摘要
研究背景和目的
越来越多的实验证据[1-4]表明肿瘤是一种干细胞疾病,即只有小部分肿瘤干细胞才有致瘤及维持恶性表型的作用。对这一小部分细胞不同的学者有不同的命名,有称为“肿瘤干细胞”(CSCs)、“肿瘤源性癌细胞”、“肿瘤始动细胞”,都是要表明只有这部分细胞移植入免疫缺陷小鼠体内时才能产生新生的肿瘤。经过对肿瘤细胞亚群的分选、特异的表面分子标志及移植动物的功能性实验,已在血液、脑肿瘤、乳腺癌、前列腺癌、肝癌、胰腺癌、鼻咽癌和结肠癌等中分离出各自的肿瘤干细胞[5-12]。2003年,Al Hajj等[13]应用FACS技术从乳腺癌组织中分离出表型为CD44+CD24-ESA+ LIN-的细胞具有干细胞特性,200个即可在小鼠体内成瘤。2005年,Ponti等[14]证实乳腺癌干细胞可以在体外繁殖成非贴壁的球囊细胞,这为以后研究乳腺癌干细胞及分子靶向特异干扰乳腺癌干细胞的自我更新和存活机制提供了合适的模型。
肿瘤干细胞数量稀少,它不仅是肿瘤的来源,而且与肿瘤的进展和转移有关,因此,尽管已经治愈了原有疾病,但肿瘤干细胞tumor stem cell,TSC)的存活会最终导致肿瘤的复发,这预示着只有杀死所有肿瘤干细胞,抗肿瘤治疗才能治愈肿瘤。放疗是乳腺癌的重要治疗手段之一,术后放疗可以杀灭残留的肿瘤细胞,术前放疗可以缩小肿块的大小。已有研究发现电离辐射后的肿瘤干细胞的凋亡率明显低于普通肿瘤细胞,提示肿瘤放射抵抗可能与肿瘤干细胞有关[15-18]。随着分子生物学的发展,一些与肿瘤放疗抵抗相关的基因被鉴定出来,使人们能够通过各种手段作用于这些新的靶点以提高肿瘤的放射敏感性。
PI3K-AKT信号传导通路在经受电离辐射后被一些外源性刺激因子激活,促进肿瘤细胞存活,已有研究证实其与放射抵抗相关[19,20]。十余年前已被发现的AKT1是PI3K的重要的下游靶因子,目前已发现3个AKT家族成员,AKT1、AKT2和AKT3。AKT/蛋白激酶B信号作用通道调控细胞增殖和生长,促进肿瘤发生[21,22][19,23,24],已有许多证据证实PI3K-AKT是乳腺癌的致癌基因[25-27]。
转录信号传导子与激活子(STAT)通路在十多年前作为干扰素诱导的基因表达介质被发现的,包括7个成员,可以被多种细胞因子、生长因子等分子激活,与细胞增殖、分化及凋亡密切相关[28,29],该通路持续激活可导致细胞异常增殖、恶性转化和阻断细胞凋亡。目前已发现STAT1与头颈部癌的放射抵抗相关[30]。AKT-1和STAT-1是否在乳腺癌干细胞中特异性表达将关系到他们是否可以成为乳腺癌放疗和化疗的新靶点。
本研究通过对乳腺癌干细胞进行51种相关辐射抵抗基因的qRT-PCR筛选,并针对AKT-1和STAT-1与乳腺癌干细胞的辐射抵抗关系进行探讨。
方法
1、悬浮培养联合放射线富集具放射抵抗的乳腺癌干细胞
MCF-7细胞复苏后加入完全培养基,分别用有无血清的培养基中培养,至对数生长期时制成单细胞悬液,准备流式染色。一部分悬浮培养细胞经varian2100C/D以300-500cGy/min的剂量率照射8Gy后继续悬浮培养,分别检测第二代、第四代、第五代、第六代、第七代的CD44+/CD24-/low的含量。
2、对乳腺癌干细胞进行51种与肿瘤增殖及周期调控有关的基因表达微阵列分析
采用Primer premier 5软件设计引物,由invitrogen公司合成51种与肿瘤增殖及周期调控有关的基因微阵列组成(具体见正文)。对MCF-7普通培养和悬浮培养的球囊细胞照射前后分别提取细胞RNA,应用荧光定量逆转录聚合酶链式反应(qRT-PCR)进行51种与肿瘤增殖及周期调控有关的基因表达微阵列分析。
3、STAT1与AKT1在乳腺癌始动细胞CD44+/CD24-/low的表达水平
对MCF-7普通培养和悬浮培养的球囊细胞照射前后应用qRT-PCR重复检测STAT1与AKT1的mRNA表达水平,并应用SPSS 13.0软件进行统计学处理,采用student's t检验分析STAT1与AKT1在MCF-7与球囊细胞照射前后的表达水平;
4、加用STAT1与AKT1抑制剂后对乳腺癌始动细胞CD44+/CD24-/low放射生物学特性的影响
分别在MCF-7和球囊细胞中加用STAT1和AKT1抑制剂后,对标本照射前后Annexin V-100 FITC/PI流式细胞仪法检测细胞凋亡、检测克隆形成率和生存曲线,并应用SPSS 13.0软件和3rap hPad Prism(4.0)统计软件处理。
5、统计方法:所有数据均以均值x±SD表示,应用student's t检验分析,P≤0.05为有统计学差异。
结果
1、流式细胞仪检测结果
MCF-7普通培养的含量是0.1%-1.8%,照射后是4.8%-8.3%,悬浮培养后55.5%-69.1%,照射后第二代是75.5%-83.1%,第四代是96.5%-98.8%,第五代是98.3-98.7%,第六代是93.3-95.7%,第七代是90.3%-93.9%。,单纯悬浮培养比普通培养含量高,差异具有显著性(P<0.001);悬浮联合放射线照射后培养比单纯悬浮培养含量高,差异具有统计学意义(P<0.001)。悬浮联合放射线照射后培养第四代到第五代细胞球CD44+CD24-含量最高,其后逐渐稳定。
2、51种与肿瘤增殖及周期调控有关的基因表达微阵列分析
应用qRT-PCR高通量筛选相关基因,分组为MCF-7组、球囊空白组和球囊照射8Gy组,分别检测了51种基因mRNA的表达水平,可见悬浮培养的球囊细胞经过放射线照射后ABL1、AKT1、APC、BAX、BAX3、BRAF、BRCA1、BTK、E2F1、MDAC1、IGFIR、BAF1、SIVA1-1、SIVA1-2、STAT1、TGFB1、TIMP3、TRADD、VDAC1、CDK4、CDK5、CDKN1A、CDKN2B、CDKN2D、ING1、JUN、LCK、GNAS-1、ING1、JUN、LCK、GNAS-1表达明显上调,而CDKN1B、FGR、NFKB1变化不明显,ATM、CDKN1C明显下降。综上所述,BCl-2, BRCA-1,Statl,Ing1,E2F1,AKT1,HDAC1,Kit在照射后的乳腺癌干细胞CD44+/CD24-的mRNA表达水平比照射前升高。
3、STAT1与AKT1在乳腺癌始动细胞CD44+/CD24-/low照射后的表达水平明显上调。
AKT1在乳腺癌MCF-7细胞株照射后和照射前的表达水平无明显差别(P=0.20),但是在球囊细胞CD44+/CD24-/low照射后比照射前明显上调(P<0.01);STAT1在乳腺癌MCF-7细胞株照射后比照射前的表达水平稍有上调(P=0.02),但是在球囊细胞CD44+/CD24-/low照射后比照射前明显上调(P<0.01)。
4、加用STAT1与AKT1抑制剂后对乳腺癌始动细胞CD44+/CD24-/low放射生物学特性的影响
在MCF-7和球囊细胞中分别加用AKT1抑制剂后,照射前后的凋亡率、生存分数改变无明显差异(P>0.05),球囊细胞加用抑制剂后未照射组的凋亡率、生存分数改变无明显差异(P>0.05),加用抑制剂后照射组的凋亡率上升,生存分数减少,差异具有统计学意义(P<0.05);
同样,在MCF-7和球囊细胞中分别加用STAT1抑制剂后,照射前后的凋亡率、生存分数改变无明显差异(P>0.05),球囊细胞加用抑制剂后未照射组的凋亡率、生存分数改变无明显差异(P>0.05),加用抑制剂后照射组的凋亡率上升,生存分数减少,差异具有统计学意义(P<0.05);
结论
1.悬浮培养联合放射线照射得到的干细胞比例高达98%,稳定于90%左右。
2.BCl-2, BRCA-1,Sta1,Ing1,E2F1,AKT1,HDAC1,Kit与乳腺癌干细胞的放疗抵抗可能有关.
3.AKT1和STAT1与乳腺癌干细胞的放射抵抗密切相关,阻断各自的通路有可能加强乳腺癌的放疗效果。
本研究的创新之处
1、利用悬浮培养联合放射线照射成功富集了乳腺癌干细胞,比之前所有的分离方法得率都高。
2、第一次应用qRTPCR的方法定量检测乳腺癌干细胞的相关放疗抵抗基因,发现BCl-2, BRCA-1, Stat1,Ing1,E2F1,AKT1,HDAC1,Kit与有关;
3、分别应用AKT1和STAT1特异性抑制剂来抑制PI3K/Akt、JAK/STAT通路的活化,进一步验证了AKT1、STAT1与乳腺癌干细胞CD44+/CD24-/low的放射抵抗相关。
4、乳腺癌干细胞的放射抵抗基因研究目前尚未有报道,本研究填补了空白。
Background and Objection:
In recent years, a growing body of evidence has been reported supporting the hypothesis that tumors are driven and maintained by a minority subpopulation of cells that have the capacity to self-renew (i.e.,give rise to progeny with similar properties as themselves) and to generate the more differentiated progeny which make up the bulk of a tumor.The minority population has been termed cancer stem cells (CSCs), tumorigenic cancer cells, or tumor-initiating cells, by various investigators, to indicate that only they can give rise to new tumors when transplanted into immunodeficient animals. Tumor-initiating cells have been identified in blood, brain, breast cancers,prostate, liver, pancreas, and colon cancers (4-6) through an experimental strategy that combines sorting of tumor cell subpopulations, identified on the basis of the different expression of surface markers, with functional transplantation into appropriate animal models. The subpopulation with the CD24-/low/CD44+ phenotype was shown to identify breast cancer-initiating cells in primary patient specimens which 200 cells were transplanted into NOD/SCID mice and formed tumor by Al Hajj using fluorescence-activated cell sorting (FACS) etc in 2003.Then in 2005,Ponti etc showed that breast tumorigenic cells with stem/progenitor cell properties can be propagated in vitro as nonadherent mammospheres and represent a suitable in vitro model to study breast cancer-initiating cells and to challenge them with molecularly targeted agents specifically interfering with breast cancer-initiating cells self-renewal and survival.
According to the hypothesis, CSCs limited number within the bulk of the tumor are not only the source of the tumor but also may be responsible for tumor progression,metastasis,thus leading to subsequent tumor relapse although the primary lesion is eradicated and resistance to therapy; Radiotherapy, as an integral part of the current comprehensive breast cancer treatment regimen, may be used to eradicate remaining cancer cells in the breast, chest wall, or axilla after surgery or to reduce the size of an advanced tumor before surgery.hence, it is imperative to understand the molecular mechanism underlying this radioresistance. A number of candidate genes have been implicated in the response of eukaryotic cells to ionizing radiation, including cell cycle, checkpoint, and DNA repair genes, as well as mediators of apoptosis, such as p53,Bax,Bcl-2, and so forth.
PI3K-AKT signaling is an important cascade to promote cell survival in response to exogenous stress factors such as exposure to ionizing radiation. It has been shown that PI-3K pathway plays a critical role in mediating radioresistance.One of the best characterized downstream targets for the PI-3K lipid products is Aktl, which was discovered about a decade ago and which is now known to be a member of a family of closely related, highly conserved cellular homologues that consists of at least two additional members (Akt2 and Akt3). Akt has been shown to be a critical player in oncogenesis. There is increasing evidence indicating that PI-3K/Akt plays an important role in breast cancer tumorigenesis. An elevated level of Akt activity was associated with increased cellular resistance to the treatment with doxorubicin or tamoxifen in breast cancer cell lines.
STAT transcription factors were discovered 10 years ago as mediators of interferon-induced gene expression. They now form an important group, comprising seven members, that are activated by virtually every cytokine and growth factor. Cell culture work has further delineated their importance in cellular proliferation, transformation, apoptosis, differentiation and growth control. STAT1,STAT3 and STAT5 may play an important role in breast carcinogenesis. Recently, signal transducer and activator of transcription 1(STAT1)has been implicated in the presence of radioresistance,first demonstrated in a head-and-neck cancer cell line (4).
To date, AKT1 and STAT1 involvement in CD24-/low/CD44+ breast cancer-initiating cells in general and their specific relationship with radioresistance of CD24-/low/CD44+ breast cancer-initiating cells have never been explored.
This study intend to discover the genes which may play a key role in radioresistence of CD24-/low/CD44+ breast cancer-initiating cells by qRT-PCR and investigate the effects of AKT1 and STAT1 in radiosensitivity by the additional of specific inhibitors.
METHODS AND MATERIALS
1.Suspension culture combined with radiation to enrich CD24-/low/CD44+breast cancer-initiating cells that have more radioresistance.
MCF-7 cultured in SSM and SFM respectively. Then some mammaspheres were cultured with radiation at a dose rate of 300-500 cGy/min by varian2100 C/D. The cells cultured to achieve 80-90% confluence on the day of experiments were stained with CD24-PE and CD44-FITC and analyzed the percentage of CD44+CD24- by flow cytometry.
2. The 51 genes chip was designed by Primer 5 software and syntheses by Invitrogen Co.Real-time quantitative reverse transcription polymerase chain reactions performed to detect the mRNA level of 51 genes in MCF-7 and mammaspheres before and after radiation.
3.AKT1 and STAT1 mRNA levels were examined d by qRT-PCR repeatedly.
4. Evaluated apoptosis by flow cytometrywith Annexin-V100 FITC/PI double staining method and calculated the plating efficiency by the number of colonies/cells seeded and fitted the surviving fraction by GraphPad Prism(4.0) in MCF-7 and mammaspheres before and after AKT1 or STAT1 inhibitor before and after radiation..
5.All data are represented as means and differences of the means with 95% confidence intervals (CIs). P values of.05 or less, calculated using a paired two-sided Student's t test or chi-square test, were considered to indicate statistically significant differences.
RESULTS
1.Flow-cytometric analysis.
The percentage of cells from suspension culture combined with radiation higher than those in single suspension culture without radiation and the difference was determined to be statistically significant(P<0.001)
2.It was revealed that-mRNA levels of BC1-2, BRCA-1,Statl,Ing1, E2F1, AKT1,HDAC1 and Kit were higher after radiation than those before radiation in mammaspheres.
3.The mRNA levels of STAT1 and AKT1 were higher after radiation than those before radiation in mammaspheres and the difference was determined to be statistically significant(P<0.001),whereas,there was no difference between MCF-7 before and after radiation.
4. Apoptosis and the surviving fraction had no difference in MCF-7 treated with AKT1 inhibitor or STAT1 inhibitor respectively before and after radiation and mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively before radiation(P>0.05).However, Apoptosis was enhanced and the surviving fraction decreased in mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively after radiation.The difference was determined to be statistically significant(P<0.001).
Conclusion
1.The percentage of CD44+CD24-through suspension culture combined with radiation is up to 98%,stable at about 90%。
2.Our data suggest that BC1-2, BRCA-1,Stat1,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low.
3.AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and manipulation of this pathway may enhance the efficacy of radiotherapy.
Innovations of our study:
1.The enrichment of CD44+CD24-through suspension culture combined with radiation is successful than all the methods previous。
2.This is the first study reporting the mRNA level of genes which may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low by qRT-PCR and suggesting that BC1-2, BRCA-1,Statl,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance.
3.The blockage of PI3K/AKT and JAK/STAT by the addition of a STAT1-specific inhibitor, fludarabine, and AKT1-specific inhibitor, significantly increased the fraction of apoptotic cells.These results confimed that AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and the targeting of STAT1 and AKT1 could prove to be a promising new therapeutic strategy against breast cancer.
4.Genetic studies of radioresistence in breast cancer stem cell has been repoted yet and our study provide a valuable result.
引文
[1]Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med,1997. 3(7):p.730-7.
[2]Miyamoto, T.,I.L. Weissman and K. Akashi, AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8,21 chromosomal translocation. Proc Natl Acad Sci U S A,2000.97(13):p. 7521-6.
[3]Reya, T., et al.,Stem cells, cancer, and cancer stem cells. Nature,2001. 414(6859):p.105-11.
[4]Greaves, M., Cancer stem cells:Back to Darwin?. Semin Cancer Biol,2010.
[5]Bhatia, M.,et al.,Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A,1997. 94(10):p.5320-5.
[6]Singh, S.K.,et al., Identification of human brain tumour initiating cells. Nature,2004.432(7015):p.396-401.
[7]Dick, J.E.,Breast cancer stem cells revealed. Proc Natl Acad Sci U S A,2003. 100(7):p.3547-9.
[8]Brown, M.D., et al., Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations. Prostate,2007.67(13):p.1384-96.
[9]Li, C., et al., Identification of pancreatic cancer stem cells. Cancer Res,2007. 67(3):p.1030-7.
[10]Wang, J.,et al.,Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res,2007.67(8):p. 3716-24.
[11]Yi, S.Y.and K.J. Nan, Tumor-initiating stem cells in liver cancer. Cancer Biol Ther,2008.7(3):p.325-30.
[12]Ricci-Vitiani,L.,et al., Identification and expansion of human colon-cancer-initiating cells. Nature,2007.445(7123):p.111-5.
[13]Al-Hajj,M.,et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A,2003.100(7):p.3983-8.
[14]Ponti, D., et al.,Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res,2005.65(13):p. 5506-11.
[15]Jemal, A.,et al., Cancer statistics,2004. CA Cancer J Clin,2004.54(1):p. 8-29.
[16]Diehn, M. and M.F. Clarke, Cancer stem cells and radiotherapy:new insights into tumor radioresistance. J Natl Cancer Inst,2006.98(24):p.1755-7.
[17]O'Brien, C.S.,et al.,Are stem-like cells responsible for resistance to therapy in breast cancer?. Breast Dis,2008.29:p.83-9.
[18]Vlashi, E., W.H. McBride and F. Pajonk, Radiation responses of cancer stem cells. J Cell Biochem,2009.108(2):p.339-42.
[19]Wendel, H.G.,et al.,Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature,2004.428(6980):p.332-7.
[20]Rossi, D.J. and I.L. Weissman, Pten, tumorigenesis, and stem cell self-renewal. Cell,2006.125(2):p.229-31.
[21]Tanno, S., et al.,AKT activation up-regulates insulin-like growth factor Ⅰ receptor expression and promotes invasiveness of human pancreatic cancer cells. Cancer Res,2001.61(2):p.589-93.
[22]Kim, D., et al.,Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J,2001.15(11):p.1953-62.
[23]Grille, S.J.,et al., The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res,2003.63(9):p.2172-8.
[24]Bleau, A.M.,et al.,PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell,2009.4(3):p.226-35.
[25]Horn, G.,et al., ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1. Exp Cell Res,2009.315(8):p.1490-504.
[26]Hutchinson, J.N.,et al.,Activation of Akt-1 (PKB-alpha) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res,2004.64(9):p.3171-8.
[27]Arboleda, M.J.,et al., Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res,2003.63(1):p.196-206.
[28]Carlesso, N.,D.A. Frank and J.D. Griffin, Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med,1996. 183(3):p.811-20.
[29]Sheng, X.R.,et al.,Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis. Dev Biol,2009.334(2):p. 335-44.
[30]Lee, M.Y.,et al.,Phosphorylation and activation of STAT proteins by hypoxia in breast cancer cells. Breast,2006.15(2):p.187-95.
[31]Reya, T.,et al.,Stem cells, cancer, and cancer stem cells. Nature,2001. 414(6859):p.105-11.
[32]Dontu, G., et al., Stem cells in normal breast development and breast cancer. Cell Prolif,2003.36 Suppl 1:p.59-72.
[33]Kondo, T., T. Setoguchi and T. Taga, Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A, 2004.101(3):p.781-6.
[34]Zhou, S.,et al., The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med,2001.7(9):p.1028-34.
[35]Telford, W.G. and E.G. Frolova, Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes. Cytometry A,2004.57(1):p.45-52.
[36]Patrawala, L.,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(14):p.6207-19.
[37]Hirschmann-Jax, C.,et al.,A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A,2004. 101(39):p.14228-33.
[38]Youn, B.S., et al.,Scale-up of breast cancer stem cell aggregate cultures to suspension bioreactors. Biotechnol Prog,2006.22(3):p.801-10.
[39]Staud, F. and P. Pavek, Breast cancer resistance protein (BCRP/ABCG2). Int J Biochem Cell Biol,2005.37(4):p.720-5.
[40]Rosner, A., et al.,Pathway pathology:histological differences between ErbB/Ras and Wnt pathway transgenic mammary tumors. Am J Pathol,2002. 161(3):p.1087-97.
[41]Jr Cohen, M.M.,The hedgehog signaling network Am J Med Genet A,2003. 123A(1):p.5-28.
[42]Dontu, G., et al., Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res,2004.6(6):p. R605-15.
[43]Shimada, Y.,et al.,Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung. Cancer Sci,2009.100(11):p.2054-9.
[44]Harrison, H., et al., Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res,2010.70(2):p.709-18.
[45]Cariati, M. and A.D. Purushotham, Stem cells and breast cancer. Histopathology,2008.52(1):p.99-107.
[46]Wang, X.Y.,et al.,Musashil modulates mammary progenitor cell expansion through proliferin-mediated activation of the Wnt and Notch pathways. Mol Cell Biol,2008.28(11):p.3589-99.
[47]Li, Y.,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(26):p.15853-8.
[48]Li, Y.,W.P. Hively and H.E. Varmus, Use of MMTV-Wnt-1 transgenic mice for studying the genetic basis of breast cancer. Oncogene,2000.19(8):p. 1002-9.
[49]Gunther, E.J., et al.,Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis. Genes Dev,2003.17(4):p.488-501.
[50]Morrison, S.J.,N.M. Shah and D.J. Anderson, Regulatory mechanisms in stem cell biology. Cell,1997.88(3):p.287-98.
[51]Liu, B.Y., et al.,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(12):p.4158-63.
[52]Moon, R.T.,et al., The promise and perils of Wnt signaling through beta-catenin. Science,2002.296(5573):p.1644-6.
[53]Chen, M.S.,et al., Wnt/beta-catenin mediates radiation resistance of Scal+ progenitors in an immortalized mammary gland cell line. J Cell Sci,2007. 120(Pt 3):p.468-77.
[54]Veeck, J., et al., Epigenetic inactivation of the secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis. Carcinogenesis,2008.29(5):p.991-8.
[55]Lewis, M.T., et al.,The Gli2 transcription factor is required for normal mouse mammary gland development. Dev Biol,2001.238(1):p.133-44.
[56]Liu, S.,et al.,Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res,2006.66(12):p. 6063-71.
[57]Beachy, P.A.,S.S.Karhadkar and D.M. Berman, Tissue repair and stem cell renewal in carcinogenesis. Nature,2004.432(7015):p.324-31.
[58]Kubo, M.,et al.,Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res,2004.64(17):p.6071-4.
[59]Dave, B.and J. Chang, Treatment resistance in stem cells and breast cancer. J Mammary Gland Biol Neoplasia,2009.14(1):p.79-82.
[60]Debeb, B.G.,W. Xu and W.A. Woodward, Radiation resistance of breast cancer stem cells:understanding the clinical framework.J Mammary Gland Biol Neoplasia,2009.14(1):p.11-7.
[61]Phillips, T.M., W.H. McBride and F. Pajonk, The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst,2006.98(24):p.1777-85.
[62]Woodward, W.A.,et al., WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A,2007.104(2):p. 618-23.
[63]Katso, R.,et al.,Cellular function of phosphoinositide 3-kinases:implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol,2001.17: p.615-75.
[64]Brunet, A.,et al.,Ak promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell,1999.96(6):p.857-68.
[65]Danilkovitch, A.,et al.,Two independent signaling pathways mediate the antiapoptotic action of macrophage-stimulating protein on epithelial cells. Mol Cell Biol,2000.20(6):p.2218-27.
[66]Eves, E.M.,et al.,Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol Cell Biol,1998.18(4):p. 2143-52.
[67]Cantley, L.C., The phosphoinositide 3-kinase pathway. Science,2002. 296(5573):p.1655-7.
[68]Staal,S.P.,Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2:amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A,1987.84(14):p.5034-7.
[69]Carpten, J.D., et al.,A transforming mutation in the pleckstrin homology domain ofAKT1 in cancer. Nature,2007.448(7152):p.439-44.
[70]Singh, S.R.,X. Chen and S.X. Hou, JAK/STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila. Cell Res,2005. 15(1):p.1-5.
[71]Jiang, H.,et al., Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell,2009.137(7):p.1343-55.
[72]Levine, R.L. and G. Wernig, Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders. Hematology Am Soc Hematol Educ Program, 2006:p.233-9,510.
[73]Aalinkeel, R.,et al.,Genomic Analysis Highlights the Role of the JAK-STAT Signaling in the Anti-proliferative Effects of Dietary Flavonoid'Ashwagandha' in Prostate Cancer Cells. Evid Based Complement Alternat Med,2008.
[74]Atreya, R. and M.F. Neurath, Signaling molecules:the pathogenic role of the IL-6/STAT-3 trans signaling pathway in intestinal inflammation and in colonic cancer. Curr Drug Targets,2008.9(5):p.369-74.
[75]Clevenger, C.V.,Roles and regulation of stat family transcription factors in human breast cancer. Am J Pathol,2004.165(5):p.1449-60.
[76]Lee, M.Y., et al., Phosphorylation and activation of STAT proteins by hypoxia in breast cancer cells. Breast,2006.15(2):p.187-95.
[77]Tang, J.Z., et al., Signal transducer and activator of transcription (STAT)-5A and STAT5B differentially regulate human mammary carcinoma cell behavior. Endocrinology,2010.151(1):p.43-55.
[78]Ho, J.N., et al., Bcl-X(L) and STAT3 mediate malignant actions of gamma-irradiation in lung cancer cells. Cancer Sci,2010.
[79]Catlett-Falcone, R., et al.,Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity,1999.10(1): p.105-15.
[80]Oshiro, M.M., et al., Inhibition of JAK kinase activity enhances Fas-mediated apoptosis but reduces cytotoxic activity of topoisomerase II inhibitors in U266 myeloma cells. Clin Cancer Res,2001.7(12):p.4262-71.
[81]Vaclavicek, A., et al., Polymorphisms in the Janus kinase 2 (JAK)/signal transducer and activator of transcription (STAT) genes:putative association of the STAT gene region with familial breast cancer. Endocr Relat Cancer, 2007.14(2):p.267-77.
[82]Garcia, R., et al., Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene,2001.20(20):p.2499-513.
[83]Klampfer, L.,Signal transducers and activators of transcription (STATs): Novel targets of chemopreventive and chemotherapeutic drugs. Curr Cancer Drug Targets,2006.6(2):p.107-21.
[84]Bowman, T.,et al., STATs in oncogenesis. Oncogene,2000.19(21):p. 2474-88.
[85]Singh, S.R., X. Chen and S.X. Hou, JAK/STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila. Cell Res,2005. 15(1):p.1-5.
[86]Benekli, M,et al., Signal transducer and activator of transcription proteins in leukemias. Blood,2003.101(8):p.2940-54.
[87]Demoulin, J.B.,et al.,STAT5 activation is required for interleukin-9-dependent growth and transformation of lymphoid cells. Cancer Res,2000. 60(14):p.3971-7.
[88]Terui,K.,et al.,Hypoxia/re-oxygenation-induced,redox-dependent activation of STAT1 (signal transducer and activator of transcription 1) confers resistance to apoptotic cell death via hsp70 induction. Biochem J,2004. 380(Pt1):p.203-9.
[89]Hui, Z.,et al.,Radiosensitization by inhibiting STAT1 in renal cell carcinoma. Int J Radiat Oncol Biol Phys,2009.73(1):p.288-95.
[90]Ailles, L.E. and I.L. Weissman, Cancer stem cells in solid tumors. Curr Opin Biotechnol,2007.18(5):p.460-6.
[91]Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med,1997. 3(7):p.730-7.
[92]Dalerba, P., R.W. Cho and M.F. Clarke, Cancer stem cells:models and concepts. Annu Rev Med,2007.58:p.267-84.
[93]Smalley, M. and A. Ashworth, Stem cells and breast cancer:A field in transit. Nat Rev Cancer,2003.3(11):p.832-44.
[94]Pardal, R.,M.F. Clarke and S.J. Morrison, Applying the principles of stem-cell biology to cancer. Nat Rev Cancer,2003.3(12):p.895-902.
[95]Cho, R.W. and M.F. Clarke, Recent advances in cancer stem cells. Curr Opin Genet Dev,2008.18(1):p.48-53.
[96]Alvi, A.J., et al., Functional and molecular characterisation of mammary side population cells. Breast Cancer Res,2003.5(1):p. R1-8.
[97]Chen, A.Y., et al.,A new mammalian DNA topoisomerase I poison Hoechst 33342:cytotoxicity and drug resistance in human cell cultures. Cancer Res, 1993.53(6):p.1332-7.
[98]Singh, S.,B.S.Dwarakanath and T.L. Mathew, DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines. J Photochem Photobiol B,2004.77(1-3):p.45-54.
[99]Hemmati, H.D., et al.,Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A,2003.100(25):p.15178-83.
[100]Singh, S.K., et al.,Identification of a cancer stem cell in human brain tumors. Cancer Res,2003.63(18):p.5821-8.
[101]Singh, S.K., et al., Identification of human brain tumour initiating cells. Nature,2004.432(7015):p.396-401.
[102]Dontu, G.,et al.,In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev,2003.17(10):p.1253-70.
[103]Dontu, G. and M.S.Wicha, Survival of mammary stem cells in suspension culture:implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia,2005.10(1):p.75-86.
[104]Li, H.Z., T.B.Yi and Z.Y. Wu, Suspension culture combined with anticancer regimens for screening breast cancer stem cells. Med Hypotheses,2007. 68(5):p.988-90.
[105]Ross, D.D. and T. Nakanishi, Impact of breast cancer resistance protein on cancer treatment outcomes. Methods Mol Biol,2010.596:p.251-90.
[106]Ponti, D., et al., Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res,2005.65(13):p. 5506-11.
[107]Diehn, M. and M.F.Clarke, Cancer stem cells and radiotherapy:new insights into tumor radioresistance. J Natl Cancer Inst,2006.98(24):p.1755-7.
[108]Dean, M.,T. Fojo and S.Bates, Tumour stem cells and drug resistance. Nat Rev Cancer,2005.5(4):p.275-84.
[109]Yu, F.,et al.,let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell,2007.131(6):p.1109-23.
[110]Britten, R.A.,et al.,Intratumoral heterogeneity as a confounding factor in clonogenic assays for tumour radioresponsiveness. Radiother Oncol,1996. 39(2):p.145-53.
[111]Pelevina, I.I., et al., [Properties of progeny of irradiated cells]. Tsitologiia, 1998.40(5):p.467-77.
[112]Mitsuhashi, N., et al., A radioresistant variant cell line, NMT-1R, isolated from a radiosensitive rat yolk sac tumour cell line, NMT-1:differences of early radiation-induced morphological changes, especially apoptosis. Int J Radiat Biol,1996.69(3):p.329-36.
[113]Wei, K.,R. Kodym and C. Jin, Radioresistant cell strain of human fibrosarcoma cells obtained after long-term exposure to x-rays. Radiat Environ Biophys,1998.37(2):p.133-7.
[114]Smith, A.B., et al.,Rapid detection of influenza A and B viruses in clinical specimens by Light Cycler real time RT-PCR. J Clin Virol,2003.28(1):p. 51-8.
[115]Woodward, W.A., et al.,On mammary stem cells. J Cell Sci,2005.118(Pt 16): p.3585-94.
[116]Cheung, A.M. and T.W. Mak, PTEN in the haematopoietic system and its therapeutic indications. Trends Mol Med,2006.12(11):p.503-5.
[117]Martin-Belmonte, F., et al.,PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell, 2007.128(2):p.383-97.
[118]Enomoto, A., et al.,Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell,2005.9(3):p.389-402.
[119]Yoeli-Lerner, M.,et al.,Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT. Mol Cell,2005.20(4):p.539-50.
[120]Ju, X., et al., Aktl governs breast cancer progression in vivo. Proc Natl Acad Sci U S A,2007.104(18):p.7438-43.
[121]Liu, H., et al.,Mechanism of Aktl inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2. Proc Natl Acad Sci U S A,2006. 103(11):p.4134-9.
[122]Karhadkar, S.S.,et al., Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature,2004.431(7009):p.707-12.
[123]Hollestelle, A.,et al.,Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines. Mol Cancer Res,2007.5(2):p. 195-201.
[124]Neve, R.M.,et al., A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell,2006.10(6):p.515-27.
[125]Arboleda, M.J.,et al., Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res,2003.63(1):p.196-206.
[126]Yu, H. and R. Jove, The STATs of cancer--new molecular targets come of age. Nat Rev Cancer,2004.4(2):p.97-105.
[127]Miranda, C., et al.,Role of STAT3 in in vitro transformation triggered by TRK oncogenes. PLoS One,2010.5(3):p. e9446.
[128]Turkson, J., STAT proteins as novel targets for cancer drug discovery. Expert Opin Ther Targets,2004.8(5):p.409-22.