Understanding normal and cancer stem cells should provide insights into the origin of prostate cancer and their mechanisms of resistance to current treatment strategies. In this study, we isolated and characterized stem-like cells present in the immortalized human prostate cell line, RWPE-1. We used a reporter system with green fluorescent protein (GFP) driven by the promoter of s-SHIP (for stem-SH2-domain-containing 5′-inositol phosphatase) whose stem cell-specific expression has been previously shown. We observed that s-SHIP-GFP-expressing RWPE-1 cells showed stem cell characteristics such as increased expression of stem cell surface markers (CD44, CD166, TROP2) and pluripotency transcription factors (Oct4, Sox2), and enhanced sphere-forming capacity and resistance to arsenite-induced cell death. Concomitant increased expression of the long noncoding RNA H19 was observed, which prompted us to investigate a putative role in stemness for this oncofetal gene. Targeted suppression of H19 with siRNA decreased Oct4 and Sox2 gene expression and colony-forming potential in RWPE-1 cells. Conversely, overexpression of H19 significantly increased gene expression of these two transcription factors and the sphere-forming capacity of RWPE-1 cells. Analysis of H19 expression in various prostate and mammary human cell lines revealed similarities with Sox2 expression, suggesting that a functional relationship may exist between H19 and Sox2. Collectively, we provide the first evidence that s-SHIP-GFP promoter reporter offers a unique marker for the enrichment of human stem-like cell populations and highlight a role in stemness for the long noncoding RNA H19.
Get full access to this article
View all access options for this article.
References
1.
JemalA, BrayF, CenterMM, FerlayJ, WardE and FormanD. (2011). Global cancer statistics. CA Cancer J Clin, 61:69–90.
2.
GoldsteinAS, StoyanovaT and WitteON. (2010). Primitive origins of prostate cancer: in vivo evidence for prostate-regenerating cells and prostate cancer-initiating cells. Mol Oncol, 4:385–396.
3.
MikiJ and RhimJS. (2008). Prostate cell cultures as in vitro models for the study of normal stem cells and cancer stem cells. Prostate Cancer Prostatic Dis, 11:32–39.
4.
LitvinovIV, Vander GriendAJ, XuY, AntonyL, DalrympleSL and IsaacsJT. (2006). Low-calcium serum-free defined medium selects for growth of normal prostatic epithelial stem cells. Cancer Res, 66:8598–8607.
5.
MaY, LiangD, LiuJ, AxcronaK, KvalheimG, StokkeT, NeslandJM and SuoZ. (2011). Prostate cancer cell lines under hypoxia exhibit greater stem-like properties. PLoS One, 6:e29170.
6.
RichardsonGD, RobsonCN, LangSH, NealDE, MaitlandNJ and CollinsAT. (2004). CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci, 117(Pt16):3539–3545.
7.
GuoC, LiuH, ZhangBH, CadaneanuRM, MayleAM and GarrawayIP. (2012). Epcam, CD44, and CD49f distinguish sphere-forming human prostate basal cells from a subpopulation with predominant tubule initiation capability. PLoS One, 7:e34219.
8.
RajasekharVK, StuderL, GeraldW, SocciND and ScherHI. (2011). Tumor-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling. Nat Commun, 2:162.
9.
JiaoJ, HindoyanA, WangS, TranLM, GoldsteinAS, LawsonD, ChenD, LiY, GuoC, et al. (2012). Identification of CD166 as a surface marker for enriching prostate stem/progenitor and cancer initiating cells. PLoS One, 7:e42564.
10.
GoldsteinAS, LawsonDA, ChengD, SunW, GarrawayIP and WitteON. (2008). Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proc Natl Acad Sci U S A, 105:20882–20887.
11.
van der HoogenC, van der HorstG, CheungH, BuijsJT, LippittJM, Guzman-RamirezN, HamdyFC, EatonCL, ThalmannGN, et al. (2010). High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. Cancer Res, 70:5163–5173.
12.
FosterBA, GangavarapuKJ, MathewG, AzabdaftariG, MorrisonCD, MillerA and HussWJ. (2013). Human prostate side population cells demonstrate stem cell properties in recombination with urogenital sinus mesenchyme. PLoS One, 8:e55062.
13.
GangavarapuKJ, AzabdaftariG, MorrisonCD, MillerA, FosterBA and HussWJ. (2013). Aldhehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity. Stem Cell Res Ther, 4:132.
14.
JeterCR, LiuB, LiuX, ChenX, LiuC, Calhoun-DavisT, RepassJ, ZaehresH, ShenJJ and TangDG. (2011). NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene, 30:3833–3845.
15.
LiangS, FuruhashiM, NakaneR, NakazawaS, GoudarziH, HamadaJ and IizasaH. (2013). Isolation and characterization of human breast cancer cells with SOX2 promoter activity. Biochem Biophys Res Commun, 437:205–211.
16.
TuZ, NinosJM, MaZ, WangJW, LemosMP, DespontsC, GhansahT, HowsonJM and KerrWG. (2001). Embryonic and hematopoietic stem cells express a novel SH2-containing inositol 5′-phosphatase isoform that partners with the Grb2 adapter protein. Blood, 98:2028–2038.
17.
DespontsC, NinosJM and KerrWG. (2006). s-SHIP associates with receptor complexes essential for pluripotent stem cell growth and survival. Stem Cells Dev, 15:641–646.
18.
RohrschneiderLR, CustodioJM, AndersonTA, MillerCP and GuH. (2005). The intron-5/6 promoter region of the ship1 gene regulates expression in stem/progenitor cells of the mouse embryo. Dev Biol, 283:503–521.
19.
BaiL and RohrschneiderLR. (2010). s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev, 24:1882–1892.
20.
BelloDB, WebberMM, KleinmanHK, WartingerDD and RhimJS. (1997). Androgen responsive adult human prostatic epithelial cell lines immortalysed by human papillomavirus 18. Carcinogenesis, 18:1215–1223.
21.
TokarEJ, AncrileBB, CunhaGR and WebberMM. (2005). Stem/progenitor and intermediate cells and the origin of human prostate cancer. Differentiation, 73:463–473.
22.
TokarEJ, QuW, LiuJ, LiuW, WebberMM, PhangJM and WaalkesMP. (2010). Arsenic-specific stem cell selection during malignant transformation. J Natl Cancer Inst, 102:638–649.
23.
VenninC, DahmaniF, SpruytN and AdriaenssensE. (2013). Role of non-coding RNA in cells: example of the H19/IGF2 locus. Adv Biosci Biotechnol, 4:34–44.
24.
ShiX, GippJ and BushmanW. (2007). Anchorage-independent culture maintains prostate stem cells. Dev Biol, 312:396–406.
25.
XinL, LukacsRU, LawsonDA, ChengD and WitteON. (2007). Self-renewal and multilineage differentiation in vitro from murine prostate stem cells. Stem Cells, 25:2760–2769.
26.
NiJ, CozziP, HaoJ, DuanW, GrahamP, KearsleyJ and LiY. (2014). Cancer stem cells in prostate cancer chemoresistance. Curr Cancer Drug Targets, 14:225–240.
27.
CalloniR, CorderoEA, HenriquesJA and BonattoD. (2013). Reviewing and updating the major molecular markers for stem cells. Stem Cells Dev, 22:1455–1476.
28.
PignonJC, GrisanzioC, GengY, SongJ, ShivdasaniRA and SignorettiS. (2013). P63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia. Proc Natl Acad Sci U S A, 110:8105–8110.
29.
BerteauxN, LottinS, AdriaenssensE, Van CoppenolleF, LeroyX, CollJ, DugimontT and CurgyJJ. (2004). Hormonal regulation of H19 gene expression in prostate epithelial cells. J Endocrinol, 183:69–78.
30.
PrajapatiA, GuptaS, MistryB and GuptaS. (2013). Prostate stem cells in the development of benign prostate hyperplasia and prostate cancer: emerging role and concepts. Biomed Res Int, 2013:107954.
31.
HuoY and MacaraIG. (2014). The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance. Nat Cell Biol, 16:526–534.
32.
KogataN, OliemullerE, WansburyO and HowardBA. (2014). Neuregulin-3 regulates epithelial progenitor cell positioning and specifies mammary phenotype. Stem Cells Dev, 23:2758–2770.
33.
KavanaughWM, PotDA, ChinSM, Deuter-ReinhartM, JeffersonAB, NorrisFA, MasiarzFR, CousensLS, MajerusPW and WilliamsLT. (1996). Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2. Curr Biol, 6:438–445.
34.
FuC-H, LinR-J, YuJ, ChangW-W, LiaoG-S, ChangW-Y, TsengL-M, TsaiY-F, YuJ-C and YuAL. (2014). A novel oncogenic role of inositol phosphatase SHIP2 in ER-negative breast cancer stem cells: involvement of JNK/vimentin activation. Stem Cells, 32:2048–2060.
35.
BartolomeiMS, ZemelS and TilghmanSM. (1991). Parental imprinting of the mouse H19 gene. Nature, 351:153–155.
36.
BrannanCI, DeesEC, IngramRS and TilghmanSM. (1990). The product of the H19 gene may function as an RNA. Mol Cell Biol, 10:28–36.
37.
CaiX and CullenBR. (2007). The imprinted H19 noncoding RNA is a primary microRNA precursor. RNA, 13:313–316.
38.
LiuJ, KahriAI, HeikkiläP, IlvesmäkiV and VoutilainenR. (1995). H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab, 80:492–496.
39.
ArielI, WeinsteinD, VoutilainenR, SchneiderT, Lustig-YarivO, de GrootN and HochbergA. (1997). Genomic imprinting and the endometrial cycle. The expression of the imprinted gene H19 in the human female reproductive organs. Diagn Mol Pathol, 6:17–25.
40.
AdriaenssensE, DumontL, LottinS, BolleD, LeprêtreA, DelobelleA, BoualiF, DugimontT, CollJ and CurgyJJ. (1998). H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression. Am J Pathol, 153:1597–1607.
41.
AdriaenssensE, LottinS, DugimontT, FauquetteW, CollJ, DupouyJP, BoillyB and CurgyJJ. (1999). Steroid hormones modulate H19 gene expression in both mammary gland and uterus. Oncogene, 18:4460–4473.
42.
LottinS, AdriaenssensE, DupressoirT, BerteauxN, MontpellierC, CollJ, DugimontT and CurgyJJ. (2002). Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells. Carcinogenesis, 23:1885–1895.
43.
BerteauxN, LottinS, MontéD, PinteS, QuatannensB, CollJ, HondermarckH, CurgyJJ, DugimontT and AdriaenssensE. (2005). H19 mRNA-like noncoding RNA promotes breast cancer cell proliferation through positive control by E2F1. J Biol Chem, 280:29625–29636.
NgJH and NgHH. (2010). LincRNAs join the pluripotency alliance. Nat Genet, 42:1035–1036.
46.
ZhangA, ZhouN, HuangJ, LiuQ, FukudaK, MaD, LuZ, BaiC, WatabeK and MoYY. (2013). The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage. Cell Res, 23:340–350.
47.
CicaleseA, BonizziG, PasiCE, FarettaM, RonzoniS, GiuliniB, BriskenC, MinucciS, Di FiorePP and PelicciPG. (2009). The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell, 138:1083–1095.
48.
BonizziG, CicaleseA, InsingaA and PelicciPG. (2012). The emerging role of p53 in stem cells. Trends Mol Med, 18:6–12.
49.
WangY, XuZ, JiangJ, XuC, KangJ, XiaoL, WuM, XiongJ, GuoX and LiuH. (2013). Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal. Dev Cell, 25:69–80.
50.
YangF, BiJ, XueX, ZhengL, ZhiK, HuaJ and FangG. (2012). Up-regulated long non-coding RNA H19 contributes to proliferation of gastric cancer cells. FEBS J, 279:3159–3165.
YuF, YaoH, ZhuP, ZhangX, PanQ, GongC, HuangY, HuX, SuF, LiebermanJ and SongE. (2007). let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell, 131:1109–1123.
53.
KongD, HeathE, ChenW, CherML, PowellI, HeilbrunL, LiY, AliS, SethiS, et al. (2012). Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM. PLoS One, 7:e33729.
54.
LuoM, LiZ, WangW, ZengY, LiuZ and QiuJ. (2013). Long non-coding RNA H19 increases bladder cancer metastasis by associating with EZH2 and inhibiting E-cadherin expression. Cancer Lett, 333:213–221.
55.
YinY, WangH, LiuK, WangF, YeX, LiuM, XiangR, LiuN and LiuL. (2014). Knockdown of H19 enhances differentiation capacity to epidermis of parthenogenetic embryonic stem cells. Curr Mol Med, 14:737–748.
56.
ZimmermanDL, BoddyCS and SchoenherrCS. (2013). Oct4/Sox2 binding sites contribute to maintaining hypomethylation of the maternal igf2/h19 imprinting control region. PLoS One, 8:e81962.
57.
Abi HabibW, AzziS, BrioudeF, SteunouV, ThibaudN, NevesCD, Le JuleM, Chantot-BastaraudS, KerenB, et al. (2014). Extensive investigation of the IGF2/H19 imprinting control region reveals novel OCT4/SOX2 binding site defects associated with specific methylation patterns in Beckwith-Wiedemann syndrome. Hum Mol Genet, 23:5763–5773.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
