TIP30基因敲除诱发小鼠乳腺癌的相关研究
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摘要
乳腺癌是女性发病率最高的恶性肿瘤之一,全世界每年约有138万妇女患乳腺癌,约45万人死于乳腺癌。在全球,乳腺癌发病率仍呈逐年上升趋势；其中欧美国家近25年来乳腺癌死亡率有所下降,但在亚太及非洲很多国家乳腺癌死亡率仍在上升中。近年来,尽管乳腺癌的诊断和治疗均有了长足的进展,但关于乳腺癌的发病机理目前仍未明确。雌激素受体(Estrogen receptor, ER)在乳腺的发育及乳腺癌的发生发展中均起了重要的作用。在人类乳腺癌中,ER阳性乳腺癌就高达60-70%。临床上已将ER检测作为评估患者预后和选择治疗方案的指标。尽管ER阳性乳腺癌的治疗效果及预后较好,但目前对于这种最常见类型乳腺癌发病机理的认识仍有限。在本研究中,我们应用TIP30基因敲除小鼠模型探讨了该亚型乳腺癌发生发展的可能机制。
TIP30基因,又称CC3或Htatip2,是1998年肖华等在研究人类免疫缺陷病毒(Human immunodeficiency virus, HIV)的体外转录时发现一种分子量为30kD的Tat(Transactivator of transcription)结合蛋白(Tat interactive protein)。研究发现TIP30在多种肿瘤组织中的表达异常,如肺癌、乳腺癌、胃癌、结肠癌、肝细胞癌和前列腺癌等,提示TIP30与肿瘤之间存在密切联系。Hua等研究发现,TIP30基因敲除可以自发形成多种肿瘤。这些研究提示TIP30是一种肿瘤抑制基因。TIP30具有多种功能,可以作为一种转录共分子参与凋亡及增殖相关基因的调控,它还可以作为细胞核转运抑制因子调节细胞凋亡。在乳腺和乳腺癌细胞中,TIP30还可以抑制ERa调节的c-myc转录。近来研究发现,在肝癌和肺癌中,TIP30可以调控EGFR细胞浆内转运和下游信号通路的活性。
我们先前的研究发现,TIP30基因敲除可以诱发小鼠乳腺导管增生,而且随着年龄的增长,增生程度越明显。同时,我们在有Erbb2(Neu)转基因背景的FVB小鼠中敲除TIP30,发现TIP30基因缺失可以加速小鼠乳腺癌发生,而且所有自发性乳腺癌均为ER阳性乳腺癌。而在本研究中,我们排除了Neu基因的影响,发现在Balb/c背景的小鼠中敲除TIP30便能诱发小鼠乳腺癌。为了进一步研究其可能的机制,我们对该小鼠模型EGFR下游信号分子进行了初步的探讨,并在人乳腺癌细胞株中研究了TIP30基因在EGFR降解中的调节作用。
由于乳腺癌是异质性相当大的肿瘤,其病理和分子学特点具有多样性复杂性。近年来,乳腺肿瘤形成的干细胞模型为乳腺癌的异质性及不同乳腺癌亚型的起源提供了解释。这一模型认为不同乳腺肿瘤亚型是起源于不同的乳腺干细胞或祖细胞,而特异性的突变可使这些细胞发生恶性转化,从而使其自我更新失控及异常分化,最终诱发肿瘤发生。此外,在小鼠的研究中发现,不同基因对不同亚群乳腺上皮细胞影响不同,而且会影响乳腺癌亚型的发生。比如,乳腺癌病毒(mmTv)-Wnt-1小鼠可以扩增乳腺干细胞亚群,同时形成luminal型和basal型乳腺癌。而nmTv-Neu小鼠可以促进luminal细胞群扩增,仅产生luminal型且ER/PR阴性乳腺癌。因此通过在小鼠动物模型中研究特定基因对乳腺细胞亚群及肿瘤类型形成的影响将有助于增强对乳腺癌发病机理的了解。此外,我们先前的研究发现,TIP30基因敲除后可以使Neu转基因小鼠乳腺癌的表型由ER-/PR-阴性转变为ER+/PR-,提示TIP30基因不只是个抑癌基因,它在乳腺癌表型的决定上也可能起了一定的作用。因此,本研究在干细胞层面探讨了TIP30基因缺失对乳腺干细胞／祖细胞亚群及其分化倾向方面可能存在的作用,并为该动物模型形成的限制性ER阳性乳腺癌提供了一定的解释。
第一部分TIP30基因敲除诱发小鼠乳腺癌及活化EGFR下游通路的研究
一、材料与方法
1.通过杂交的方法,获得TIP30基因敲除的Balb/c小鼠,连续传代7代以上,获得Balb/c纯合背景的TIP30基因敲除小鼠。
2.所有Balb/c小鼠饲养至满78周时,二氧化碳吸入处死,详细检查小鼠大脑、乳腺、胸腔脏器、腹腔脏器、生殖器官等肉眼可见部位有无肿瘤和其它疾病出现。常规组织固定、石蜡包埋并切片。部分组织丢入液氮速冻,最后于-80℃保存。
3.应用苏木精-伊红(H&E)染色法检测组织器官及乳腺肿瘤组织形态结构。切片由两位病理学家阅片并分析。计算发生乳腺癌的小鼠百分比。
4.应用免疫组织化学(IHC)法检测乳腺组织或肿瘤组织CK8、αSMA、pAkt、 pERK表达。
5.应用免疫荧光染色(IF)法检测乳腺肿瘤组织ER及PR表达情况。
6.应用shRNA-TIP30及shRNA-CON质粒包装慢病毒颗粒,感染MCF-7细胞,puromycin筛选,并经Western-blot检测验证基因干扰效果。
7.应用细胞免疫荧光方法检测经EGF诱导的MCF-7细胞在不同时间点EGF与EGFR、EGFR与EEA1及TIP30与EEA1的共表达情况。
8.统计处理：实验结果以均数±标准差(x±s)表示,数据处理采用SPSS13.0统计软件。两组的肿瘤发生率比较采用χ2检验；两组及多组间均数比较分别采用独立样本t检验和单向方差分析；两组不同时间点均数比较采用重复测量方差分析；多重比较采用LSD检验或Dunnett检验。P50.05(双侧)表示差异有显著性。
二、结果
1.TIP30基因敲除可导致Balb/c小鼠自发性乳腺肿瘤出现
经过18个月的密切观察,共有47只雌性未孕小鼠完成实验观察。28只Tip30-/-小鼠中有8只发现乳腺自发肿瘤(发病率28.6%),而19只Tip30+/+小鼠中均未发现有乳腺自发肿瘤(0%)。Tip30"/-小鼠乳腺自发肿瘤的发生率显著高于Tip30+/+小鼠,x2=6.269,P=0.012。
2.TIP30基因敲除致Balb/c小鼠乳腺肿瘤均为ER/PR阳性luminal型
我们应用luminal标志CK8(?)basal标志αSMA对乳腺肿瘤组织行免疫组织化学染色,结果提示,所有肿瘤组织CK8染色呈强阳性,而aSMA染色阴性,提示Tip30-/-小鼠自发乳腺肿瘤均为luminal型。进一步的ER及PR免疫荧光染色,结果显示所有肿瘤均为ER阳性和PR阳性乳腺癌。提示Tip30-/-小鼠自发乳腺肿瘤为ER/PR阳性luminal型肿瘤。
3.TIP30基因敲除可致EGFR下游MAPK及P13K信号通路的活化
我们应用了免疫组化对乳腺及肿瘤组织pErk1/2及pAkt进行检测。Tip30-/-小鼠乳腺上皮细胞pErk1/2及pAkt阳性率分别为27.83±8.46%和30.83±6.65%(n=5),显著高于rip30+/+小鼠乳腺上皮细胞的12.58±5.87%和14.94±5.77%(n=5),P值分别0.02和0.011。而Tip30-/-小鼠乳腺肿瘤细胞pErk1/2及pAkt阳性率分别为51.68±8.57%和56.08±8.44%(n=5),则显著高于Tip30-/-小鼠乳腺上皮细胞的27.83±8.46%和30.83±6.65%,P分别为0.002和0.001。
4.TIP30表达下调可延迟EGF诱导的人乳腺癌细胞中EGFR的降解。
4.1TIP30-SH1、TIP30-SH2乳腺细胞株MCF-7的TIP30蛋白明显抑制
乳腺癌细胞株MCF-7通过shRNA干扰后,以内参[3-actin为标准,TIP30-SH1和TIP30-SH2细胞TIP30蛋白表达相对值分别为13.12±3.95%和11.54±2.68%(n=3),显著低于shRNA-CON细胞的84.3±8.66%(n=3),P均<0.001。TIP30-SH1及TIP30-SH2细胞TIP30蛋白表达分别下降达71.18%及72.76%,这些结果提示基因转染后TIP30-SH1、TIP30-SH2细胞TIP30表达被显著抑制。
4.2TIP30在乳腺癌细胞中可促进EGF诱导的EGFR降解
TIP30-SH组细胞在经EGF作用后,10min和1h时EGF及EGFR的荧光强度与shRNA-CON组相比,均无统计学差异；4h时两组EGF及EGFR荧光强度均显著下降,其中TIP30-SH组细胞EGF及EGFR的荧光强度分别为1.19±0.18和1.33±0.13,显著性高于shRNA-CON组0.68±0.08和0.76±0.10,t值分别为4.563和6.132,P值分别为0.01和0.004,以上结果提示细胞的TIP30基因干扰后,EGF及EGFR降解速度减慢,于4h时仍有大量EGF及EGFR未降解。
同时检测EGF及EGFR共定位情况时发现,两组细胞EGF及EGFR在10min时Pearson's共定位值最高,随时间延长逐渐降低。于4h时,TIP30-SH组共定位值为35.07±5.33,显著性高于shRNA-CON组的10.76±2.28,t=7.270,P=0.002。提示相对于shRNA-CON组,TIP30-SH组细胞EGF及EGFR推迟分离降解,有更多的EGF/EGFR结合物存在于细胞内。
4.3TIP30基因下调可延长EGFR在早期内涵体内聚集时间
TIP30-SH及shRNA-CON细胞在经EGF处理2小时后,shRNA-CON细胞EGFR绿色荧光与EEA1红色荧光Pearson's共定位值为27.31±9.12%,显著性低于TIP30-SH细胞组的56.39±14.08%,t=3.003,P=0.04。提示TIP30基因干扰后,EGFR的推迟从早期内涵体分离。
4.4TIP30可聚集于早期内涵体内,参与EGFR代谢
EGF处理shRNA-CON细胞10分钟后,激光共聚焦显微镜下TIP30绿色荧光与EEA1红色荧光明显重叠,提示TIP30在早期进入内涵体参与EGFR转运。
三、结论
1.TIP30基因敲除的Balb/c小鼠可自发乳腺癌。
2.TIP30基因敲除的Balb/c小鼠所发生乳腺癌均为ER/PR阳性luminal型肿瘤。
3.TIP30基因敲除可以激活乳腺及乳腺癌中EGFR下游MAPK及PI3K信号通路分子。
4.TIP30基因抑制可干扰EGFR自内涵体分离,抑制EGF诱导的EGFR降解,导致EGFR下游信号分子持续激活。
第二部分TIP30基因敲除诱导小鼠乳腺干/祖细胞扩增并影响其分化倾向的研究
一、材料与方法
1.通过杂交的方法,获得TIP30基因敲除的Balb/c及Neu+/+FVB小鼠,连续传代7代以上,获得各种系纯合背景的TIP30基因敲除小鼠。
2.解剖收集5-6月龄小鼠乳腺组织并剪碎,用无血清胶原酶溶液消化,获得乳腺上皮细胞悬液。
3.应用细胞悬浮球形培养方法检测各基因表型小鼠乳腺上皮细胞乳腺小球形成能力。显微镜下拍照并计算乳腺小球数量及大小。
4.应用体外克隆形成试验检测各基因表型小鼠乳腺上皮细胞克隆形成能力。显微镜下拍照并计算克隆形成数及大小
5.应用流式细胞仪分析各基因表型小鼠乳腺上皮细胞CD24HighCD49flow (MRUs)及CD24HighCD49flow (Ma-CFCs)百分比例,并分析CD24HighCD49low群中Scal+及Scal-比例。
6.应用流式细胞仪分选出Balb/c小鼠各基因表型乳腺上皮细胞中祖细胞群CD24HighCD49flow并行体外克隆形成试验,收集克隆并行ER/PR免疫荧光染色。
7.应用免疫荧光法检测Balb/c小鼠各基因表型乳腺组织ER/PR表达情况。
8.应用qPCR检测Balb/c小鼠各基因表型乳腺组织中与乳腺干/祖细胞分化相关的因子表达情况。
9.应用Western-blot检测Balb/c各基因表型小鼠乳腺组织FOXA1及RANKL表达。
10.应用shRNA-FOXA1及shRNA-CON质粒包装慢病毒颗粒,感染乳腺细胞,puromycin筛选,并经Western-blot检测验证基因干扰效果后,行体外克隆形成试验并行克隆ER免疫荧光染色。
11.统计处理：实验结果以均数±标准差(x±s)表示,数据处理采用SPSS13.0统计软件,两组及多组间均数比较分别采用独立样本t检验和单向方差分析；多重比较采用LSD检验或Dunnett检验。P<0.05(双侧)表示差异有显著性。
二、结果
1.TIP30基因敲除可促进乳腺干/祖细胞群扩增
1.1TIP30基因敲除可增强乳腺上皮细胞体外乳腺小球形成能力
小鼠乳腺上皮细胞经体外悬浮无血清培养9天后,Tip30-/-Balb/c小鼠每5000个乳腺上皮细胞形成乳腺小球数目为31.75±5.85个,显著性多于Tip30+/+Balb/c小鼠的19.75±4.79个,t=3.174,P=0.019。而Tip30-/-Neu+/+FVB及Tip30+/+Neu+/+FVB小鼠的乳腺小球形成数目分别为31.25±5.56个/5000细胞及17.5±4.04个/5000细胞,具有显著性差异(t=4.001,P=0.007)。
同时检测乳腺小球直径,Tip30-/-Balb/c小鼠乳腺小球直径为156.80±29.28μm,显著性大于Tip30+/+Balb/c小鼠的97.59±20.66gm,t=3.305,P=0.016;而在Tip30-/-Neu+/+FVB小鼠乳腺小球的直径为128.29±27.83μm,虽然高于Tip30+/+Neu+/+FVB小鼠的105.53±14.19μm,但无显著性差异(t=1.457,P=0.195)。
1.2TIP30基因敲除可增强乳腺上皮细胞体外克隆形成能力
将从小鼠乳腺组织获得的乳腺上皮细胞单细胞悬液按照2000细胞每孔种植于铺有Matrigel孔板中,培养10天。Tip30-/-'Balb/c小鼠的体外克隆形成数目为33.33±5.13,显著性多于Tip30+/+Balb/c小鼠的19.00±6.00,t=3.144,P=0.035。而Tip30-/-Neu+/+FVB小鼠的体外克隆形成数目亦显著性高于Tip30+/+Neu+/+FVB小鼠(33.00±5.29vs.18.00±4.36,t值3.709,P值为0.019)。
在检测克隆大小时发现,Tip30-/-Neu+/+FVB小鼠的克隆直径虽然大于Tip30+/+Neu+/+FVB小鼠(126.68±25.42μm vs.97.41±13.13μm),但无显著差异(t=1.772,P=0.151)。而Balb/C背景的Tip30基因敲除小鼠的克隆大小显著性高于Tip30+/+Balb/C小鼠(137.9±16.17gm vs.90.79±22.89μm),t值为2.914,P值为0.044。
1.3TIP30基因敲除可扩增小鼠乳腺干细胞
MRUs即乳腺再繁殖单位(CD24HighCD49flow),该细胞群富集乳腺干细胞。流式细胞仪检测结果显示,Tip30基因敲除的Balb/C小鼠的MRUs百分比为2.83±0.51%,显著性高于Tip30+/+Balb/C小鼠的1.27±0.51%,t值为3.739,P值为0.02。在有Neu转基因背景的FVB小鼠中亦得到相似结果,Tip30-/-Neu+/+FVB小鼠MRUs的百分比为6.73±0.85%,显著性高于Tip30+/+Neu+/+FVB小鼠的1.30±0.60%,t=9.042,P=0.001。
1.4TIP30基因敲除可扩增小鼠乳腺祖细胞
Ma-CFCs即乳腺克隆形成细胞(CD24HighCD49flow),该细胞群富集乳腺祖细胞。同样应用流式细胞仪检测,Tip30-/-Balb/C小鼠的Ma-CFCs百分比(3.23±0.49%)显著性高于Tip30+/+Balb/C小鼠(0.87±0.38%),t值为6.592,P值为0.003。Tip30-/-Neu+/+FVB小鼠的Ma-CFCs百分比为3.07±0.76%,显著性高于Tip30+/+Neu+/+FVB小鼠的1.43±0.31%,t=3.465,P=0.026。
2. TIP30基因敲除可导致乳腺ER+祖细胞比例增多
在Balb/c小鼠中,应用Scal对乳腺祖细胞再划分为CD24HighCD49flowScal+及CD24HighCD49flowSca1两个亚群,分别代表ER+祖细胞及ER-祖细胞。流式细胞仪检测结果显示：Tip30-/-Balb/c小鼠乳腺ER+祖细胞占祖细胞的30.67±2.45%,显著性高于Tip30+/+Balb/c小鼠的17.73±1.76%,t=7.415,P=0.002；而Tip30-/-Neu+/+FVB小鼠ER+祖细胞占祖细胞的23.80±5.27%,显著性高于Tip30+/+Neu+/+FVB的13.57±2.70%,t=2.994,P=0.04。
3.TIP30基因敲除可导致乳腺祖细胞倾向ER+细胞分化
我们运用流式细胞仪分选出乳腺祖细胞亚群(CD24HighCD49flow),使其在Matrigel上分化生长形成克隆,并进行ER/PR免疫荧光染色。结果如图及表示,Tip30-/-Balb/c小鼠的乳腺祖细胞形成的克隆中ER阳性细胞数占45.35±7.80%,显著性高于Tip30+/+Balb/c小鼠的31.77±6.38%,t=2.698,P=0.036。而Tip30-/-Balb/c和Tip30+/+Balb/c小鼠的乳腺祖细胞形成的克隆中PR阳性细胞数分别为38.70±8.45%及33.76±6.33%,无显著性差异(t=0.936,P=0.385)。
为了进一步验证TIP30基因对ER+细胞分化的影响,我们对Balb/c小鼠乳腺组织行ER/PR免疫荧光染色,结果示Tip30-/-Balb/c和Tip30+/+Balb/c小鼠乳腺组织ER阳性细胞分别占21.13±3.59%和13.00±3.716%,t=3.15,P=0.020,有显著性差异；而PR阳性细胞分别占18.53±2.83%和16.77±3.16%,t=0.832,P=0.437,无显著性差异。
4.TIP30基因敲除可上调乳腺组织FOXA1表达
我们在mRNA水平检测了与乳腺干/祖细胞分化相关的信号通路分子Notch. Hedgehog.Wnt及GATA3.FOXA1的表达情况,应用独立样本t检验,结果仅有FOXA1在TIP30基因敲除的乳腺组织中高表达,其相对表达值为3.05±0.90,显著性高于Tip30+/+Balb/c小鼠的1.00±0.17,t=3.879,P=0.018。进一步我们对其蛋白水平进行检测,发现,Tip30-/-Balb/c小鼠乳腺组织FOXA1蛋白表达相对值为66.24±5.93%,亦显著性高于Tip+/+Balb/c小鼠的40.27±8.18%,t=4.454,P=0.011。提示TIP30基因的缺失可能使FOXA1的表达的上调,从而诱导更多的ER阳性细胞分化。
5.下调乳腺细胞FOXA1表达可减少ER阳性细胞分化
为了进一步验证TIP30基因敲除导致的ER阳性细胞分化增多是否通过升高FOXA1表达而实现。我们从Tip30-/-Balb/c小鼠分离出乳腺上皮细胞,应用FOXA1-SH1、FOXA1-SH2及shRNA-CON基因干扰后,应用Western-blot检测验证FOXA1表达明显示受抑制后,行体外克隆形成试验,并进行ER免疫荧光染色。结果示FOXA1-SH1、FOXA1-SH2组形成的克隆中ER阳性细胞分别占31.00±2.85%及29.52±5.67%,显著性低于shRNA-CON组的39.40±6.16%,P值分别为0.042及0.018。该结果提示,TIP30基因敲除后,通过(至少部分通过)升高FOXA1的表达来促进乳腺祖细胞倾向ER阳性细胞分化。
6. TIP30基因敲除可以上调乳腺组织RANKL的表达
我们在进一步研究TIP30基因敲除小鼠乳腺干/祖细胞扩增可能的原因时发现,TIP30基因敲除可以使小鼠乳腺组织RANKL表达增高,其相对表达值为16.91±1.13,显著性高于野生型小鼠的7.94±4.47,t=3.370,P=0.028。提示TIP30基因敲除小鼠可能通过依靠增多的性激素受体细胞增强分泌RANKL,并通过旁分泌的方式作用于乳腺干/祖细胞,从而使其扩增。
三、结论
1.TIP30基因敲除可以扩增小鼠乳腺干/祖细胞。
2.TIP30基因敲除可增加小鼠乳腺ER阳性祖细胞比例,并通过上调FOXA1表达使祖细胞倾向于ER阳性细胞分化。
3.TIP30基因敲除可能通过依靠增多的性激素受体阳性细胞分泌更多的RANKL,并通过旁分泌的方式使乳腺干／祖细胞扩增。
Background and objective
Breast cancer is one of the most frequently diagnosed cancers in females worldwide, accounting for23%(1.38million) of the total new cancer cases and14%(458,400) of the total cancer deaths in2008. In general, incidence rates of breast cancer are still increasing year by year, but breast cancer death rates have been decreasing in North America and several European countries over the past25years. However, in many African and Asian countries, both incidence and mortality rates are rising. Although, a great improvement of dignosis and treatment of breast cancer have been made, the mechanism underlining breast cancer remains unclear. Estrogen receptor alpha (ERa) plays important roles in mammary gland development and carcinogenesis. ER positive breast cancer subtype is the most common breast cancer subtype, approximately accounting for60-70%. ER has been a preditive marker for patients'prognosis and a good marker for therapy chosen. ER positive breast cancer has better effect of therapy and prognosis in clinic, however, the machanism of this subtype of breast cancer is still unkown. This study generated an TIP30knockout mouse model and focused on exploring its limited development of ER positive breast cancer.
TIP30, also known as CC3, is a30-kDa human cellular protein that was purified as a HIV-1Tat interacting protein and its expression is found altered in human liver, lung, and breast cancers etal. Our previous studies showed that Tip30-deficient mice spontaneously develop tumors in several tissues and mammary preneoplastic lesions, suggesting that TIP30acts as a tumor suppressor. Its tumor suppressor activity is likely due to its actions in multiple cellular processes. TIP30can function as a transcription cofactor to repress expression of genes that are involved in proliferation and apoptosis. TIP30can act as a repressor of ERa-mediated c-Myc transcription in mammary glands and breast cancer cells. Moreover, data about TIP30controling endocytic downregulation of the EGFR signaling pathway in primary hepatocytes、 hepatocellular carcinoma cells and lung cancer cells also have been highlighted recently.
Our previous data showed that deleting the TIP30gene in mice leads to ductal hyperplasia in mammary glands early in life and extensive mammary hyperplasia with age. In addition, we also showed that deletion of TIP30in MMTV-Neu mouse can accelerate the onset of mammary tumors and resulted in ER+/PR-mammary tumors. In this study, we generated a TIP30knockout Balb/c mice model without the interference of Neu gene, and found that TIP30deletion in mice developed mammary tumors. We futher analysed the EGFR downstream cell signaling in this model to study the possible machanism of breast cancer development and used human breast cancer cell line to explore the role of TIP30in regulating EGFR degration.
Breast cancer has heterogeneous pathologies and molecular profiles and in this respect there are many molecular subtypes of breast cancer. The stem cell model of mammary carcinogenesis provides an explanation for the cellular heterogeneity within tumors, in addition to the phenotypic diversity seen in different subsets of patients. This model predicts that different subsets of mammary carcinoma arise from different cells and/or the particular mutations required to transform these cells. These mutations might result in uncontrolled self-renewal of the cancer stem cell, driving tumorigenicity, and aberrant differentiation, with this last property generating most of the cells within a tumor. In addition, studies in mice have clearly shown that different genes exert their influence in different cell subpopulations. For example, mouse mammary tumour virus (mmTv)-Wnt-1results in tumours that are composed of both luminal and myoepithelial cells and an expansion of the MaSC pool. However, mmTv-Erbb2(Neu) mice can generate luminal cell-restricted tumours and display an expansion of the luminal cell compartment.In our study, we are trying to explore effects of TIP30deletion on mammary stem/progenitor cells pool and their cell fate, which can provide some explainations for our mouse model which spontaneously developed ER positive breast cancers.
Part I:Loss of TIP30drives development of mouse breast cancer and activates EGFR downstream signal molecules.
Methods
1. TIP30knockout Balb/c mice were obtained by backcross TIP30knockout C57J6mice with Balb/c mice. After over7generations continuous passages, we finally established TIp30-/-Balb/c mice.
2. After being observed for78weeks, mice were sacrificed for relevant researches. Check the mouse organs such as brain, mammary, lung, etal to see any tumor or desease happened in them. Part of tissues were fixed, dehydrated and embeded in paraffin for futher analysis. Rest of tissues were freshly frozen in liquid nitrogen and were kept in-80℃for longterm storage.
3. H&E staining were used to detect the tissue section including the organs and tumors. Slides were examined by two pathologists for tumorgenesis or other aberrant histological changes. After that, incidence of breast carcinoma was caculated.
4. Immunohistochemistry (IHC) was performed to analyse expression of CK8, aSMA, pAkt and pERK in mammary gland and mammary tumor.
5. Immunofluorescence was used to detect expression of ER and PR in mamary tumor.
6. Lentiviruses were generated using shRNA-TIP30and shRNA-CON plasmids and then transfected MCF-7cells following puromycin selection. Transfection efficiencies were analyzed by western-blot.
7. Immunofluorescence was used to analyse the colocolization of EGF and EGFR, EGFR and EEA1as well as TIP30and EEA1in MCF-7cells after treated with EGF for indicated times.
8. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Incidence rate of breast cancer between two groups was analysed by x2test. Comparisons of two groups'or three groups'mean values were analyseed by independent sampe t test or One-way ANOVA followed by LSD or Dunnett multiple comparison test. P-Values were considered to be significant at <0.05.
Results
1. Knockout of TIP30results in spontaneous mammary tumor in Balb/c mice.
After18months observeation,47female mice reached the analysis end point.8of28Tip30-'-mice were found having mammary tumor, but no tumor found in19Tip30+/+mice (0%). Incidence rate of mammary tumor in Tip30-/-mice (28.6%) is significantly higher than Tip30+/+mice (0%), x2=6.269, P=0.012.
2. Mammary tumor induced by TIP30deletion mice were all ER/PR postive luminal type.
Mammary tumor tissue section were stained with K8and by IHC and results show edthat all the tumors are K8strongly posive, but aSMA negative, which implied that mammary tumors found in Tip30-/-mice were Luminal subtype. Then we used immunofluorescence to dectect ER/PR expression and found that all tumor sections are ER postive and PR positive. All these data indicated that Tip30-'-mice spontaneous developed ER/PR positive, Luminal type breast cancer.
3. TIP30deletion leads to EGFR pathways such as MAPK and PI3K activation.
IHC was used to dectect pErkl/2and pAkt expression in mammary gland and mammary tumor sections. Positive rates of pErkl/2and pAkt in Tip30-/-mice mammary gland were27.83±8.46%and30.83±6.65%(n=5), which were significantly higher than12.58±5.87%and14.94±5.77%in Tip30+/+mice respectively, P values were0.02and0.011. Moreover, positive rates of pErkl/2and pAkt in Tip30-/-mice mammary tumor were51.68±8.57%and56.08±8.44%, significantly higher than mammary gland, P values were0.002and0.001 respectively.
4. TIP30knockdown could prolong activation of EGFR downstream signal molecules of mamary tumor cells induced by EGF.
4.1TIP30-SH1and TIP30-SH2transfection respectively downregulated the expression of TIP30in MCF-7cells.
After transfection and anibiotic selection, relative TIP30expression of TIP30-SHland TIP30-SH2cells are13.12±3.95%and11.54±2.68%(n=3), significantly lower than shRNA-CON (84.3±8.66%, n=3) respectively, both of P values less than0.001. In general, TIP30expression of TIP30-SH1and TIP30-SH2cells were downregulated by71.18%and72.76%respectively, which implicated that the transfecting efficience was well.
4.2TIP30could promote degration of EGFR induced by EGF
After treated with EGF, EGF and EGFR expressions were analysed by immunofluorescence at indicated times1Omin,1h,4h. We found that both intencity of EGF and EGFR fluorescence were no difference between TIP30-SH and shRNA-CON cells at10min and1h after treated with EGF, and the Pearson's colocalization of EGF and EGFR were not different either. However, at4h after treated with EGF, both intencity of EGF and EGFR fluorescence in TIP30-SH cells were1.19±0.18and1.33±0.13, significantly higher than0.68±0.08and0.76±0.10in shRNA-CON cells respectively (1=4.563and6.132, P=0.01and0.004); at the same time, the Pearson's colocalization of EGF and EGFR in TIP30-SH cells was significantly higher than shRNA-CON cells (35.07±5.33vs.10.76±2.28; t=7.270, P=0.002).
4.3TIP30knockdown could stagnate EGFR in early endosomes.
2h after treated with EGF, EGFR and early endosome marker EEA1expressions in cells were analysed by immunofluorescence. The Pearson's colocalization value of EGFR and EEA1in TIP30-SH was56.39±14.08%, significantly higher than27.31±9.12%of shRNA-CON.
4.4TIP30could assemble in early endosome and regulate EGFR degration.
We also used immunofluorescence to dectect colocalization of TIP30and EEA1, and found that TIP30are colocalized with EEA1at10min after shRNA-CON cells treated with EGF.
Conclusion
1. TIP30knockout Balb/c mice can spontaneously develop mammary tumor.
2. All the mammary tumor developed by TIP30knockout Balb/c mice are ER/PR+Luminal type.
3. TIP30deletion results in EGFR downstream pathways such as MAPK and PI3K activation.
4. Inhibition of Tip30could delay EGF-induced degradation of EGFR, thus prolong EGFR downstream signal molecules and promote the proliferation of breast cancer cells.
Part Ⅱ:TIP30deletion promotes mouse mammary stem/progenitor cells expansion and regulates progenitor cell fate
Methods
1. Gained Tip30-/-Balb/c and Tip30-/-Neu+/+FVB mice by crossbreeding. After over7generations continuous passages, we finally established TIP30knockout mice with different backgrounds.
2.5-6month old mouse mammary tissues were dissociated mechanically and enzymatically. Finally the mammary cell suspensions were obtained.
3. Mammosphere culture was used to detect the mammosphere forming capacity of mammary cells from different genotype mice. Caculated the mammosphere's number and size under microscope.
4. Colony forming assay in vitro was performed to analyse the colony generation capacity of mammary cells from different genotype mice. Colony number and size were caculated under microscope.
5. Flow cytometry was used to analyse the population of CD24lighCD49flow (MRUs) and CD24HighCD49flow (Ma-CFCs) in mammary cells from different genotype mice, then analyse the subpopulation of Scal+and Seal-in CD24HighCD49flow population.
6. Flow cytometry was used to sort out CD24HighCD49flow population which were gown in vitro using colony forming assay, then were stianed with ER/PR by immunofluorescence.
7. Immunofluorescence was used to detect ER/PR expression of the mammary glands.
8. QPCR was used to the mRNA levels of factors which related to mammary stem/progenitor cells fate decision.
9. Used Western-blot to detect FOXA1and RANKL expression of mammary tissues.
10. Lentiviruses were generated using shRNA-FOXAl and shRNA-CON plasmids and then transfected TIP30-/-cells following puromycin selection. After the transfection efficiencies were confirmed by western-blot. The cells were grown in vitro to form colonies and then stained with ER.
11. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Comparisons of two groups'or three groups'mean values were analyseed by independent sampe t test or One-way ANOVA followed by Dunnett multiple comparison test. P-Values were considered to be significant at≤0.05.
Results
1. Loss of TIP30can promote mammary stem/progenitor cells expansion.
1.1TIP30deletion could increase mammosphere forming capacity.
After9d mammosphere culture in serum free medium, mammosphere forming number of Tip30-/-Balb/c was31.75±5.85/5000cells seeded, significantly higher than19.75±4.79/5000cells of7ip30+/+Balb/c, t=3.174, P=0.019. We found a similar result in Neu+/+FVB background mice. Tip30-/-Neuw+/+FVB mice had more mammospheres than Tip30+/+Neu+/+FVB mice (31.25±5.56/5000cells vs.17.5±4.04/5000cells), t=4.001, P=0.007.
We also measured the size of mammospheres and found that mammospheres formed by by mammary cells of Tip30-''Balb/c mice had larger size than that in Tip30+/+Balb/c mice.(156.80±29.28μm vs.97.59±20.66μm; t=3.305, P=0.016). But we can't find any difference in size between Tip30-/-Neu+/+FVB (128.29±27.83μm) and Tip30+/+Neu+/+FVB mice(105.53±14.19μm),t=1.457,P=0.195.
1.2TIP30deletion could enhance colony forming capacity in vitro.
2000Mammary cells/well from different mice were seeded on matrigel precoated well for10d.The colony number of Tip30-/-Balb/c(33.33±5.13)was significantly higher compaired with Tip30+/+Balb/c mice(19.00±6.00),t=3.144,P=0.035. Moreover,mammary cells from and Tip30-/-Neu+/+FVB also formed more colonies than those from Tip30+/+Neu+/+FVB respectively(33.00±5.29vS.18.00±4.36),t=3.709,P=0.019.
When measuring the size of colonies,we found that Tip30-/-Balb/c(137.9±16.17μm) has larger colony than Tip30+/+Balb/c(90.79±22.89¨m),t=2.914,P=0.044.However, there was no difference in size between Tip30-/-Neu+/+FVB(126.68±25.42μm)and Tip30+/+Neu+/+FVB mice(97.41±13.13Um),t=1.772,P=0.151.
1.3TIP30deletion can promote MRUs expansion.
Flow cytometry analysis showed that MRUs population of Tip30-/-Balb/c accouting for2.83±0.51%was significantly higher than Tip30+/+Balb/c which wasl.27±0.51%, t=3.739.P=0.02.We got a silimar result which showed that TIP30deletion can significantly increase the MRUs population from1.30±0.60%to6.73±0.85%in Neu+/+FVB mice,t=9.042,P=0.001.
1.4TIP30deletion can increase Ma-CFCs.
Flow cytometry analyse also showed that Ma-CFCs population of Tip30-/-Balb/c was3.23±0.49%,significantly higher than0.87±0.38%of Tip30+/+Balb/C,t=6.592, P=0.003.For Neu+/+FVB mice,TIP30deletion resulted in Ma-CFCs population increasing from1.43±0.31％to3.07±O.76&,t=3.465,P=0.026.
2.TIP30deletion in mice can increase ER+mammary progenitor cells.
We used balb/c and Neu+/+FVB mice for further analysis.Sca-1was used for discriminate mammary progenitors cells to two suppoulations,CD24HighCD49flow Scal+and CD24HighCD49flowScal-which represent ER+and ER-progenitor cells respectively.Flow cytometry analysis showed that TIP30deletion can significantly increase the CD24HighCD49flowScal+population from17.73±1.76%to30.67±2.45%, t=7.415, P=0.002. In addition, Tip30-/-Neu+/+FVB mice also had more ER+progenitor cells compared with Tip30+/+Neu+/+B mice (23.80±5.27%vs.13.57±2.70%), t=2.994, P=0.04。
3. TIP30deletion can predispose mammary progenitor cells to ER+cell fate.
Flow cytometry was used to sort out the mammary progenitor cell population (CD24HighCD49flow), and the cells were plated on the matrigel precoated well and cultured to form colonies then stained with ER/PR by immunofluorescence. We found that colonies formed by Tip30-'-Balb/c mice have more ER positive cells than colonies formed by Tip+/+Balb/c mice (45.35±7.80%vs.31.77±6.38%), t=2.698, P=0.036. But we can't find difference in number of PR positive cells in colonies form by Tip30-/-and Tip30+/+Balb/c mice (38.70±8.45%vs.33.76±6.33%), t=0.936, P=0.385.
To further comfirm the influence of TIP30deletion on ER positive cell differentiation of mammary cells, we checked the ER/PR expression of mouse mammary gland by immunofluorescence. Tip30-/-Bdlb/c mammary glands had more ER positive cells than Tip30+/+Balb/c mammary gland (21.13±3.59%vs.13.00±3.716%, t=3.15, P=0.020). However, there was no difference of PR positive cells between Tip30-/-Balb/c and Tip30+/+Balb/c mammary glands.
4. Loss of TIP30upregulated FOXA1expression of mammary glands.
We next measured some signaling pathway (Notch, Hedgehog, Wnt) or genes (GATA3、FOXA1) related to cell fate decision at mRNA levels. Only FOXA1mRNA level was found markedly upregulated in Tip30-/-Balb/c mouse mammary glands compared with Tip30+/+Balb/c mice (3.05±0.90vs.1.00±0.17), t=3.879, P=0.018. We further analysised the protein level of FOXA1. The relative expression of FOXA1in Tip30-/-Ba\b/c mammary glands was66.24±5.93%, significantly higher than40.27±8.18%in Tip30+/+Balb/c mammary glands, t=4.454, P=0.011.
5. Down regulation of FOXA1in Tip30-/-mammary cells decreased ER+cell differentiation.
To determine whether upregulation of FOXA1in Tip30-/-mammary cells was responsible for more differentiation of ER+cell than Tip30+/+mammary cells. We harvested the mammary cells from Tip30-/-balb/c mice, and knocked down the FOXA1expression. After confirmation of knockdown of FOXA1by western blot, cells were grown in matrigel to form colonies which were then stianed with ER. The percentage of ER+cells in colonies formed by FOXA1-SHland FOXA1-SH2cells were31.00±2.85%and29.52±5.67%respectively, both of which were significantly lower than39.40±6.16%in colonies formed by shRNA-CON; P values were0.042and0.018respectively.
6. TIP30deletion upregulated RANKL expression of mammary glands.
To determine why TIP30deletion can result in expansion of mammary stem/progenitor cells, expression of RANKL was measured by western blot. We found that the levels of RANKL were markedly increased in Tip30-'-Ba\b/c mammary glands than in Tip30+/+Balb/c mammary glands (16.91±1.13vs.7.94±4.47, t=3.370, P=0.028) which indicated that TIP30deletion may promote mamamry stem/progenitor cells expansion through upregulaion of RANKL in a paracrine manner.
Conclusion
1. TIP30deletion can promote mammary stem/progenitor cells expansion in mice.
2. TIP30deletion can increase ER positive progenitor cells, and predispose mammary progenitor cells to ER+cell fate through upregulation of FOXA1.
3. TIP30deletion may induce secretion of RANKL from hormone receptor positive cells and promote the mammry stem/progenitor cells expansion by paracrine way.
TIP30基因,又称CC3或Htatip2,是1998年肖华等在研究人类免疫缺陷病毒(Human immunodeficiency virus, HIV)的体外转录时发现一种分子量为30kD的Tat(Transactivator of transcription)结合蛋白(Tat interactive protein)。研究发现TIP30在多种肿瘤组织中的表达异常,如肺癌、乳腺癌、胃癌、结肠癌、肝细胞癌和前列腺癌等,提示TIP30与肿瘤之间存在密切联系。Hua等研究发现,TIP30基因敲除可以自发形成多种肿瘤。这些研究提示TIP30是一种肿瘤抑制基因。TIP30具有多种功能,可以作为一种转录共分子参与凋亡及增殖相关基因的调控,它还可以作为细胞核转运抑制因子调节细胞凋亡。在乳腺和乳腺癌细胞中,TIP30还可以抑制ERa调节的c-myc转录。近来研究发现,在肝癌和肺癌中,TIP30可以调控EGFR细胞浆内转运和下游信号通路的活性。
我们先前的研究发现,TIP30基因敲除可以诱发小鼠乳腺导管增生,而且随着年龄的增长,增生程度越明显。同时,我们在有Erbb2(Neu)转基因背景的FVB小鼠中敲除TIP30,发现TIP30基因缺失可以加速小鼠乳腺癌发生,而且所有自发性乳腺癌均为ER阳性乳腺癌。而在本研究中,我们排除了Neu基因的影响,发现在Balb/c背景的小鼠中敲除TIP30便能诱发小鼠乳腺癌。为了进一步研究其可能的机制,我们对该小鼠模型EGFR下游信号分子进行了初步的探讨,并在人乳腺癌细胞株中研究了TIP30基因在EGFR降解中的调节作用。
由于乳腺癌是异质性相当大的肿瘤,其病理和分子学特点具有多样性复杂性。近年来,乳腺肿瘤形成的干细胞模型为乳腺癌的异质性及不同乳腺癌亚型的起源提供了解释。这一模型认为不同乳腺肿瘤亚型是起源于不同的乳腺干细胞或祖细胞,而特异性的突变可使这些细胞发生恶性转化,从而使其自我更新失控及异常分化,最终诱发肿瘤发生。此外,在小鼠的研究中发现,不同基因对不同亚群乳腺上皮细胞影响不同,而且会影响乳腺癌亚型的发生。比如,乳腺癌病毒(mmTv)-Wnt-1小鼠可以扩增乳腺干细胞亚群,同时形成luminal型和basal型乳腺癌。而nmTv-Neu小鼠可以促进luminal细胞群扩增,仅产生luminal型且ER/PR阴性乳腺癌。因此通过在小鼠动物模型中研究特定基因对乳腺细胞亚群及肿瘤类型形成的影响将有助于增强对乳腺癌发病机理的了解。此外,我们先前的研究发现,TIP30基因敲除后可以使Neu转基因小鼠乳腺癌的表型由ER-/PR-阴性转变为ER+/PR-,提示TIP30基因不只是个抑癌基因,它在乳腺癌表型的决定上也可能起了一定的作用。因此,本研究在干细胞层面探讨了TIP30基因缺失对乳腺干细胞／祖细胞亚群及其分化倾向方面可能存在的作用,并为该动物模型形成的限制性ER阳性乳腺癌提供了一定的解释。
第一部分TIP30基因敲除诱发小鼠乳腺癌及活化EGFR下游通路的研究
一、材料与方法
1.通过杂交的方法,获得TIP30基因敲除的Balb/c小鼠,连续传代7代以上,获得Balb/c纯合背景的TIP30基因敲除小鼠。
2.所有Balb/c小鼠饲养至满78周时,二氧化碳吸入处死,详细检查小鼠大脑、乳腺、胸腔脏器、腹腔脏器、生殖器官等肉眼可见部位有无肿瘤和其它疾病出现。常规组织固定、石蜡包埋并切片。部分组织丢入液氮速冻,最后于-80℃保存。
3.应用苏木精-伊红(H&E)染色法检测组织器官及乳腺肿瘤组织形态结构。切片由两位病理学家阅片并分析。计算发生乳腺癌的小鼠百分比。
4.应用免疫组织化学(IHC)法检测乳腺组织或肿瘤组织CK8、αSMA、pAkt、 pERK表达。
5.应用免疫荧光染色(IF)法检测乳腺肿瘤组织ER及PR表达情况。
6.应用shRNA-TIP30及shRNA-CON质粒包装慢病毒颗粒,感染MCF-7细胞,puromycin筛选,并经Western-blot检测验证基因干扰效果。
7.应用细胞免疫荧光方法检测经EGF诱导的MCF-7细胞在不同时间点EGF与EGFR、EGFR与EEA1及TIP30与EEA1的共表达情况。
8.统计处理：实验结果以均数±标准差(x±s)表示,数据处理采用SPSS13.0统计软件。两组的肿瘤发生率比较采用χ2检验；两组及多组间均数比较分别采用独立样本t检验和单向方差分析；两组不同时间点均数比较采用重复测量方差分析；多重比较采用LSD检验或Dunnett检验。P50.05(双侧)表示差异有显著性。
二、结果
1.TIP30基因敲除可导致Balb/c小鼠自发性乳腺肿瘤出现
经过18个月的密切观察,共有47只雌性未孕小鼠完成实验观察。28只Tip30-/-小鼠中有8只发现乳腺自发肿瘤(发病率28.6%),而19只Tip30+/+小鼠中均未发现有乳腺自发肿瘤(0%)。Tip30"/-小鼠乳腺自发肿瘤的发生率显著高于Tip30+/+小鼠,x2=6.269,P=0.012。
2.TIP30基因敲除致Balb/c小鼠乳腺肿瘤均为ER/PR阳性luminal型
我们应用luminal标志CK8(?)basal标志αSMA对乳腺肿瘤组织行免疫组织化学染色,结果提示,所有肿瘤组织CK8染色呈强阳性,而aSMA染色阴性,提示Tip30-/-小鼠自发乳腺肿瘤均为luminal型。进一步的ER及PR免疫荧光染色,结果显示所有肿瘤均为ER阳性和PR阳性乳腺癌。提示Tip30-/-小鼠自发乳腺肿瘤为ER/PR阳性luminal型肿瘤。
3.TIP30基因敲除可致EGFR下游MAPK及P13K信号通路的活化
我们应用了免疫组化对乳腺及肿瘤组织pErk1/2及pAkt进行检测。Tip30-/-小鼠乳腺上皮细胞pErk1/2及pAkt阳性率分别为27.83±8.46%和30.83±6.65%(n=5),显著高于rip30+/+小鼠乳腺上皮细胞的12.58±5.87%和14.94±5.77%(n=5),P值分别0.02和0.011。而Tip30-/-小鼠乳腺肿瘤细胞pErk1/2及pAkt阳性率分别为51.68±8.57%和56.08±8.44%(n=5),则显著高于Tip30-/-小鼠乳腺上皮细胞的27.83±8.46%和30.83±6.65%,P分别为0.002和0.001。
4.TIP30表达下调可延迟EGF诱导的人乳腺癌细胞中EGFR的降解。
4.1TIP30-SH1、TIP30-SH2乳腺细胞株MCF-7的TIP30蛋白明显抑制
乳腺癌细胞株MCF-7通过shRNA干扰后,以内参[3-actin为标准,TIP30-SH1和TIP30-SH2细胞TIP30蛋白表达相对值分别为13.12±3.95%和11.54±2.68%(n=3),显著低于shRNA-CON细胞的84.3±8.66%(n=3),P均<0.001。TIP30-SH1及TIP30-SH2细胞TIP30蛋白表达分别下降达71.18%及72.76%,这些结果提示基因转染后TIP30-SH1、TIP30-SH2细胞TIP30表达被显著抑制。
4.2TIP30在乳腺癌细胞中可促进EGF诱导的EGFR降解
TIP30-SH组细胞在经EGF作用后,10min和1h时EGF及EGFR的荧光强度与shRNA-CON组相比,均无统计学差异；4h时两组EGF及EGFR荧光强度均显著下降,其中TIP30-SH组细胞EGF及EGFR的荧光强度分别为1.19±0.18和1.33±0.13,显著性高于shRNA-CON组0.68±0.08和0.76±0.10,t值分别为4.563和6.132,P值分别为0.01和0.004,以上结果提示细胞的TIP30基因干扰后,EGF及EGFR降解速度减慢,于4h时仍有大量EGF及EGFR未降解。
同时检测EGF及EGFR共定位情况时发现,两组细胞EGF及EGFR在10min时Pearson's共定位值最高,随时间延长逐渐降低。于4h时,TIP30-SH组共定位值为35.07±5.33,显著性高于shRNA-CON组的10.76±2.28,t=7.270,P=0.002。提示相对于shRNA-CON组,TIP30-SH组细胞EGF及EGFR推迟分离降解,有更多的EGF/EGFR结合物存在于细胞内。
4.3TIP30基因下调可延长EGFR在早期内涵体内聚集时间
TIP30-SH及shRNA-CON细胞在经EGF处理2小时后,shRNA-CON细胞EGFR绿色荧光与EEA1红色荧光Pearson's共定位值为27.31±9.12%,显著性低于TIP30-SH细胞组的56.39±14.08%,t=3.003,P=0.04。提示TIP30基因干扰后,EGFR的推迟从早期内涵体分离。
4.4TIP30可聚集于早期内涵体内,参与EGFR代谢
EGF处理shRNA-CON细胞10分钟后,激光共聚焦显微镜下TIP30绿色荧光与EEA1红色荧光明显重叠,提示TIP30在早期进入内涵体参与EGFR转运。
三、结论
1.TIP30基因敲除的Balb/c小鼠可自发乳腺癌。
2.TIP30基因敲除的Balb/c小鼠所发生乳腺癌均为ER/PR阳性luminal型肿瘤。
3.TIP30基因敲除可以激活乳腺及乳腺癌中EGFR下游MAPK及PI3K信号通路分子。
4.TIP30基因抑制可干扰EGFR自内涵体分离,抑制EGF诱导的EGFR降解,导致EGFR下游信号分子持续激活。
第二部分TIP30基因敲除诱导小鼠乳腺干/祖细胞扩增并影响其分化倾向的研究
一、材料与方法
1.通过杂交的方法,获得TIP30基因敲除的Balb/c及Neu+/+FVB小鼠,连续传代7代以上,获得各种系纯合背景的TIP30基因敲除小鼠。
2.解剖收集5-6月龄小鼠乳腺组织并剪碎,用无血清胶原酶溶液消化,获得乳腺上皮细胞悬液。
3.应用细胞悬浮球形培养方法检测各基因表型小鼠乳腺上皮细胞乳腺小球形成能力。显微镜下拍照并计算乳腺小球数量及大小。
4.应用体外克隆形成试验检测各基因表型小鼠乳腺上皮细胞克隆形成能力。显微镜下拍照并计算克隆形成数及大小
5.应用流式细胞仪分析各基因表型小鼠乳腺上皮细胞CD24HighCD49flow (MRUs)及CD24HighCD49flow (Ma-CFCs)百分比例,并分析CD24HighCD49low群中Scal+及Scal-比例。
6.应用流式细胞仪分选出Balb/c小鼠各基因表型乳腺上皮细胞中祖细胞群CD24HighCD49flow并行体外克隆形成试验,收集克隆并行ER/PR免疫荧光染色。
7.应用免疫荧光法检测Balb/c小鼠各基因表型乳腺组织ER/PR表达情况。
8.应用qPCR检测Balb/c小鼠各基因表型乳腺组织中与乳腺干/祖细胞分化相关的因子表达情况。
9.应用Western-blot检测Balb/c各基因表型小鼠乳腺组织FOXA1及RANKL表达。
10.应用shRNA-FOXA1及shRNA-CON质粒包装慢病毒颗粒,感染乳腺细胞,puromycin筛选,并经Western-blot检测验证基因干扰效果后,行体外克隆形成试验并行克隆ER免疫荧光染色。
11.统计处理：实验结果以均数±标准差(x±s)表示,数据处理采用SPSS13.0统计软件,两组及多组间均数比较分别采用独立样本t检验和单向方差分析；多重比较采用LSD检验或Dunnett检验。P<0.05(双侧)表示差异有显著性。
二、结果
1.TIP30基因敲除可促进乳腺干/祖细胞群扩增
1.1TIP30基因敲除可增强乳腺上皮细胞体外乳腺小球形成能力
小鼠乳腺上皮细胞经体外悬浮无血清培养9天后,Tip30-/-Balb/c小鼠每5000个乳腺上皮细胞形成乳腺小球数目为31.75±5.85个,显著性多于Tip30+/+Balb/c小鼠的19.75±4.79个,t=3.174,P=0.019。而Tip30-/-Neu+/+FVB及Tip30+/+Neu+/+FVB小鼠的乳腺小球形成数目分别为31.25±5.56个/5000细胞及17.5±4.04个/5000细胞,具有显著性差异(t=4.001,P=0.007)。
同时检测乳腺小球直径,Tip30-/-Balb/c小鼠乳腺小球直径为156.80±29.28μm,显著性大于Tip30+/+Balb/c小鼠的97.59±20.66gm,t=3.305,P=0.016;而在Tip30-/-Neu+/+FVB小鼠乳腺小球的直径为128.29±27.83μm,虽然高于Tip30+/+Neu+/+FVB小鼠的105.53±14.19μm,但无显著性差异(t=1.457,P=0.195)。
1.2TIP30基因敲除可增强乳腺上皮细胞体外克隆形成能力
将从小鼠乳腺组织获得的乳腺上皮细胞单细胞悬液按照2000细胞每孔种植于铺有Matrigel孔板中,培养10天。Tip30-/-'Balb/c小鼠的体外克隆形成数目为33.33±5.13,显著性多于Tip30+/+Balb/c小鼠的19.00±6.00,t=3.144,P=0.035。而Tip30-/-Neu+/+FVB小鼠的体外克隆形成数目亦显著性高于Tip30+/+Neu+/+FVB小鼠(33.00±5.29vs.18.00±4.36,t值3.709,P值为0.019)。
在检测克隆大小时发现,Tip30-/-Neu+/+FVB小鼠的克隆直径虽然大于Tip30+/+Neu+/+FVB小鼠(126.68±25.42μm vs.97.41±13.13μm),但无显著差异(t=1.772,P=0.151)。而Balb/C背景的Tip30基因敲除小鼠的克隆大小显著性高于Tip30+/+Balb/C小鼠(137.9±16.17gm vs.90.79±22.89μm),t值为2.914,P值为0.044。
1.3TIP30基因敲除可扩增小鼠乳腺干细胞
MRUs即乳腺再繁殖单位(CD24HighCD49flow),该细胞群富集乳腺干细胞。流式细胞仪检测结果显示,Tip30基因敲除的Balb/C小鼠的MRUs百分比为2.83±0.51%,显著性高于Tip30+/+Balb/C小鼠的1.27±0.51%,t值为3.739,P值为0.02。在有Neu转基因背景的FVB小鼠中亦得到相似结果,Tip30-/-Neu+/+FVB小鼠MRUs的百分比为6.73±0.85%,显著性高于Tip30+/+Neu+/+FVB小鼠的1.30±0.60%,t=9.042,P=0.001。
1.4TIP30基因敲除可扩增小鼠乳腺祖细胞
Ma-CFCs即乳腺克隆形成细胞(CD24HighCD49flow),该细胞群富集乳腺祖细胞。同样应用流式细胞仪检测,Tip30-/-Balb/C小鼠的Ma-CFCs百分比(3.23±0.49%)显著性高于Tip30+/+Balb/C小鼠(0.87±0.38%),t值为6.592,P值为0.003。Tip30-/-Neu+/+FVB小鼠的Ma-CFCs百分比为3.07±0.76%,显著性高于Tip30+/+Neu+/+FVB小鼠的1.43±0.31%,t=3.465,P=0.026。
2. TIP30基因敲除可导致乳腺ER+祖细胞比例增多
在Balb/c小鼠中,应用Scal对乳腺祖细胞再划分为CD24HighCD49flowScal+及CD24HighCD49flowSca1两个亚群,分别代表ER+祖细胞及ER-祖细胞。流式细胞仪检测结果显示：Tip30-/-Balb/c小鼠乳腺ER+祖细胞占祖细胞的30.67±2.45%,显著性高于Tip30+/+Balb/c小鼠的17.73±1.76%,t=7.415,P=0.002；而Tip30-/-Neu+/+FVB小鼠ER+祖细胞占祖细胞的23.80±5.27%,显著性高于Tip30+/+Neu+/+FVB的13.57±2.70%,t=2.994,P=0.04。
3.TIP30基因敲除可导致乳腺祖细胞倾向ER+细胞分化
我们运用流式细胞仪分选出乳腺祖细胞亚群(CD24HighCD49flow),使其在Matrigel上分化生长形成克隆,并进行ER/PR免疫荧光染色。结果如图及表示,Tip30-/-Balb/c小鼠的乳腺祖细胞形成的克隆中ER阳性细胞数占45.35±7.80%,显著性高于Tip30+/+Balb/c小鼠的31.77±6.38%,t=2.698,P=0.036。而Tip30-/-Balb/c和Tip30+/+Balb/c小鼠的乳腺祖细胞形成的克隆中PR阳性细胞数分别为38.70±8.45%及33.76±6.33%,无显著性差异(t=0.936,P=0.385)。
为了进一步验证TIP30基因对ER+细胞分化的影响,我们对Balb/c小鼠乳腺组织行ER/PR免疫荧光染色,结果示Tip30-/-Balb/c和Tip30+/+Balb/c小鼠乳腺组织ER阳性细胞分别占21.13±3.59%和13.00±3.716%,t=3.15,P=0.020,有显著性差异；而PR阳性细胞分别占18.53±2.83%和16.77±3.16%,t=0.832,P=0.437,无显著性差异。
4.TIP30基因敲除可上调乳腺组织FOXA1表达
我们在mRNA水平检测了与乳腺干/祖细胞分化相关的信号通路分子Notch. Hedgehog.Wnt及GATA3.FOXA1的表达情况,应用独立样本t检验,结果仅有FOXA1在TIP30基因敲除的乳腺组织中高表达,其相对表达值为3.05±0.90,显著性高于Tip30+/+Balb/c小鼠的1.00±0.17,t=3.879,P=0.018。进一步我们对其蛋白水平进行检测,发现,Tip30-/-Balb/c小鼠乳腺组织FOXA1蛋白表达相对值为66.24±5.93%,亦显著性高于Tip+/+Balb/c小鼠的40.27±8.18%,t=4.454,P=0.011。提示TIP30基因的缺失可能使FOXA1的表达的上调,从而诱导更多的ER阳性细胞分化。
5.下调乳腺细胞FOXA1表达可减少ER阳性细胞分化
为了进一步验证TIP30基因敲除导致的ER阳性细胞分化增多是否通过升高FOXA1表达而实现。我们从Tip30-/-Balb/c小鼠分离出乳腺上皮细胞,应用FOXA1-SH1、FOXA1-SH2及shRNA-CON基因干扰后,应用Western-blot检测验证FOXA1表达明显示受抑制后,行体外克隆形成试验,并进行ER免疫荧光染色。结果示FOXA1-SH1、FOXA1-SH2组形成的克隆中ER阳性细胞分别占31.00±2.85%及29.52±5.67%,显著性低于shRNA-CON组的39.40±6.16%,P值分别为0.042及0.018。该结果提示,TIP30基因敲除后,通过(至少部分通过)升高FOXA1的表达来促进乳腺祖细胞倾向ER阳性细胞分化。
6. TIP30基因敲除可以上调乳腺组织RANKL的表达
我们在进一步研究TIP30基因敲除小鼠乳腺干/祖细胞扩增可能的原因时发现,TIP30基因敲除可以使小鼠乳腺组织RANKL表达增高,其相对表达值为16.91±1.13,显著性高于野生型小鼠的7.94±4.47,t=3.370,P=0.028。提示TIP30基因敲除小鼠可能通过依靠增多的性激素受体细胞增强分泌RANKL,并通过旁分泌的方式作用于乳腺干/祖细胞,从而使其扩增。
三、结论
1.TIP30基因敲除可以扩增小鼠乳腺干/祖细胞。
2.TIP30基因敲除可增加小鼠乳腺ER阳性祖细胞比例,并通过上调FOXA1表达使祖细胞倾向于ER阳性细胞分化。
3.TIP30基因敲除可能通过依靠增多的性激素受体阳性细胞分泌更多的RANKL,并通过旁分泌的方式使乳腺干／祖细胞扩增。
Background and objective
Breast cancer is one of the most frequently diagnosed cancers in females worldwide, accounting for23%(1.38million) of the total new cancer cases and14%(458,400) of the total cancer deaths in2008. In general, incidence rates of breast cancer are still increasing year by year, but breast cancer death rates have been decreasing in North America and several European countries over the past25years. However, in many African and Asian countries, both incidence and mortality rates are rising. Although, a great improvement of dignosis and treatment of breast cancer have been made, the mechanism underlining breast cancer remains unclear. Estrogen receptor alpha (ERa) plays important roles in mammary gland development and carcinogenesis. ER positive breast cancer subtype is the most common breast cancer subtype, approximately accounting for60-70%. ER has been a preditive marker for patients'prognosis and a good marker for therapy chosen. ER positive breast cancer has better effect of therapy and prognosis in clinic, however, the machanism of this subtype of breast cancer is still unkown. This study generated an TIP30knockout mouse model and focused on exploring its limited development of ER positive breast cancer.
TIP30, also known as CC3, is a30-kDa human cellular protein that was purified as a HIV-1Tat interacting protein and its expression is found altered in human liver, lung, and breast cancers etal. Our previous studies showed that Tip30-deficient mice spontaneously develop tumors in several tissues and mammary preneoplastic lesions, suggesting that TIP30acts as a tumor suppressor. Its tumor suppressor activity is likely due to its actions in multiple cellular processes. TIP30can function as a transcription cofactor to repress expression of genes that are involved in proliferation and apoptosis. TIP30can act as a repressor of ERa-mediated c-Myc transcription in mammary glands and breast cancer cells. Moreover, data about TIP30controling endocytic downregulation of the EGFR signaling pathway in primary hepatocytes、 hepatocellular carcinoma cells and lung cancer cells also have been highlighted recently.
Our previous data showed that deleting the TIP30gene in mice leads to ductal hyperplasia in mammary glands early in life and extensive mammary hyperplasia with age. In addition, we also showed that deletion of TIP30in MMTV-Neu mouse can accelerate the onset of mammary tumors and resulted in ER+/PR-mammary tumors. In this study, we generated a TIP30knockout Balb/c mice model without the interference of Neu gene, and found that TIP30deletion in mice developed mammary tumors. We futher analysed the EGFR downstream cell signaling in this model to study the possible machanism of breast cancer development and used human breast cancer cell line to explore the role of TIP30in regulating EGFR degration.
Breast cancer has heterogeneous pathologies and molecular profiles and in this respect there are many molecular subtypes of breast cancer. The stem cell model of mammary carcinogenesis provides an explanation for the cellular heterogeneity within tumors, in addition to the phenotypic diversity seen in different subsets of patients. This model predicts that different subsets of mammary carcinoma arise from different cells and/or the particular mutations required to transform these cells. These mutations might result in uncontrolled self-renewal of the cancer stem cell, driving tumorigenicity, and aberrant differentiation, with this last property generating most of the cells within a tumor. In addition, studies in mice have clearly shown that different genes exert their influence in different cell subpopulations. For example, mouse mammary tumour virus (mmTv)-Wnt-1results in tumours that are composed of both luminal and myoepithelial cells and an expansion of the MaSC pool. However, mmTv-Erbb2(Neu) mice can generate luminal cell-restricted tumours and display an expansion of the luminal cell compartment.In our study, we are trying to explore effects of TIP30deletion on mammary stem/progenitor cells pool and their cell fate, which can provide some explainations for our mouse model which spontaneously developed ER positive breast cancers.
Part I:Loss of TIP30drives development of mouse breast cancer and activates EGFR downstream signal molecules.
Methods
1. TIP30knockout Balb/c mice were obtained by backcross TIP30knockout C57J6mice with Balb/c mice. After over7generations continuous passages, we finally established TIp30-/-Balb/c mice.
2. After being observed for78weeks, mice were sacrificed for relevant researches. Check the mouse organs such as brain, mammary, lung, etal to see any tumor or desease happened in them. Part of tissues were fixed, dehydrated and embeded in paraffin for futher analysis. Rest of tissues were freshly frozen in liquid nitrogen and were kept in-80℃for longterm storage.
3. H&E staining were used to detect the tissue section including the organs and tumors. Slides were examined by two pathologists for tumorgenesis or other aberrant histological changes. After that, incidence of breast carcinoma was caculated.
4. Immunohistochemistry (IHC) was performed to analyse expression of CK8, aSMA, pAkt and pERK in mammary gland and mammary tumor.
5. Immunofluorescence was used to detect expression of ER and PR in mamary tumor.
6. Lentiviruses were generated using shRNA-TIP30and shRNA-CON plasmids and then transfected MCF-7cells following puromycin selection. Transfection efficiencies were analyzed by western-blot.
7. Immunofluorescence was used to analyse the colocolization of EGF and EGFR, EGFR and EEA1as well as TIP30and EEA1in MCF-7cells after treated with EGF for indicated times.
8. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Incidence rate of breast cancer between two groups was analysed by x2test. Comparisons of two groups'or three groups'mean values were analyseed by independent sampe t test or One-way ANOVA followed by LSD or Dunnett multiple comparison test. P-Values were considered to be significant at <0.05.
Results
1. Knockout of TIP30results in spontaneous mammary tumor in Balb/c mice.
After18months observeation,47female mice reached the analysis end point.8of28Tip30-'-mice were found having mammary tumor, but no tumor found in19Tip30+/+mice (0%). Incidence rate of mammary tumor in Tip30-/-mice (28.6%) is significantly higher than Tip30+/+mice (0%), x2=6.269, P=0.012.
2. Mammary tumor induced by TIP30deletion mice were all ER/PR postive luminal type.
Mammary tumor tissue section were stained with K8and by IHC and results show edthat all the tumors are K8strongly posive, but aSMA negative, which implied that mammary tumors found in Tip30-/-mice were Luminal subtype. Then we used immunofluorescence to dectect ER/PR expression and found that all tumor sections are ER postive and PR positive. All these data indicated that Tip30-'-mice spontaneous developed ER/PR positive, Luminal type breast cancer.
3. TIP30deletion leads to EGFR pathways such as MAPK and PI3K activation.
IHC was used to dectect pErkl/2and pAkt expression in mammary gland and mammary tumor sections. Positive rates of pErkl/2and pAkt in Tip30-/-mice mammary gland were27.83±8.46%and30.83±6.65%(n=5), which were significantly higher than12.58±5.87%and14.94±5.77%in Tip30+/+mice respectively, P values were0.02and0.011. Moreover, positive rates of pErkl/2and pAkt in Tip30-/-mice mammary tumor were51.68±8.57%and56.08±8.44%, significantly higher than mammary gland, P values were0.002and0.001 respectively.
4. TIP30knockdown could prolong activation of EGFR downstream signal molecules of mamary tumor cells induced by EGF.
4.1TIP30-SH1and TIP30-SH2transfection respectively downregulated the expression of TIP30in MCF-7cells.
After transfection and anibiotic selection, relative TIP30expression of TIP30-SHland TIP30-SH2cells are13.12±3.95%and11.54±2.68%(n=3), significantly lower than shRNA-CON (84.3±8.66%, n=3) respectively, both of P values less than0.001. In general, TIP30expression of TIP30-SH1and TIP30-SH2cells were downregulated by71.18%and72.76%respectively, which implicated that the transfecting efficience was well.
4.2TIP30could promote degration of EGFR induced by EGF
After treated with EGF, EGF and EGFR expressions were analysed by immunofluorescence at indicated times1Omin,1h,4h. We found that both intencity of EGF and EGFR fluorescence were no difference between TIP30-SH and shRNA-CON cells at10min and1h after treated with EGF, and the Pearson's colocalization of EGF and EGFR were not different either. However, at4h after treated with EGF, both intencity of EGF and EGFR fluorescence in TIP30-SH cells were1.19±0.18and1.33±0.13, significantly higher than0.68±0.08and0.76±0.10in shRNA-CON cells respectively (1=4.563and6.132, P=0.01and0.004); at the same time, the Pearson's colocalization of EGF and EGFR in TIP30-SH cells was significantly higher than shRNA-CON cells (35.07±5.33vs.10.76±2.28; t=7.270, P=0.002).
4.3TIP30knockdown could stagnate EGFR in early endosomes.
2h after treated with EGF, EGFR and early endosome marker EEA1expressions in cells were analysed by immunofluorescence. The Pearson's colocalization value of EGFR and EEA1in TIP30-SH was56.39±14.08%, significantly higher than27.31±9.12%of shRNA-CON.
4.4TIP30could assemble in early endosome and regulate EGFR degration.
We also used immunofluorescence to dectect colocalization of TIP30and EEA1, and found that TIP30are colocalized with EEA1at10min after shRNA-CON cells treated with EGF.
Conclusion
1. TIP30knockout Balb/c mice can spontaneously develop mammary tumor.
2. All the mammary tumor developed by TIP30knockout Balb/c mice are ER/PR+Luminal type.
3. TIP30deletion results in EGFR downstream pathways such as MAPK and PI3K activation.
4. Inhibition of Tip30could delay EGF-induced degradation of EGFR, thus prolong EGFR downstream signal molecules and promote the proliferation of breast cancer cells.
Part Ⅱ:TIP30deletion promotes mouse mammary stem/progenitor cells expansion and regulates progenitor cell fate
Methods
1. Gained Tip30-/-Balb/c and Tip30-/-Neu+/+FVB mice by crossbreeding. After over7generations continuous passages, we finally established TIP30knockout mice with different backgrounds.
2.5-6month old mouse mammary tissues were dissociated mechanically and enzymatically. Finally the mammary cell suspensions were obtained.
3. Mammosphere culture was used to detect the mammosphere forming capacity of mammary cells from different genotype mice. Caculated the mammosphere's number and size under microscope.
4. Colony forming assay in vitro was performed to analyse the colony generation capacity of mammary cells from different genotype mice. Colony number and size were caculated under microscope.
5. Flow cytometry was used to analyse the population of CD24lighCD49flow (MRUs) and CD24HighCD49flow (Ma-CFCs) in mammary cells from different genotype mice, then analyse the subpopulation of Scal+and Seal-in CD24HighCD49flow population.
6. Flow cytometry was used to sort out CD24HighCD49flow population which were gown in vitro using colony forming assay, then were stianed with ER/PR by immunofluorescence.
7. Immunofluorescence was used to detect ER/PR expression of the mammary glands.
8. QPCR was used to the mRNA levels of factors which related to mammary stem/progenitor cells fate decision.
9. Used Western-blot to detect FOXA1and RANKL expression of mammary tissues.
10. Lentiviruses were generated using shRNA-FOXAl and shRNA-CON plasmids and then transfected TIP30-/-cells following puromycin selection. After the transfection efficiencies were confirmed by western-blot. The cells were grown in vitro to form colonies and then stained with ER.
11. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Comparisons of two groups'or three groups'mean values were analyseed by independent sampe t test or One-way ANOVA followed by Dunnett multiple comparison test. P-Values were considered to be significant at≤0.05.
Results
1. Loss of TIP30can promote mammary stem/progenitor cells expansion.
1.1TIP30deletion could increase mammosphere forming capacity.
After9d mammosphere culture in serum free medium, mammosphere forming number of Tip30-/-Balb/c was31.75±5.85/5000cells seeded, significantly higher than19.75±4.79/5000cells of7ip30+/+Balb/c, t=3.174, P=0.019. We found a similar result in Neu+/+FVB background mice. Tip30-/-Neuw+/+FVB mice had more mammospheres than Tip30+/+Neu+/+FVB mice (31.25±5.56/5000cells vs.17.5±4.04/5000cells), t=4.001, P=0.007.
We also measured the size of mammospheres and found that mammospheres formed by by mammary cells of Tip30-''Balb/c mice had larger size than that in Tip30+/+Balb/c mice.(156.80±29.28μm vs.97.59±20.66μm; t=3.305, P=0.016). But we can't find any difference in size between Tip30-/-Neu+/+FVB (128.29±27.83μm) and Tip30+/+Neu+/+FVB mice(105.53±14.19μm),t=1.457,P=0.195.
1.2TIP30deletion could enhance colony forming capacity in vitro.
2000Mammary cells/well from different mice were seeded on matrigel precoated well for10d.The colony number of Tip30-/-Balb/c(33.33±5.13)was significantly higher compaired with Tip30+/+Balb/c mice(19.00±6.00),t=3.144,P=0.035. Moreover,mammary cells from and Tip30-/-Neu+/+FVB also formed more colonies than those from Tip30+/+Neu+/+FVB respectively(33.00±5.29vS.18.00±4.36),t=3.709,P=0.019.
When measuring the size of colonies,we found that Tip30-/-Balb/c(137.9±16.17μm) has larger colony than Tip30+/+Balb/c(90.79±22.89¨m),t=2.914,P=0.044.However, there was no difference in size between Tip30-/-Neu+/+FVB(126.68±25.42μm)and Tip30+/+Neu+/+FVB mice(97.41±13.13Um),t=1.772,P=0.151.
1.3TIP30deletion can promote MRUs expansion.
Flow cytometry analysis showed that MRUs population of Tip30-/-Balb/c accouting for2.83±0.51%was significantly higher than Tip30+/+Balb/c which wasl.27±0.51%, t=3.739.P=0.02.We got a silimar result which showed that TIP30deletion can significantly increase the MRUs population from1.30±0.60%to6.73±0.85%in Neu+/+FVB mice,t=9.042,P=0.001.
1.4TIP30deletion can increase Ma-CFCs.
Flow cytometry analyse also showed that Ma-CFCs population of Tip30-/-Balb/c was3.23±0.49%,significantly higher than0.87±0.38%of Tip30+/+Balb/C,t=6.592, P=0.003.For Neu+/+FVB mice,TIP30deletion resulted in Ma-CFCs population increasing from1.43±0.31％to3.07±O.76&,t=3.465,P=0.026.
2.TIP30deletion in mice can increase ER+mammary progenitor cells.
We used balb/c and Neu+/+FVB mice for further analysis.Sca-1was used for discriminate mammary progenitors cells to two suppoulations,CD24HighCD49flow Scal+and CD24HighCD49flowScal-which represent ER+and ER-progenitor cells respectively.Flow cytometry analysis showed that TIP30deletion can significantly increase the CD24HighCD49flowScal+population from17.73±1.76%to30.67±2.45%, t=7.415, P=0.002. In addition, Tip30-/-Neu+/+FVB mice also had more ER+progenitor cells compared with Tip30+/+Neu+/+B mice (23.80±5.27%vs.13.57±2.70%), t=2.994, P=0.04。
3. TIP30deletion can predispose mammary progenitor cells to ER+cell fate.
Flow cytometry was used to sort out the mammary progenitor cell population (CD24HighCD49flow), and the cells were plated on the matrigel precoated well and cultured to form colonies then stained with ER/PR by immunofluorescence. We found that colonies formed by Tip30-'-Balb/c mice have more ER positive cells than colonies formed by Tip+/+Balb/c mice (45.35±7.80%vs.31.77±6.38%), t=2.698, P=0.036. But we can't find difference in number of PR positive cells in colonies form by Tip30-/-and Tip30+/+Balb/c mice (38.70±8.45%vs.33.76±6.33%), t=0.936, P=0.385.
To further comfirm the influence of TIP30deletion on ER positive cell differentiation of mammary cells, we checked the ER/PR expression of mouse mammary gland by immunofluorescence. Tip30-/-Bdlb/c mammary glands had more ER positive cells than Tip30+/+Balb/c mammary gland (21.13±3.59%vs.13.00±3.716%, t=3.15, P=0.020). However, there was no difference of PR positive cells between Tip30-/-Balb/c and Tip30+/+Balb/c mammary glands.
4. Loss of TIP30upregulated FOXA1expression of mammary glands.
We next measured some signaling pathway (Notch, Hedgehog, Wnt) or genes (GATA3、FOXA1) related to cell fate decision at mRNA levels. Only FOXA1mRNA level was found markedly upregulated in Tip30-/-Balb/c mouse mammary glands compared with Tip30+/+Balb/c mice (3.05±0.90vs.1.00±0.17), t=3.879, P=0.018. We further analysised the protein level of FOXA1. The relative expression of FOXA1in Tip30-/-Ba\b/c mammary glands was66.24±5.93%, significantly higher than40.27±8.18%in Tip30+/+Balb/c mammary glands, t=4.454, P=0.011.
5. Down regulation of FOXA1in Tip30-/-mammary cells decreased ER+cell differentiation.
To determine whether upregulation of FOXA1in Tip30-/-mammary cells was responsible for more differentiation of ER+cell than Tip30+/+mammary cells. We harvested the mammary cells from Tip30-/-balb/c mice, and knocked down the FOXA1expression. After confirmation of knockdown of FOXA1by western blot, cells were grown in matrigel to form colonies which were then stianed with ER. The percentage of ER+cells in colonies formed by FOXA1-SHland FOXA1-SH2cells were31.00±2.85%and29.52±5.67%respectively, both of which were significantly lower than39.40±6.16%in colonies formed by shRNA-CON; P values were0.042and0.018respectively.
6. TIP30deletion upregulated RANKL expression of mammary glands.
To determine why TIP30deletion can result in expansion of mammary stem/progenitor cells, expression of RANKL was measured by western blot. We found that the levels of RANKL were markedly increased in Tip30-'-Ba\b/c mammary glands than in Tip30+/+Balb/c mammary glands (16.91±1.13vs.7.94±4.47, t=3.370, P=0.028) which indicated that TIP30deletion may promote mamamry stem/progenitor cells expansion through upregulaion of RANKL in a paracrine manner.
Conclusion
1. TIP30deletion can promote mammary stem/progenitor cells expansion in mice.
2. TIP30deletion can increase ER positive progenitor cells, and predispose mammary progenitor cells to ER+cell fate through upregulation of FOXA1.
3. TIP30deletion may induce secretion of RANKL from hormone receptor positive cells and promote the mammry stem/progenitor cells expansion by paracrine way.
引文
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