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Abstract：
BACKGROUND: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.
METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.
RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.
CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.
METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.
RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.
CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.
摘要：
背景：Bcl-2和Bcl-xL是Bcl-2家族蛋白中研究最多的抗凋亡成员。我们以前描述过他们两个，不仅因为它们在调节细胞凋亡和抵抗癌细胞治疗中的作用，而且对于它们的非规范函数，主要包括促进癌症进展，转移，血管生成,并参与癌细胞和肿瘤微环境成分之间的串扰。我们的目标是鉴定由Bcl-2特异性调节的转录特征和新的细胞途径。
方法：我们在人黑色素瘤细胞中进行了siRNA介导的Bcl-2或Bcl-xL瞬时敲低的RNAseq分析和基因本体论分析，以鉴定特定的Bcl-2转录特征。在人黑色素瘤中验证了由Bcl-2调节并与Hippo途径相关的基因的表达，通过qRT-PCR检测乳腺癌和非小细胞肺癌细胞系。进行Western印迹分析以分析YAP的上游调节因子的蛋白表达以及与不同水平的Bcl-2蛋白的关系。在相对于不同硬度条件的迁移和细胞活力测定中评估了YAP沉默在Bcl-2过表达癌细胞中的作用。体外伤口愈合测定和共培养物用于评估癌症特异性Bcl-2激活成纤维细胞的能力。
结果：我们通过作用于上游YAP调节剂，证明了来自不同肿瘤类型的癌细胞系中Hippo通路的Bcl-2依赖性调节。YAP抑制消除了Bcl-2在高刚性培养条件下增加肿瘤细胞迁移和增殖的能力，刺激体外成纤维细胞迁移并诱导成纤维细胞活化。
结论：我们发现Bcl-2在不同肿瘤类型中调节Hippo通路，促进细胞迁移，适应较高的刚度培养条件和成纤维细胞活化。我们的数据表明，应进一步研究Bcl-2抑制剂以抵消促癌机制。
方法：我们在人黑色素瘤细胞中进行了siRNA介导的Bcl-2或Bcl-xL瞬时敲低的RNAseq分析和基因本体论分析，以鉴定特定的Bcl-2转录特征。在人黑色素瘤中验证了由Bcl-2调节并与Hippo途径相关的基因的表达，通过qRT-PCR检测乳腺癌和非小细胞肺癌细胞系。进行Western印迹分析以分析YAP的上游调节因子的蛋白表达以及与不同水平的Bcl-2蛋白的关系。在相对于不同硬度条件的迁移和细胞活力测定中评估了YAP沉默在Bcl-2过表达癌细胞中的作用。体外伤口愈合测定和共培养物用于评估癌症特异性Bcl-2激活成纤维细胞的能力。
结果：我们通过作用于上游YAP调节剂，证明了来自不同肿瘤类型的癌细胞系中Hippo通路的Bcl-2依赖性调节。YAP抑制消除了Bcl-2在高刚性培养条件下增加肿瘤细胞迁移和增殖的能力，刺激体外成纤维细胞迁移并诱导成纤维细胞活化。
结论：我们发现Bcl-2在不同肿瘤类型中调节Hippo通路，促进细胞迁移，适应较高的刚度培养条件和成纤维细胞活化。我们的数据表明，应进一步研究Bcl-2抑制剂以抵消促癌机制。
