Breast cancer stem cells: potential therapeutic targets.

Introduction

There is now a large body of evidence showing that leukaemias originate from cancer stem cells (CSCs) [1,2]. However, it remains a matter of considerable debate as to whether cancer initiation is secondary to mutations within the normal haematopoietic stem cell (HSC) compartment or in more differentiated progenitors that then acquire stem cell characteristics such as the capacity to self-renew [3]. Many groups have extrapolated the cancer stem cell hypothesis from the haematopoietic system to solid cancers and although the evidence for CSCs in solid cancers is in its infancy compared to the haematopoietic field, the evidence is growing rapidly. In a recent workshop on CSCs, a consensus definition of the CSC was proposed. This states that CSCs are '... those cells within a tumour that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumour. Cancer stem cells can thus only be defined experimentally by their ability to recapitulate the generation of a continuously growing tumour.' [4]. The evidence for the existence of breast CSCs and their regulation by the Notch pathway are reviewed in this article.

Breast CSCs

Al-Hajj et al. were the first to identify a highly tumorigenic subpopulation of human breast cancer cells using a set of cell surface markers identified by fluorescence-activated cell sorting (FACS) [5]. Cells that were CD[44.sup.+], CD[24.sup.low], ES[A.sup.+] and [lineage.sup.-] (cells lacking markers CD2, DC3, CD10, CD16, CD18, CD31, CD64 and CD140b), isolated from one primary breast cancer and eight malignant pleural effusion samples, were able to form heterogeneous tumours containing both the CD[44.sup.+], CD[24.sup.low], ES[A.sup.+] and [lineage.sup.-] tumour initiating cells and also the phenotypically diverse nontumorigenic cells that comprised the bulk of tumours (see stem cell definition above). As few as 1000 CD[44.sup.+]/CD[24.sup.low]/ES[A.sup.+]/[lineage.sup.-] cells implanted directly into immune-deficient mice could form tumours with 100% efficiency, whereas no tumours formed using 800,000 cells from the CD[44.sup.-]/CD[24.sup.low]/ES[A.sup.+] cell population [5]. In a subsequent study using a suspension sphere culture technique that has been used to enrich for normal breast stem cells, three long-term primary cultures from invasive breast cancer specimens were generated [6,7]. The sphere-forming cells were found to have self-renewing capacity, could differentiate into the different breast lineages and over 96-98% had the CD[44.sup.+]/CD[24.sup.-] phenotype. The same group also developed a long-term sphere culture of the MCF-7 breast cancer cell line, termed MCF-S, and CD[44.sup.+]/CD[24.sup.-] cells from the MCF-S or parental MCF7 cells were implanted into the mammary fat pad of immune-deficient mice. The MCF7 cells only gave rise to tumours when at least 1 million were implanted; however, the MCF-S cells gave rise to tumours with smaller numbers of cells ([10.sup.5], [10.sup.4] and [10.sup.3]) with at least 60% efficiency. Thus both the mammosphere culture system and cell surface marker selection enriches for tumour-initiating cells. However, cells with self-renewal capacity only accounted for 0.1-20% of the total cell number in these studies, highlighting the need for additional markers or techniques to refine the isolation of the true CSC.

One such technique, originally pioneered by Goodell et al. for the isolation of HSCs, is the selection of the Hoechst effluxing 'side population' (SP) of cells by FACS [8]. Such an SP has been demonstrated in MCF7 cells (~0.2%) and been found to have a greater tumorigenic capacity than non-SP cells in immune-deficient mice [9]. The MCF7 SP also expressed higher levels of Notch1 and [beta]-catenin mRNA, suggesting that the SP cancer cells have some intrinsic properties of stem cells (see below). Notch signalling has also been implicated in p53-mediated resistance to chemotherapy in MCF-7 cells and Woodward et al. recently showed that irradiation of MCF7 cells induced enrichment of SP progenitors suggesting that this CSC-like population may be resistant to radiotherapy [10,11]. Further support for this theory comes from a study in which MCF7 and MDA-MB-231 breast cancer cells grown as mammospheres had elevated numbers of CD[44.sup.+]/[CD[24.sup.-]/.sup.low] cells, and were more radioresistant than cells grown in monolayer [12]. A comparable but more extensive study in glioblastomas showed not only that the putative CSCs (isolated by virtue of their expression of CD133) were more radioresistant than the CD[133.sup.-] cells, but also that ionising radiation increased the proportion of CD[133.sup.+] cells from glioblastoma specimens [13]. The CD[133.sup.+] population preferentially activated the DNA damage checkpoint response. A specific checkpoint kinase inhibitor reversed this radioresistance and repaired radiation-induced DNA damage more effectively than CD[133.sup.-] cells, suggesting that CSCs could be the source of tumour recurrence in patients after radiation. A specific Chk1 and Chk2 inhibitor used in this study was found to reverse this radioresistance in vitro and in vivo, indicating that targeting DNA damage checkpoints may disrupt this resistance mechanism and improve tumour control with radiation treatment. It remains to be seen whether the mechanism for radioresistance is similar in breast CSCs.

Breast CSCs and the Notch pathway

Many cancers have dysregulated signalling pathways involved in normal mammary stem cell regulation, including WNT [14,15], Hedgehog [16], Notch [17,18], transforming growth factor (TGF)[beta] and epidermal growth factor (EGF) families [6,19,20] and the steroid hormones oestrogen and progesterone [21]. These pathways have been the focus of many studies as potential targets for therapeutic intervention. The Notch signalling pathway has been shown to act as a mediator of cell-cell communication in many tissues and is thought to play a role in stem cell self-renewal as well as cell fate, apoptosis, proliferation and migration [22,23]. Notch signalling in mammals, involves the interaction of Delta/Serrate/Lag-2 (DSL) ligands, of which there are five (Delta-like-1, -3 and -4, and Jagged1 and 2), with one of the four Notch receptors (Notch 1-4) [22]. The extracellular domain (NECD) of the Notch receptor is pulled away from the intracellular domain (NICD) exposing a site at which two proteolytic cleavage events occur. The first, by members of the A disintegrin and metalloproteinases (ADAM) protease family, and the second, by [gamma]-secretase, result in the release of the NICD and its subsequent translocation to the nucleus [15]. The NICD then interacts with members of the CSL (CBF1, suppressor of hairless, LAG2) superfamily activating a number of target genes in the Hes and Hey superfamilies that play major roles in cell fate determination and regulation of differentiation [24].

Notch signalling has been shown to be important in the development of the normal breast. Transgenic mice expressing a constitutively active form of Notch 4 show retarded mammary gland development with the subsequent emergence of mammary tumours [25-27]. In in vitro studies using the mammosphere culture system, Dontu et al. demonstrated that activation of Notch signalling, via a DSL peptide, promoted self-renewal and branching morphogenesis in three-dimensional matrigel cultures, which could be completely inhibited by a Notch 4 blocking antibody or a [gamma]-secretase inhibitor [17]. These results suggest that Notch signalling may regulate ductal branching during normal mammary gland development and that unregulated Notch signalling prevents terminal differentiation of the mammary epithelial cells. Aberrant Notch signalling may thus maintain the epithelial cells in a proliferative state that leads to extensive ductal dysplasia and an associated risk of carcinoma. An expanding number of studies in human breast cancer have shown evidence of aberrant Notch signalling. Our group have reported the accumulation of NICD and Hey-1 with corresponding decreases in the Notch pathway inhibitor Numb, in both breast cancer cell lines and invasive breast cancer tissue, compared to normal breast cell lines and normal breast tissue, respectively [18]. These results complemented previous studies in which loss of Numb was observed in ~50% of 321 human breast cancers, determined by immunohistochemistry [28], and high-level expression of Jagged 1 or Notch 1 was associated with a significantly worse overall survival (5-year survival rate, 42%) compared with patients expressing low levels of these genes (5-year survival rate, 65%). Moreover, a synergistic effect of high-level Jagged 1 and Notch 1 co-expression reduced the overall 5-year survival rate to 32% [29].

Further in vitro studies have suggested a link with Notch signalling and increased self-renewal in the mammosphere culture system in both MCF7 cells and primary normal and breast cancer tissue, where downregulation of Jagged 1 and Notch 3 mRNA reduced the capacity of first-generation mammospheres to produce a secondary generation [30]. In addition, Notch 3 was also found to be upregulated in CD[44.aup.+] populations of normal and breast cancer cells via serial analysis of gene expression (SAGE) analysis, linking the CD[44.sup.+]/CD[24.sup.-] population of cells with increased Notch gene expression and signalling [7,31]. Most recently, primary ductal carcinoma in situ (DCIS) mammospheres have been grown in a culture system analogous to that used to enrich for normal and invasive CSCs [32]. High-grade DCIS had an increased mammosphere-forming efficiency (MFE) compared to low-grade DCIS, which is consistent with findings from both brain and colon cancers where the most aggressive clinical samples have tumour stem cells with the highest sphere formation and self-renewal capacity [33,34]. Notch signalling was shown to be highly activated in DCIS compared to normal breast tissue and positive immunohistochemical staining for NICD predicted early recurrence 5 years after breast surgery (n = 50; two-sided log rank test, P = 0.012). Inhibition of Notch signalling using a [gamma]-secretase inhibitor (DAPT) or a Notch 4 neutralising antibody reduced the MFE of DCIS cells by up to 100%, with complete inhibition in two of six cases [32]. The [gamma]-secretase inhibitor MK-0752 is currently in early phase clinical studies in advanced breast cancer and other solid tumours [35].

Summary

Recent results have increased our understanding of normal stem cells and the signalling pathways that regulate them during the development of the mammary gland. Tumours in many tissues are now thought to develop from dysregulated stem or progenitor cells that may be recognised by cell surface protein expression or patterns of biological behaviour such as dye efflux or the capacity to grow as tumours in vivo or spheres in vitro. Many stem cell pathways are dysregulated in cancers, including the Notch signalling pathway, which is involved in the development of the breast and is aberrantly activated in invasive and pre-invasive breast cancers. Recent studies have shown a connection with increased levels of Notch pathway activation and enrichment for breast CSCs, and stem cell self-renewal pathways such as Notch may represent novel therapeutic targets in both pre-invasive and invasive breast cancer.

References

[1.] Bonnet D and Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997, 3, 730-737.

[2.] Reya T, Morrison SJ, Clarke MF and Weissman IL. Stem cells, cancer, and cancer stem cells. Nature, 2001, 414, 105-111.

[3.] Somervaille TCP and Cleary ML. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell, 2006, 10, 257-268.

[4.] Clarke M, Dick JE, Dirks PB et al. Cancer stem cells-perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res, 2006, 66, 9339-9344.

[5.] Al-Hajj M, Wicha MS, Benito-Hernandez A et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA, 2003, 100, 3983-3988.

[6.] Dontu G, Abdallah WM, Foley JM et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev, 2003, 17, 1253-1270.

[7.] Ponti D, Costa A, Zaffaroni N et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res, 2005, 65, 5506-5511.

[8.] Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med, 1996, 183, 1797-1806.

[9.] Patrawala L, Calhoun T, Schneider-Brousard R et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res, 2005, 65, 6207-6219.

[10.] Woodward WA, Chen MS, Behbod F et al. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A, 2007, 104, 618-623.

[11.] Mungamuri SK, Yang X, Thor AD and Somasundaram K. Survival signaling by Notch1: mammalian target of rapamycin (mTOR)-dependent inhibition of p53. Cancer Res, 2006, 66, 4715-4724.

[12.] Phillips TM, McBride WH and Pajonk F. The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst, 2006, 98, 1777-1785.

[13.] Bao S, Wu Q, McLendon RE et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature, 2006, 444, 756-760.

[14.] Li Y, Welm B, Podsypanina K et al. Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells. Proc Natl Acad Sci U S A, 2003, 100, 15853-15858.

[15.] Liu BY, McDermott SP, Khwaja SS and Alexander CM. 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, 2004, 101, 4158-4163.

[16.] Dontu G and Wicha MS. Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia, 2005, 10, 75-86.

[17.] Dontu G, Jackson KW, McNicholas E et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res, 2004, 6, R605-615.

[18.] Stylianou S, Clarke RB and Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res, 2006, 66, 1517-1525.

[19.] Ewan KB, Oketch-Rabah SA, Ravini SA et al. Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice. Am J Pathol, 2005, 167, 409-417.

[20.] Boulanger CA, Wagner KU and Smith GH. Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-beta1 expression. Oncogene, 2005, 24, 552-560.

[21.] Clarke RB, Spence K, Anderson E et al. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol, 2005, 277, 443-456.

[22.] Lai EC. Notch signaling: control of cell communication and cell fate. Development, 2004, 131, 965-973.

[23.] Politi K, Feirt N and Kitajewski J. Notch in mammary gland development and breast cancer. Semin Cancer Biol, 2004, 14, 341-347.

[24.] Ross DA, Rao PK and Kadesch T. Dual roles for the Notch target gene Hes-1 in the differentiation of 3T3-L1 preadipocytes. Mol Cell Biol, 2004, 24, 3505-3513.

[25.] Smith GH, Gallahan D, Diella F et al. Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development. Cell Growth Differ, 1995, 6, 563-577.

[26.] Gallahan D, Jhappan C, Robinson G et al. Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. Cancer Res, 1996, 56, 1775-1785.

[27.] Jhappan C, Gallahan D, Stahle C et al. Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands. Genes Dev, 1992, 6, 345-355.

[28.] Pece S, Serresi M, Santolini E et al. Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J Cell Biol, 2004, 167, 215-221.

[29.] Reedijk M, Ordorcic C, Chang L et al. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res, 2005, 65, 8530-8537.

[30.] Sansone P, Storci G, Giovannini C et al. p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres. Stem Cells, 2007, 25, 807-815.

[31.] Shipitsin M, Campbell LL, Argani P et al. Molecular definition of breast tumor heterogeneity. Cancer Cell, 2007, 11, 259-273.

[32.] Farnie G, Clarke RB, Spence K et al. Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst, 2007, 99, 616-627.

[33.] Ricci-Vitiani L, Lombardi DG, Pilozzi E et al. Identification and expansion of human colon-cancer-initiating cells. Nature, 2007, 445, 111-115.

[34.] Singh SK, Clarke ID, Terasaki M et al. Identification of a cancer stem cell in human brain tumors. Cancer Res, 2003, 63, 5821-5828.

[35.] Krop IE, Kosh M, Fearen I et al. Phase I pharmacokinetic, and pharmacodynamic trial of the novel oral notch inhibitor MK-0752 in patients with advanced breast cancer and other solid tumors. San Antonio Breast Cancer Symposium, 2006, Abstr. 6094.

Sacha J Howell, Gillian Farnie and Robert B Clarke

University of Manchester, Christie Hospital NHS Foundation Trust, Manchester, UK

Correspondence to: Sacha Howell, Breast Biology Group, Division of Cancer Studies, Faculty of Medicine and Human Sciences, University of Manchester, Christie Hospital NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK (email: [e-mail])