Mechanical induction of the tumorigenic
β-catenin pathway by tumour growth pressure
The
tumour microenvironment may contribute to tumorigenesis owing to mechanical
forces such as fibrotic stiffness or mechanical pressure caused by the expansion
of hyper-proliferative cells. Here we explore the contribution of the
mechanical pressure exerted by tumour growth onto non-tumorous adjacent
epithelium. In the early stage of mouse colon tumour development in the Notch+Apc+/1638Nmouse model, we observed mechanistic pressure stress in the non-tumorous
epithelial cells caused by hyper-proliferative adjacent crypts overexpressing
active Notch, which is associated with increased Ret and β-catenin signalling.
We thus developed a method that allows the delivery of a defined mechanical
pressure in vivo, by subcutaneously inserting a magnet close to the mouse
colon. The implanted magnet generated a magnetic force on ultra-magnetic
liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding
colonic crypts after intravenous injection. The magnetically induced pressure
quantitatively mimicked the endogenous early tumour growth stress in the order
of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound
strain imaging and shear wave elastography. The exertion of pressure mimicking
that of tumour growth led to rapid Ret activation and downstream
phosphorylation of β-catenin on Tyr654, imparing its interaction with the
E-cadherin in adherens junctions, and which was followed by β-catenin nuclear
translocation after 15 days. As a consequence, increased expression of
β-catenin-target genes was observed at 1 month, together with crypt enlargement
accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation
of the tumorigenic β-catenin pathway suggests unexplored modes of tumour
propagation based on mechanical signalling pathways in healthy epithelial cells
surrounding the tumour, which may contribute to tumour heterogeneity.
【独家解读】Nature：肿瘤生长产生机械压力诱导
发布时间：2015-06-25 00:00 文章来源： 作者：
由过度增生细胞扩充所产生的纤维性僵直或者机械压力将引起肿瘤微环境的改变进而引发肿瘤的形成。研究人员在早期探索了肿瘤在癌旁上皮细胞上生长所产生的机械压力，并通过观察在小鼠模型结肠肿瘤的早期生长过程，发现癌旁上皮细胞产生了机械压力，同时会过表达Notch，并伴随着RET和β-连环蛋白通路的增强。同时，研究人员也构建出了一种方法，即通过在小鼠皮下结肠附近插入一个磁体使得在小鼠体内可以产生一个确定的机械压力。植入的磁体可以在磁性脂质体上产生磁力，静脉注射后，在结肠间隐窝周围的结缔组织中的间充质干细胞中稳定。磁力诱导的压力可以定量地模仿内源性肿瘤早期生长产生的压力（1200 pa），发现没有产生纤维性僵直（排除肿瘤晚期纤维性僵直的微环境的影响）。模仿肿瘤生长产生的压力引起的RET会快速活化以及β-连环蛋白Tyr654位点的磷酸化，这减弱了它与E-钙黏蛋白在黏着连接中的相互作用，而15天后β-连环蛋白则发生核易位。实验结果显示，1个月后，β-连环蛋白信号途径靶基因的表达得到增强，同时发现隐窝扩大及早期肿瘤异常隐窝病灶形成。在肿瘤周围的正常上皮细胞中基于机械信号途径的肿瘤增生模型，可能对肿瘤异质性有促进作用。
为了检测早期肿瘤生长对癌旁上皮细胞所产生的机械压力，研究人员首先用旋转盘共聚焦显微镜观察了体内的结肠间隐窝肿瘤组织的形态变化，随后利用过度增生隐窝过表达Notch的Notch/Apc小鼠模型，并依据胡克定律来进行弹性测量。结果显示，到2周的时候，压力达到0-2.4 kPa；1个月后，压力达到0.4 -5.6 kPa。为了模仿应用体内过度增生的结肠隐窝所产生的同样的机械压力，研究人员通过一种基于周边包围有超顺磁性氧化铁粒子的磁性脂质体（以便于静脉注射）的方法，将3mm圆筒状磁石插入到皮下结肠组织前方。静脉注射一周后，激光共聚焦显微镜观察到结肠周围的间质细胞中脂质体负载，组织免疫荧光分析显示结肠组织周围间质细胞中脂质体内化是由波形蛋白标记所决定的。这些数据和肿瘤生长产生的病理压力相一致。它们与在2周至1个月在Notch/Apc突变体小鼠异常隐窝中产生机械压力的测量也是相同的。
随后，研究人员将磁力诱导的压力应用到Apc单一突变的小鼠结肠隐窝中，结果显示，1个月后β-连环蛋白靶基因Myc, Axin2和Zeb1的表达上调。定量PCR结果表明三个靶基因上调表达倍数分别达到2.3, 2.5和3.5倍。与这些转录应答相一致的是产生压力2周后，发现β-连环蛋白Tyr654位点的磷酸化水平提高进而阻碍了其与E-钙黏蛋白之间的互作，检测到磷酸化的β-连环蛋白与绒毛蛋白共定位于刷状缘组织。同时，研究人员在Apc单一突变的小鼠结肠隐窝中也发现了核内和胞质中均有β-连环蛋白的富集，证实了相关研究。
通过小动物内窥结肠镜检查，在Apc异质结合遗传小鼠应用压力5天后，明显看到异常隐窝病灶的出现，15天后引发腺瘤增生。因此，模仿体内肿瘤生长产生的压力活化了Wnt/β-catenin信号通路。针对肿瘤生长机械压力中具体分子机制，体内机械压力是否可活化表达Ret基因的研究，在野生型小鼠和Apc突变体小鼠结肠隐窝中均有进展和发现。当应用压力为1.194±0.660 kPa时，RET快速活化、β-连环蛋白Tyr654位点磷酸化以及β-连环蛋白出现核转运；在2月、3月观察到内部生长压力引起的Myc过表达和异常隐窝病灶的形成。此外，研究人员还发现了Akt信号依赖的Ret上游GSK-3β基因的失活，GSK-3β蛋白可抑制β-连环蛋白的核聚集及向胞质中的释放。
最后，研究人员在Notch/Apc突变体小鼠结肠中检验了过度增生诱导的内源性压力是否可以激活动力传导途径。首先发现，突变体结肠隐窝中，Ret和β-连环蛋白均多数发生了磷酸化。一个月后同样检测到β-连环蛋白的核、胞质富集，2.5个月后，Myc发生过表达。此外，Ret和β-连环蛋白磷酸化等上述多种现象不仅发生在Notch信号激活的结肠隐窝中，同样也出现在Notch信号失活的癌旁隐窝中。
总的来说，在野生型和Notch/Apc小鼠模型的结肠上皮组织中过度增生生长压力均可引发肿瘤发生早期过程中Ret/β-catenin信号途径的活化机制。同时发现，这样的动力转导途径同样也存在于Apc异质接合的癌旁隐窝细胞中。通过以上研究，可以推测出，各种类型肿瘤形成的信号途径产生的机械压力刺激很有可能在癌旁正常组织中也会发生，构建出原癌基因表达、肿瘤形成诱导以及对于肿瘤生长和扩散的增强相互之间不稳定的正反馈循环。（来源：百替生物 作者：付绪亮）
原文摘要：
Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure
The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch+Apc+/1638Nmouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and β-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of β-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by β-catenin nuclear translocation after 15 days. As a consequence, increased expression of β-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic β-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.
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