.
[26]
Howe LR, Brown AM (2004) Wnt signaling and breast cancer. Cancer Biol Ther 3: 36–41.
[27]
Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade OI, et al. (2010) Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol 176: 2911–2920.
[28]
Klarmann GJ, Decker A, Farrar WL (2008) Epigenetic gene silencing in the Wnt pathway in breast cancer. Epigenetics 3: 59–63.
[29]
Lindvall C, Evans NC, Zylstra CR, Li Y, Alexander CM, et al. (2006) The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis. J Biol Chem 281: 35081–35087.
[30]
Liu BY, McDermott SP, Khwaja SS, Alexander CM (2004) The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A 101: 4158–4163.
[31]
Nusse R (2005) Wnt signaling in disease and in development. Cell Res 15: 28–32.
[32]
Suzuki H, Toyota M, Carraway H, Gabrielson E, Ohmura T, et al. (2008) Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer. Br J Cancer 98: 1147–1156.
[33]
Ugolini F, Charafe-Jauffret E, Bardou VJ, Geneix J, Adelaide J, et al. (2001) WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type. Oncogene 20: 5810–5817.
[34]
Wong GT, Gavin BJ, McMahon AP (1994) Differential transformation of mammary epithelial cells by Wnt genes. Mol Cell Biol 14: 6278–6286.
[35]
Chang HY, Nuyten DS, Sneddon JB, Hastie T, Tibshirani R, et al. (2005) Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A 102: 3738–3743.
[36]
Hallett RM, Dvorkin A, Gabardo CM, Hassell JA (2010) An algorithm to discover gene signatures with predictive potential. J Exp Clin Cancer Res 29: 120.
[37]
Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8: 486–498.
[38]
Reynolds BA, Rietze RL (2005) Neural stem cells and neurospheres–re-evaluating the relationship. Nat Methods 2: 333–336.
[39]
Clevers H (2011) The cancer stem cell: premises, promises and challenges. Nat Med 17: 313–319.
[40]
Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, et al. (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148: 3–15.
[41]
Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423: 409–414.
[42]
Binnerts ME, Tomasevic N, Bright JM, Leung J, Ahn VE, et al. (2009) The first propeller domain of LRP6 regulates sensitivity to DKK1. Mol Biol Cell 20: 3552–3560.
[43]
Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, et al. (1998) Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391: 357–362.
[44]
Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, et al. (2003) GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10: 1255–1266.
[45]
Lepourcelet M, Chen YN, France DS, Wang H, Crews P, et al. (2004) Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex. Cancer Cell 5: 91–102.
[46]
Stingl J (2008) Detection and analysis of mammary gland stem cells. J Pathol.
[47]
Bloom HJ, Richardson WW (1957) Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer 11: 359–377.
[48]
Liu JC, Deng T, Lehal RS, Kim J, Zacksenhaus E (2007) Identification of tumorsphere- and tumor-initiating cells in HER2/Neu-induced mammary tumors. Cancer Res 67: 8671–8681.
[49]
Zhang M, Behbod F, Atkinson RL, Landis MD, Kittrell F, et al. (2008) Identification of tumor-initiating cells in a p53-null mouse model of breast cancer. Cancer Res 68: 4674–4682.
[50]
Cho RW, Wang X, Diehn M, Shedden K, Chen GY, et al. (2008) Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors. Stem Cells 26: 364–371.
[51]
Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, et al. (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138: 645–659.
[52]
Guzman ML, Rossi RM, Karnischky L, Li X, Peterson DR, et al. (2005) The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells. Blood 105: 4163–4169.
[53]
Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, et al. (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1: 555–567.
[54]
Lindvall C, Bu W, Williams BO, Li Y (2007) Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Rev 3: 157–168.
[55]
Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255: 1707–1710.
[56]
Pullan Sachs (1996) Epithelial Cell Culture. Cambridge: Cambridge University Press.
[57]
Kurpios NA, MacNeil L, Shepherd TG, Gludish DW, Giacomelli AO, et al. (2009) The Pea3 Ets transcription factor regulates differentiation of multipotent progenitor cells during mammary gland development. Dev Biol 325: 106–121.
[58]
Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, et al. (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17: 1253–1270.
[59]
Stingl J, Eaves CJ, Kuusk U, Emerman JT (1998) Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation 63: 201–213.
