Abstract
Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. They are also considered as an attractive target for advanced cancer therapy. Using a sphere culture method that favors the growth of self-renewal cells, we have isolated sphere-forming cells (SFCs) from cervical cancer cell lines HeLa and SiHa. HeLa-SFCs were resistant to multiple chemotherapeutic drugs and were more tumorigenic, as evidenced by the growth of tumors following injection of immunodeficient mice with 1 × 104 cells, compared with 1 × 106 parental HeLa cells required to grow tumors of similar size in the same time frame. These cells showed an expression pattern of CD44high/CD24low that resembles the CSC surface biomarker of breast cancer. We further demonstrated that HeLa-SFCs expressed a higher level (6.9-fold) of the human papillomavirus oncogene E6, compared with that of parental HeLa cells. Gene silencing of E6 with a lentiviral-short-hairpin RNA (shRNA) profoundly inhibited HeLa-SFC sphere formation and cell growth. The inhibition of cell growth was even greater than that for sphere formation after E6 silence, suggesting that the loss of self-renewing ability may be more important. We then measured the expression of self-renewal genes, transformation growth factor-beta (TGF-β) and leukemia-inhibitory factor (LIF), in shRNA-transduced HeLa-SFCs and found that expression of all three TGF-β isoforms was significantly downregulated while LIF remained unchanged. Expression of the Ras gene (a downstream component of TGF-β) was also markedly decreased, suggesting that the growth-inhibitory effect could be via the TGF-β pathway. The above data indicate RNA interference-based therapy may offer a new approach for CSC-targeted cancer therapy.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Abbreviations
- CSCs:
-
cancer stem cells
- HPV:
-
human papillomavirus
- shRNA:
-
short-hairpin RNA
- SFCs:
-
sphere-forming cells
- TGF-β:
-
transformation growth factor-beta
- LIF:
-
leukemia-inhibitory factor
References
Visvader JE, Lindeman GJ . Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008; 8: 755–768.
Nakai E, Park K, Yawata T, Chihara T, Kumazawa A, Nakabayashi H et al. Enhanced MDR1 expression and chemoresistance of cancer stem cells derived from glioblastoma. Cancer Invest 2009; 27: 901–908.
Lou H, Dean M . Targeted therapy for cancer stem cells: the patched pathway and ABC transporters. Oncogene 2007; 26: 1357–1360.
Kondo T . Stem cell-like cancer cells in cancer cell lines. Cancer Biomark 2007; 3: 245–250.
Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q et al. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci USA 2005; 102: 5820–5825.
Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 2004; 10: 816–820.
Qin XF, An DS, Chen IS, Baltimore D . Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci USA 2003; 100: 183–188.
Jacque JM, Triques K, Stevenson M . Modulation of HIV-1 replication by RNA interference. Nature 2002; 418: 435–438.
Schomber T, Kalberer CP, Wodnar-Filipowicz A, Skoda RC . Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells. Blood 2004; 103: 4511–4513.
Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, Meyers R et al. A status report on RNAi therapeutics. Silence 2010; 1: 14.
Borkhardt A . Blocking oncogenes in malignant cells by RNA interference--new hope for a highly specific cancer treatment? Cancer Cell 2002; 2: 167–168.
Sledz CA, Williams BR . RNA interference in biology and disease. Blood 2005; 106: 787–794.
Jiang M, Milner J . Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002; 21: 6041–6048.
Putral LN, Bywater MJ, Gu W, Saunders NA, Gabrielli BG, Leggatt GR et al. RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin. Mol Pharmacol 2005; 68: 1311–1319.
Gu W, Putral L, Hengst K, Minto K, Saunders NA, Leggatt G et al. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes. Cancer Gene Ther 2006; 13: 1023–1032.
Yamato K, Fen J, Kobuchi H, Nasu Y, Yamada T, Nishihara T et al. Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA. Cancer Gene Ther 2006; 13: 234–241.
Faltus T, Yuan J, Zimmer B, Kramer A, Loibl S, Kaufmann M et al. Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells. Neoplasia 2004; 6: 786–795.
Wang YH, Liu S, Zhang G, Zhou CQ, Zhu HX, Zhou XB et al. Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Research 2005; 7: R220–R228.
Liu TG, Yin JQ, Shang BY, Min Z, He HW, Jiang JM et al. Silencing of hdm2 oncogene by siRNA inhibits p53-dependent human breast cancer. Cancer Gene Ther 2004; 11: 748–756.
Narisawa-Saito M, Kiyono T . Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: roles of E6 and E7 proteins. Cancer Sci 2007; 98: 1505–1511.
zur Hausen H . Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002; 2: 342–350.
Chang JT, Kuo TF, Chen YJ, Chiu CC, Lu YC, Li HF et al. Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers. Cancer Gene Ther 2010; 17: 827–836.
Jonson AL, Rogers LM, Ramakrishnan S, Downs Jr LS . Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer. Gynecol Oncol 2008; 111: 356–364.
Sima N, Wang W, Kong D, Deng D, Xu Q, Zhou J et al. RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53. Apoptosis 2008; 13: 273–281.
Tang S, Tao M, McCoy Jr JP, Zheng ZM . Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells. Oncogene 2006; 25: 2094–2104.
Gu W, Cochrane M, Leggatt GR, Payne E, Choyce A, Zhou F et al. Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses. Proc Natl Acad Sci USA 2009; 106: 8314–8319.
Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003; 17: 1253–1270.
Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007; 131: 1109–1123.
Yeung TM, Gandhi SC, Wilding JL, Muschel R, Bodmer WF . Cancer stem cells from colorectal cancer-derived cell lines. Proc Natl Acad Sci USA 2010; 107: 3722–3727.
Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 2009; 69: 1302–1313.
Li F . Every single cell clones from cancer cell lines growing tumors in vivo may not invalidate the cancer stem cell concept. Mol Cells 2009; 27: 491–492.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF . Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 3983–3988.
Singh A, Settleman J . EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 2010; 29: 4741–4751.
Penuelas S, Anido J, Prieto-Sanchez RM, Folch G, Barba I, Cuartas I et al. TGF-beta increases glioma-initiating cell self-renewal through the induction of LIF in human glioblastoma. Cancer Cell 2009; 15: 315–327.
Grusch M, Petz M, Metzner T, Ozturk D, Schneller D, Mikulits W . The crosstalk of RAS with the TGF-beta family during carcinoma progression and its implications for targeted cancer therapy. Curr Cancer Drug Targets 2010; 10: 849–857.
Kang JS, Liu C, Derynck R . New regulatory mechanisms of TGF-beta receptor function. Trends Cell Biol 2009; 19: 385–394.
Heldin CH, Landstrom M, Moustakas A . Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol 2009; 21: 166–176.
Watabe T, Miyazono K . Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell Res 2009; 19: 103–115.
Bellone G, Carbone A, Tibaudi D, Mauri F, Ferrero I, Smirne C et al. Differential expression of transforming growth factors-beta1, -beta2 and -beta3 in human colon carcinoma. Eur J Cancer 2001; 37: 224–233.
Charette ST, McCance DJ . The E7 protein from human papillomavirus type 16 enhances keratinocyte migration in an Akt-dependent manner. Oncogene 2007; 26: 7386–7390.
Menges CW, Baglia LA, Lapoint R, McCance DJ . Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein. Cancer Res 2006; 66: 5555–5559.
Scheffner M, Whitaker NJ . Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system. Semin Cancer Biol 2003; 13: 59–67.
Assinder SJ, Dong Q, Kovacevic Z, Richardson DR . The TGF-beta, PI3K/Akt and PTEN pathways: established and proposed biochemical integration in prostate cancer. Biochem J 2009; 417: 411–421.
Safina AF, Varga AE, Bianchi A, Zheng Q, Kunnev D, Liang P et al. Ras alters epithelial-mesenchymal transition in response to TGFbeta by reducing actin fibers and cell-matrix adhesion. Cell Cycle 2009; 8: 284–298.
Levina V, Marrangoni AM, DeMarco R, Gorelik E, Lokshin AE . Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One 2008; 3: e3077.
Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA 2009; 106: 16281–16286.
Dean M, Fojo T, Bates S . Tumour stem cells and drug resistance. Nat Rev Cancer 2005; 5: 275–284.
Acknowledgements
We would like to acknowledge the financial supports of NHMRC of Australia (Peter Doherty Fellowship and Travelling Award to WG, project grant ID631402 to NM) and Australian Cancer Foundation. We would also like to thank Prof Judy Lieberman and Dr Fabio Petrocca at Harvard Medical School for their support and technique help. We also thank Dr Barbara Rolfe at the University of Queensland for reading and providing useful suggestions to the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Rights and permissions
About this article
Cite this article
Gu, W., Yeo, E., McMillan, N. et al. Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability. Cancer Gene Ther 18, 897–905 (2011). https://doi.org/10.1038/cgt.2011.58
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/cgt.2011.58