Asc12的RNA干扰通过microRNA-302b相关的途径抑制结肠癌前体细胞生长
摘要
研究背景及目的：
结肠癌是消化道常见的恶性肿瘤之一，预后不良，死亡率较高。结肠癌的治疗一直是一大难题，结肠癌早期诊断率低，而中晚期结肠癌侵袭和转移又使结肠癌的治疗面临着巨大的挑战。肿瘤干细胞理论的提出给结肠癌的治疗带来了新的希望。该理论认为肿瘤干细胞是一类具有自我更新能力和多向分化潜能的种子细胞，在整个肿瘤细胞中仅占少数，但由于其具有无限增殖和不对称分裂能力，是恶性肿瘤不断生长、侵袭、转移和复发的根源。肿瘤干细胞是整个肿瘤的种子和源泉，是肿瘤得以发生、发展、转移和复发的“起始细胞”或“动力细胞”，在整个肿瘤过程中起着至关重要的作用。目前研究认为，结肠癌干细胞可能起源于正常成体肠隐窝干细胞。Ascl2是一个碱性/螺旋-环-螺旋转录因子，研究已证实其控制正常成体肠隐窝干细胞“命运”。Ascl2在小鼠肠上皮中的转基因过表达诱导了肠隐窝的过度增生和肠绒毛上的异位隐窝出现；Ascl2基因在成体小鼠肠道的诱导缺失则导致Lgr5阳性的成体肠隐窝干细胞在数天后消失。并且，近来研究发现，Ascl2在人结肠癌标本中表达上调。因此，我们设想，Ascl2可能与结肠癌干/前体细胞相关。尽管如此，Ascl2在结肠癌干/前体细胞中的作用尚未知。因此，我们选择了人结肠癌HT-29细胞(CD133阳性比例为47.5-95%)和LS174T细胞(CD133阳性比例为0.45%)来对Ascl2在结肠癌干/前体细胞中的作用进行研究。
研究方法及结果：
免疫组织化学检测证实Ascl2在结肠腺癌中表达增高，而在正常结肠组织中表达仅限于隐窝基底。Ascl2干扰后，HT-29和LS174T细胞的体外增殖、克隆形成、迁移和侵袭能力均下降，同时干扰后的细胞裸鼠皮下成瘤能力也降低。Western-blot和细胞免疫荧光检测发现CD133阳性HT-29细胞中Ascl2蛋白质表达明显高于CD133阴性HT-29细胞。流式细胞仪检测进一步发现Ascl2干扰后，HT-29细胞中CD133阳性细胞比例由54.7%下降到26.2%。与干细胞“干性”相关的基因CD133、Sox2、Oct4、Lgr5、Bmi1和C-myc在Ascl2干扰后HT-29和LS174T细胞出现mRNA和蛋白质表达水平下调(LS174T细胞由于流式检测CD133阳性细胞比例极低未检测CD133表达情况)，在两株细胞的裸鼠移植瘤中检测这些“干性”相关基因也得到一致的结果。同时，Ascl2干扰后HT-29细胞的成球能力也下降，表现为所形成的肿瘤球大小和数目均减少。miRNA芯片筛选出26种miRNA在Ascl2干扰后HT-29细胞中出现2倍以上的表达上调，而58种miRNA在Ascl2干扰后HT-29细胞中出现2倍以上的表达下调。与干细胞“干性”相关的let-7b、 miRNA-124、 miRNA-125b、miRNA-17、miRNA-20a和miRNA-302b也包括在其中，并且行qPCR验证进一步证实了其表达差异。我们对这些miRNA进行进一步研究发现，用miRNA-302b mimics在Ascl2干扰后的HT-29细胞中恢复了miRNA-302b的表达后，该细胞的成球能力得到恢复，并且部分“干性”相关基因表达出现上调，同时细胞的克隆形成、迁移和侵袭能力均增强。
结论：
Ascl2的RNA干扰可以导致结肠癌细胞生长抑制。Ascl2是一个与结肠癌干/前体细胞“干性”密切相关的重要转录因子，并且可通过miRNA-302b相关的途径发挥其对结肠癌干/前体细胞“干性”的调控作用。Ascl2可能作为一个针对结肠癌干/前体细胞的潜在靶标用于结肠癌的治疗。
Background: Colon cancer is one of the common gastrointestinal cancer, with poorprognosis and a higher mortality rate. Treatment of colon cancer has been a major problem,because its early diagnosis rate is low, and treatment of advanced colon cancer withinvasion and metastasis faces enormous challenges. The cancer stem cell theory hasbrought new hope for the treatment of colon cancer. The theory is that cancer stem cells area class of seed cells, with self-renewal and multilineage differentiation potential, and theyare a minority of all tumor cells. But because of their unlimited proliferation andasymmetric division, cancer stem cells are roots of malignant tumor growth, invasion,metastasis and recurrence. Cancer stem cells are the seeds and sources of tumors, they are“initiating cells” or “power cells” in the process of tumor development, metastasis andrecurrence, they play a key role in all tumor processes. The current studies suggest thatcolon cancer stem cells may have originated in the normal adult intestinal crypt stem cells.Achaete scute-like2(Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controlsthe fate of intestinal stem cells. Ascl2transgenic overexpression in mouse inducedintestinal epithelial crypt hyperplasia and ectopic crypts in the intestinal villi; Ascl2geneknockdown in adult mouse intestinal led to disappear of Lgr5positive adult intestinal cryptstem cells within a few days. And, recent studies have found that the expression level ofAscl2is upregulated in human colon cancer specimens. Therefore, we assume that, Ascl2may be associated with colon cancer stem/progenitor cells. However, the role of Ascl2incolon cancer progenitor cells remains unknown. The cell line HT-29(47.5-95%of CD133+population) and LS174T (0.45%of CD133+population) were chosen for functionalevaluation of Ascl2in colon cancer progenitor cells after gene knockdown by RNAinterference.
Methodology/Principle Findings: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas, and Ascl2expression wasspecifically localized at the nucleus of crypt base cells of normal human colon mucosa.Downregulation of Ascl2using RNA interference in cultured colonic adenocarcinomaHT-29and LS174T cells reduced cellular proliferation, colony-forming ability, invasion andmigration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2protein level in CD133+HT-29cells was significantly higher than in CD133-HT-29cells.Ascl2blockade via shRNA interference in HT-29cells (shRNA-Ascl2/HT-29cells)resulted in26.2%of cells staining CD133+compared with54.7%in controlshRNA-Ctr/HT-29cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2,Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29andshRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft(CD133was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29cellshad inhibited abilities to form tumorspheres compared with control. The microRNA(miRNAs) microarrays, identified26up-regulated miRNAs and58down-regulatedmiRNAs in shRNA-Ascl2/HT-29cells. Expression levels of let-7b, miRNA-124,miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration ofmiRNA-302b, via its mimics, led to the restoration of shRNA-Ascl2/HT-29‘stemness’characteristics, including tumorsphere formation and ‘stemness’ associated genes levels,and the recovery of cellular behaviors, including colony-forming ability, invasion andmigration in vitro.
Conclusions/Significance: Ascl2RNA interference can lead to growth inhibition ofcolon cancer cells. Ascl2is an important transcription factor closely related to colon cancerstem/progenitor cells, and functions through a miR-302b-related mechanism. Ascl2maybe a potential target for treatment of colon cancer.
结肠癌是消化道常见的恶性肿瘤之一，预后不良，死亡率较高。结肠癌的治疗一直是一大难题，结肠癌早期诊断率低，而中晚期结肠癌侵袭和转移又使结肠癌的治疗面临着巨大的挑战。肿瘤干细胞理论的提出给结肠癌的治疗带来了新的希望。该理论认为肿瘤干细胞是一类具有自我更新能力和多向分化潜能的种子细胞，在整个肿瘤细胞中仅占少数，但由于其具有无限增殖和不对称分裂能力，是恶性肿瘤不断生长、侵袭、转移和复发的根源。肿瘤干细胞是整个肿瘤的种子和源泉，是肿瘤得以发生、发展、转移和复发的“起始细胞”或“动力细胞”，在整个肿瘤过程中起着至关重要的作用。目前研究认为，结肠癌干细胞可能起源于正常成体肠隐窝干细胞。Ascl2是一个碱性/螺旋-环-螺旋转录因子，研究已证实其控制正常成体肠隐窝干细胞“命运”。Ascl2在小鼠肠上皮中的转基因过表达诱导了肠隐窝的过度增生和肠绒毛上的异位隐窝出现；Ascl2基因在成体小鼠肠道的诱导缺失则导致Lgr5阳性的成体肠隐窝干细胞在数天后消失。并且，近来研究发现，Ascl2在人结肠癌标本中表达上调。因此，我们设想，Ascl2可能与结肠癌干/前体细胞相关。尽管如此，Ascl2在结肠癌干/前体细胞中的作用尚未知。因此，我们选择了人结肠癌HT-29细胞(CD133阳性比例为47.5-95%)和LS174T细胞(CD133阳性比例为0.45%)来对Ascl2在结肠癌干/前体细胞中的作用进行研究。
研究方法及结果：
免疫组织化学检测证实Ascl2在结肠腺癌中表达增高，而在正常结肠组织中表达仅限于隐窝基底。Ascl2干扰后，HT-29和LS174T细胞的体外增殖、克隆形成、迁移和侵袭能力均下降，同时干扰后的细胞裸鼠皮下成瘤能力也降低。Western-blot和细胞免疫荧光检测发现CD133阳性HT-29细胞中Ascl2蛋白质表达明显高于CD133阴性HT-29细胞。流式细胞仪检测进一步发现Ascl2干扰后，HT-29细胞中CD133阳性细胞比例由54.7%下降到26.2%。与干细胞“干性”相关的基因CD133、Sox2、Oct4、Lgr5、Bmi1和C-myc在Ascl2干扰后HT-29和LS174T细胞出现mRNA和蛋白质表达水平下调(LS174T细胞由于流式检测CD133阳性细胞比例极低未检测CD133表达情况)，在两株细胞的裸鼠移植瘤中检测这些“干性”相关基因也得到一致的结果。同时，Ascl2干扰后HT-29细胞的成球能力也下降，表现为所形成的肿瘤球大小和数目均减少。miRNA芯片筛选出26种miRNA在Ascl2干扰后HT-29细胞中出现2倍以上的表达上调，而58种miRNA在Ascl2干扰后HT-29细胞中出现2倍以上的表达下调。与干细胞“干性”相关的let-7b、 miRNA-124、 miRNA-125b、miRNA-17、miRNA-20a和miRNA-302b也包括在其中，并且行qPCR验证进一步证实了其表达差异。我们对这些miRNA进行进一步研究发现，用miRNA-302b mimics在Ascl2干扰后的HT-29细胞中恢复了miRNA-302b的表达后，该细胞的成球能力得到恢复，并且部分“干性”相关基因表达出现上调，同时细胞的克隆形成、迁移和侵袭能力均增强。
结论：
Ascl2的RNA干扰可以导致结肠癌细胞生长抑制。Ascl2是一个与结肠癌干/前体细胞“干性”密切相关的重要转录因子，并且可通过miRNA-302b相关的途径发挥其对结肠癌干/前体细胞“干性”的调控作用。Ascl2可能作为一个针对结肠癌干/前体细胞的潜在靶标用于结肠癌的治疗。
Background: Colon cancer is one of the common gastrointestinal cancer, with poorprognosis and a higher mortality rate. Treatment of colon cancer has been a major problem,because its early diagnosis rate is low, and treatment of advanced colon cancer withinvasion and metastasis faces enormous challenges. The cancer stem cell theory hasbrought new hope for the treatment of colon cancer. The theory is that cancer stem cells area class of seed cells, with self-renewal and multilineage differentiation potential, and theyare a minority of all tumor cells. But because of their unlimited proliferation andasymmetric division, cancer stem cells are roots of malignant tumor growth, invasion,metastasis and recurrence. Cancer stem cells are the seeds and sources of tumors, they are“initiating cells” or “power cells” in the process of tumor development, metastasis andrecurrence, they play a key role in all tumor processes. The current studies suggest thatcolon cancer stem cells may have originated in the normal adult intestinal crypt stem cells.Achaete scute-like2(Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controlsthe fate of intestinal stem cells. Ascl2transgenic overexpression in mouse inducedintestinal epithelial crypt hyperplasia and ectopic crypts in the intestinal villi; Ascl2geneknockdown in adult mouse intestinal led to disappear of Lgr5positive adult intestinal cryptstem cells within a few days. And, recent studies have found that the expression level ofAscl2is upregulated in human colon cancer specimens. Therefore, we assume that, Ascl2may be associated with colon cancer stem/progenitor cells. However, the role of Ascl2incolon cancer progenitor cells remains unknown. The cell line HT-29(47.5-95%of CD133+population) and LS174T (0.45%of CD133+population) were chosen for functionalevaluation of Ascl2in colon cancer progenitor cells after gene knockdown by RNAinterference.
Methodology/Principle Findings: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas, and Ascl2expression wasspecifically localized at the nucleus of crypt base cells of normal human colon mucosa.Downregulation of Ascl2using RNA interference in cultured colonic adenocarcinomaHT-29and LS174T cells reduced cellular proliferation, colony-forming ability, invasion andmigration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2protein level in CD133+HT-29cells was significantly higher than in CD133-HT-29cells.Ascl2blockade via shRNA interference in HT-29cells (shRNA-Ascl2/HT-29cells)resulted in26.2%of cells staining CD133+compared with54.7%in controlshRNA-Ctr/HT-29cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2,Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29andshRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft(CD133was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29cellshad inhibited abilities to form tumorspheres compared with control. The microRNA(miRNAs) microarrays, identified26up-regulated miRNAs and58down-regulatedmiRNAs in shRNA-Ascl2/HT-29cells. Expression levels of let-7b, miRNA-124,miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration ofmiRNA-302b, via its mimics, led to the restoration of shRNA-Ascl2/HT-29‘stemness’characteristics, including tumorsphere formation and ‘stemness’ associated genes levels,and the recovery of cellular behaviors, including colony-forming ability, invasion andmigration in vitro.
Conclusions/Significance: Ascl2RNA interference can lead to growth inhibition ofcolon cancer cells. Ascl2is an important transcription factor closely related to colon cancerstem/progenitor cells, and functions through a miR-302b-related mechanism. Ascl2maybe a potential target for treatment of colon cancer.
引文
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8. Zhu R, Yang Y, Tian Y, et al. Ascl2Knockdown Results in Tumor Growth Arrestby miRNA-302b-Related Inhibition of Colon Cancer Progenitor Cells. PLoS one,2012;7(2):e32170.
9. Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell,2006;124:1111-1115
10. Odoux C, FohrerH, H oppoT, et al. A stochastic model for cancer stem celloriginin metastatic colon cancer. Cancer Res,2008;68(17):6932-6941.
11. Zhu L, Gibson P, Currle DS, et al. Prominin1marks intestinal stem cells that aresusceptible to neoplastic transformation. Nature,2009;457:603-607.
12. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin ofintestinal cancer. Nature,2009;457:608-611.
13.赵璇,冉宇靓,遇珑,等.人肝癌组织中肿瘤干细胞样细胞的分离培养及鉴定[J].中国肿瘤生物治疗杂志,2009;16(5):436-441.
14. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable ofinitiating tumour growth in immunodefcient mice. Nature,2007;445:106-110.
15. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identifcation and expansion ofhuman colon-cancer-initiating cells. Nature,2007;445:111-115.
16. Todaro M, Alea MP, Di Stefano AB, et al. Colon cancer stem cells dictate tumorgrowth and resist cell death by production of interleukin-4. Cell Stem Cell,2007;1:389-402.
17. Vermeulen L, Todaro M, de Sousa MF, et al. Single-cell cloning of colon cancerstem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad SciUSA,2008;105:13427-13432.
18. Chu P, Clanton DJ, Snipas TS, et al. Characterization of a subpopulation of coloncancer cells with stem cell-like properties. International Journal of Cancer,2009;124:1312-1321.
19. Du L, Wang HY, He LY, et al. CD44is of Functional Importance for ColorectalCancer Stem Cells. Clinical Cancer Research,2008;14:6751-6760.
20. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of humancolorectal cancer stem cells. Proc Natl Acad Sci USA,2007;104:10158-10163.
21. Huang EH, H ynes M J, Zhang T, et al. Aldehyde dehydrogenase1is a markerfor normal and malignant human colonic stem cells (SC) and tracks SCoverpopulation during colon tumor-igenesis. Cancer Res,2009;69(8):3382-3389.
22. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin ofintestinal cancer. Nature,2009;457:608-U119.
23. Miraglia S, Godfrey W, Yin AH, et al. A novel five-transmembranehematopoietic stem cell antigen: isolation, characterization, and molecularcloning. Blood,1997;90(12):5013-5021.
24. Horst D, Kriegl L, Engel J, et al. CD133expression is an independent prognosticmarker for low survival in colorectal cancer. Br J Cancer,2008;99(8):1285-1289.
25. Shmelkov SV, Butler JM, Hooper AT, et al. CD133expression is not restricted tostem cells, and both CD133+and CD133-metastatic colon cancer cells initiatetumors. J Clin Invest,2008;118(6):2111-2120.
26. Haraguchi N, Ohkuma M, Sakashita H, et al. CD133CD44+populationefficiently enriches colon cancer initiating cells. Ann Surg Onco,2008;15(10):2927-2933.
27. Du L, Wang H, He L, et al. CD44is of functional importance for colorectalcancer stem cells. Clin Cancer Res,2008;14:6751-6760.
28. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinal stem cellmarker, in normal and premalignant human gastrointestinal tissue. ScientificWorld Journal,2008;23(8):1168-1176.
29. Kuhnert F, Davis CR, Wang HT, et al. Essential requirement for Wnt signaling inproliferation of adult small intestine and colon revealed by adenoviral expressionof Dickkopf-1. Proc Natl Acad Sci USA,2004;101(1):266-271.
30. van de Wetering M, Sancho E, Verweij C, et al. The beta-catenin/TCF-4compleximposes a crypt progenitor phenotype on colorectal cancer cells. Cell,2002;111(2):241-250.
31. Radtke F, Clevers H, Riccio O. From gut homeostasis to cancer. Curr Mol Med,2006;6(3):275-289.
32. de Sousa EM, Vermeulen L, Richel D, et al. Targeting Wnt signaling in coloncancer stem cells. Clin Cancer Res,2011;17(4):647-653.
33. van Es JH, van Gijn ME, Riccio O, et al. Notch/gamma-secretase inhibition turnsproliferative cells inintestinal crypts and adenomas into goblet cells. Nature,2005;435(7044):959-963.
34. Reedijk M, Odorcic S, Zhang H, et al. Activation of Notch signaling in humancolon adenocarcinoma. Int J Oncol,2008;33:1223–1229.
35. Sikandar SS, Pate KT, Anderson S, et al. NOTCH signaling is required forformation and self-renewal of tumor-initiating cells and for repression ofsecretory cell differentiation in colon cancer. Cancer Res,2010;70:1469–1478.
36. Ponnurangam S, Mammen JM, Ramalingam S, et al. Honokiol in combinationwith radiation targets Notch signaling to inhibit colon cancer stem cells. MolCancer Ther,2012;[Epub ahead of print].
37. He XC, Zhang J, Tong WG, et al. BMP signaling inhibits intestinal stem cellself-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet,2004;36(10):1117-1121.
38. Lombardo Y, Scopelliti A, Cammareri P, et al. Bone morphogenetic protein4induces differentiation of colorectal cancer stem cells and increases their responseto chemotherapy in mice. Gastroenterology,2011;140(1):297-309.
39. Vermeulen L, Todaro M, de Sousa Mello F, et al. Single-cell cloning of coloncancer stem ce lls reveals a multilineage differentiation capacity. Proc Natl AcadSci USA,2008;105(36):13427-13432.
40. Quillien A, Blanco-Sanchez B, Halluin C, et al. BMP signaling orchestratesphotoreceptor specification in the zebrafish pineal gland in collaboration withNotch. Development,2011;138(11):2293-2302.
41. Wu R, Hu TC, Rehemtulla A, et al. Preclinical testing of PI3K/AKT/mTORsignaling inhibitors in a mouse model of ovarian endometrioid adenocarcinoma.Clin Cancer Res,2011;17(23):7359-7372.
42. Sato T, van Es JH, Snippert HJ, et al. Paneth cells constitute the niche for Lgr5stem cells in intestinal crypts. Nature,2011;469(7330):415-418.
43. Barker N, van Es JH, Kuipers J,et al. Identification of stem cells in small intestineand colon by marker gene Lgr5. Nature,2007;449(7165):1003-1007.
44. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5stem cells build crypt-villusstructures in vitro without a mesenchymal niche. Nature,2009;459(7244):262-265.
45. Johnson JE, Birren SJ, Anderson DJ. Two rat homologues of Drosophilaachaete-scute specifically expressed in neuronal precursors. Nature,1990;346:858–861.
46. Guillemot F, Nagy A, Auerbach A, et al. Essential role of Mash-2inextraembryonic development. Nature,1994;371:333–336.
47. Sansom OJ, Reed KR, Hayes AJ, et al. Loss of Apc in vivo immediately perturbsWnt signaling, differentiation, and migration. Genes Dev,2004;18:1385–1390.
48. Jubb AM, Chalasani S, Frantz GD, et al. Achaete-scute like2(ascl2) is a target ofWnt signalling and is upregulated in intestinal neoplasia. Oncogene,2006;25:3445–3457.
49. van der Flier LG, Sabates-Bellver J, Oving I, et al. The Intestinal Wnt/TCFSignature. Gastroenterology,2007;132:628–632.
50. Sachdeva M, Zhu S, Wu F, et al. p53represses c-Myc through induction of thetumor suppressor miR-145. Proc Natl Acad Sci USA,2009;106(9):3207-3212.
51. Chang CJ, Chao CH, Xia W, et al. p53regulates epithelial-mesenchymaltransition and stem cell properties through modulating miRNAs. Nat Cell Biol,2011;13(3):317-323.
52. Gao P, Tchernyshyov I, Chang TC, et al. c-Myc suppression of miR-23a/benhances mitochondrial glutaminase expression and glutamine metabolism.Nature,2009;458(7239):762-765.
53. Liu H, Deng S, Zhao Z, et al. Oct4regulates the miR-302cluster in P19mouseembryonic carcinoma cells. Mol Biol Rep,2011;38(3):2155-2160.
54. Sureban SM, May R, Ramalingam S, et al. Selective blockade of DCAMKL-1results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.Gastroenterology,2009;137(2):649-659.
55. Ramasamy R, Lam EW, SoeiroI, et al. Mesenchymal stem cells inhibitproliferation and apoptosis of tumor cells: impact on in vivo tumor growth.Leukemia,2007;21(2):304-310.
56. Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors of cancerstem cells by high-throughput screening. Cell,2009;138(4):645-659.
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1. Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in2008:GLOBOCAN2008. Int J Cancer,2010;127(12):2893-2917.
2. Markowitz SD, Bertagnolli MM. Molecular origins of cancer: Molecular basis ofcolorectal cancer. N Engl J Med,2009;361:2449-2460.
3. Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med,2011;17:313-319.
4. van der Flier LG, van Gijn ME, Hatzis P, et al. Transcription factor achaetescute-like2controls intestinal stem cell fate. Cell,2009;136(5):903-912.
5. Jubb AM, Hoeflich KP, Haverty PM, et al. Ascl2and11p15.5amplification incolorectal cancer.Gut,2011;60(11):1606-1607.
6. van der Flier LG, van Gijn ME, Hatzis P, et al. Expression of an ASCL2relatedstem cell signature and IGF2in colorectal cancer liver metastases with11p15.5gain [J]. Gut,2010;59(9):1236-1244.
7. Jubb AM, Chalasani S, Frantz GD, et al. Achaete-scute like2(ascl2) is a target ofWnt signaling and is upregulated in intestinal neoplasia. Oncogene,2006;25(24):3445-3457.
8. Zhu R, Yang Y, Tian Y, et al. Ascl2Knockdown Results in Tumor Growth Arrestby miRNA-302b-Related Inhibition of Colon Cancer Progenitor Cells. PLoS one,2012;7(2):e32170.
9. Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell,2006;124:1111-1115
10. Odoux C, FohrerH, H oppoT, et al. A stochastic model for cancer stem celloriginin metastatic colon cancer. Cancer Res,2008;68(17):6932-6941.
11. Zhu L, Gibson P, Currle DS, et al. Prominin1marks intestinal stem cells that aresusceptible to neoplastic transformation. Nature,2009;457:603-607.
12. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin ofintestinal cancer. Nature,2009;457:608-611.
13.赵璇,冉宇靓,遇珑,等.人肝癌组织中肿瘤干细胞样细胞的分离培养及鉴定[J].中国肿瘤生物治疗杂志,2009;16(5):436-441.
14. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable ofinitiating tumour growth in immunodefcient mice. Nature,2007;445:106-110.
15. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identifcation and expansion ofhuman colon-cancer-initiating cells. Nature,2007;445:111-115.
16. Todaro M, Alea MP, Di Stefano AB, et al. Colon cancer stem cells dictate tumorgrowth and resist cell death by production of interleukin-4. Cell Stem Cell,2007;1:389-402.
17. Vermeulen L, Todaro M, de Sousa MF, et al. Single-cell cloning of colon cancerstem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad SciUSA,2008;105:13427-13432.
18. Chu P, Clanton DJ, Snipas TS, et al. Characterization of a subpopulation of coloncancer cells with stem cell-like properties. International Journal of Cancer,2009;124:1312-1321.
19. Du L, Wang HY, He LY, et al. CD44is of Functional Importance for ColorectalCancer Stem Cells. Clinical Cancer Research,2008;14:6751-6760.
20. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of humancolorectal cancer stem cells. Proc Natl Acad Sci USA,2007;104:10158-10163.
21. Huang EH, H ynes M J, Zhang T, et al. Aldehyde dehydrogenase1is a markerfor normal and malignant human colonic stem cells (SC) and tracks SCoverpopulation during colon tumor-igenesis. Cancer Res,2009;69(8):3382-3389.
22. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin ofintestinal cancer. Nature,2009;457:608-U119.
23. Miraglia S, Godfrey W, Yin AH, et al. A novel five-transmembranehematopoietic stem cell antigen: isolation, characterization, and molecularcloning. Blood,1997;90(12):5013-5021.
24. Horst D, Kriegl L, Engel J, et al. CD133expression is an independent prognosticmarker for low survival in colorectal cancer. Br J Cancer,2008;99(8):1285-1289.
25. Shmelkov SV, Butler JM, Hooper AT, et al. CD133expression is not restricted tostem cells, and both CD133+and CD133-metastatic colon cancer cells initiatetumors. J Clin Invest,2008;118(6):2111-2120.
26. Haraguchi N, Ohkuma M, Sakashita H, et al. CD133CD44+populationefficiently enriches colon cancer initiating cells. Ann Surg Onco,2008;15(10):2927-2933.
27. Du L, Wang H, He L, et al. CD44is of functional importance for colorectalcancer stem cells. Clin Cancer Res,2008;14:6751-6760.
28. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinal stem cellmarker, in normal and premalignant human gastrointestinal tissue. ScientificWorld Journal,2008;23(8):1168-1176.
29. Kuhnert F, Davis CR, Wang HT, et al. Essential requirement for Wnt signaling inproliferation of adult small intestine and colon revealed by adenoviral expressionof Dickkopf-1. Proc Natl Acad Sci USA,2004;101(1):266-271.
30. van de Wetering M, Sancho E, Verweij C, et al. The beta-catenin/TCF-4compleximposes a crypt progenitor phenotype on colorectal cancer cells. Cell,2002;111(2):241-250.
31. Radtke F, Clevers H, Riccio O. From gut homeostasis to cancer. Curr Mol Med,2006;6(3):275-289.
32. de Sousa EM, Vermeulen L, Richel D, et al. Targeting Wnt signaling in coloncancer stem cells. Clin Cancer Res,2011;17(4):647-653.
33. van Es JH, van Gijn ME, Riccio O, et al. Notch/gamma-secretase inhibition turnsproliferative cells inintestinal crypts and adenomas into goblet cells. Nature,2005;435(7044):959-963.
34. Reedijk M, Odorcic S, Zhang H, et al. Activation of Notch signaling in humancolon adenocarcinoma. Int J Oncol,2008;33:1223–1229.
35. Sikandar SS, Pate KT, Anderson S, et al. NOTCH signaling is required forformation and self-renewal of tumor-initiating cells and for repression ofsecretory cell differentiation in colon cancer. Cancer Res,2010;70:1469–1478.
36. Ponnurangam S, Mammen JM, Ramalingam S, et al. Honokiol in combinationwith radiation targets Notch signaling to inhibit colon cancer stem cells. MolCancer Ther,2012;[Epub ahead of print].
37. He XC, Zhang J, Tong WG, et al. BMP signaling inhibits intestinal stem cellself-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet,2004;36(10):1117-1121.
38. Lombardo Y, Scopelliti A, Cammareri P, et al. Bone morphogenetic protein4induces differentiation of colorectal cancer stem cells and increases their responseto chemotherapy in mice. Gastroenterology,2011;140(1):297-309.
39. Vermeulen L, Todaro M, de Sousa Mello F, et al. Single-cell cloning of coloncancer stem ce lls reveals a multilineage differentiation capacity. Proc Natl AcadSci USA,2008;105(36):13427-13432.
40. Quillien A, Blanco-Sanchez B, Halluin C, et al. BMP signaling orchestratesphotoreceptor specification in the zebrafish pineal gland in collaboration withNotch. Development,2011;138(11):2293-2302.
41. Wu R, Hu TC, Rehemtulla A, et al. Preclinical testing of PI3K/AKT/mTORsignaling inhibitors in a mouse model of ovarian endometrioid adenocarcinoma.Clin Cancer Res,2011;17(23):7359-7372.
42. Sato T, van Es JH, Snippert HJ, et al. Paneth cells constitute the niche for Lgr5stem cells in intestinal crypts. Nature,2011;469(7330):415-418.
43. Barker N, van Es JH, Kuipers J,et al. Identification of stem cells in small intestineand colon by marker gene Lgr5. Nature,2007;449(7165):1003-1007.
44. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5stem cells build crypt-villusstructures in vitro without a mesenchymal niche. Nature,2009;459(7244):262-265.
45. Johnson JE, Birren SJ, Anderson DJ. Two rat homologues of Drosophilaachaete-scute specifically expressed in neuronal precursors. Nature,1990;346:858–861.
46. Guillemot F, Nagy A, Auerbach A, et al. Essential role of Mash-2inextraembryonic development. Nature,1994;371:333–336.
47. Sansom OJ, Reed KR, Hayes AJ, et al. Loss of Apc in vivo immediately perturbsWnt signaling, differentiation, and migration. Genes Dev,2004;18:1385–1390.
48. Jubb AM, Chalasani S, Frantz GD, et al. Achaete-scute like2(ascl2) is a target ofWnt signalling and is upregulated in intestinal neoplasia. Oncogene,2006;25:3445–3457.
49. van der Flier LG, Sabates-Bellver J, Oving I, et al. The Intestinal Wnt/TCFSignature. Gastroenterology,2007;132:628–632.
50. Sachdeva M, Zhu S, Wu F, et al. p53represses c-Myc through induction of thetumor suppressor miR-145. Proc Natl Acad Sci USA,2009;106(9):3207-3212.
51. Chang CJ, Chao CH, Xia W, et al. p53regulates epithelial-mesenchymaltransition and stem cell properties through modulating miRNAs. Nat Cell Biol,2011;13(3):317-323.
52. Gao P, Tchernyshyov I, Chang TC, et al. c-Myc suppression of miR-23a/benhances mitochondrial glutaminase expression and glutamine metabolism.Nature,2009;458(7239):762-765.
53. Liu H, Deng S, Zhao Z, et al. Oct4regulates the miR-302cluster in P19mouseembryonic carcinoma cells. Mol Biol Rep,2011;38(3):2155-2160.
54. Sureban SM, May R, Ramalingam S, et al. Selective blockade of DCAMKL-1results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.Gastroenterology,2009;137(2):649-659.
55. Ramasamy R, Lam EW, SoeiroI, et al. Mesenchymal stem cells inhibitproliferation and apoptosis of tumor cells: impact on in vivo tumor growth.Leukemia,2007;21(2):304-310.
56. Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors of cancerstem cells by high-throughput screening. Cell,2009;138(4):645-659.