BACKGROUND The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. METHODS We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. RESULTS We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. CONCLUSION This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

中文翻译：

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p66ShcA可以作为乳腺癌转移的背景启动子。

背景技术p66ShcA氧化还原蛋白是Shc1基因的最长同工型，在乳腺癌中可变表达。响应各种应激刺激，p66ShcA在丝氨酸36上被磷酸化，使其能够从细胞质转移到线粒体，从而刺激活性氧（ROS）的形成。相互矛盾的研究表明，p66ShcA具有促癌和抗肿瘤功能，这促使我们研究了p66ShcA肿瘤细胞内在功能在乳腺癌转移中的作用。方法我们测试了p66ShcA是否会影响乳腺癌细胞的肺转移能力。将具有ErbB2 + /管腔（NIC）或基础（4T1）亚型特征的乳腺癌细胞改造为过表达p66ShcA。此外，通过Crispr / Cas9基因组编辑，将肺转移性4T1变体（4T1-537）工程化为缺乏内源性p66ShcA。然后用野生型p66ShcA或不能易位至线粒体的突变体（S36A）重建p66ShcA空细胞，从而缺乏刺激线粒体依赖性ROS产生的能力。在实验（尾静脉）转移测定中，测试了这些细胞从原发部位或种子形成自发转移并在肺中定植的能力。这些细胞的迁移率，粘着动力学和体外对阳极的抗性进一步表征。最终，在体内评估了它们在循环中存活并播种小鼠肺部的能力。结果我们显示p66ShcA通过增强乳腺癌细胞在转移级联的每个阶段中的导航能力来增加其肺癌的转移潜力。一个不可磷酸化的p66ShcA-S36A突变体，不能突变至线粒体，仍能增强乳腺癌细胞的迁移，肺部定植和继发性肺转移的生长。但是，乳腺癌细胞在循环中的生存唯一需要完整的p66ShcA S36磷酸化位点。结论这项研究提供了线粒体和非线粒体p66ShcA池在乳腺癌细胞中协同作用以促进其最大转移适应性的第一个证据。仍能增强乳腺癌细胞的迁移，肺部定植和继发性肺转移的生长。但是，乳腺癌细胞在循环中的生存唯一需要完整的p66ShcA S36磷酸化位点。结论这项研究提供了线粒体和非线粒体p66ShcA池在乳腺癌细胞中协同作用以促进其最大转移适应性的第一个证据。仍能增强乳腺癌细胞的迁移，肺部定植和继发性肺转移的生长。但是，乳腺癌细胞在循环中的生存唯一需要完整的p66ShcA S36磷酸化位点。结论这项研究提供了线粒体和非线粒体p66ShcA池在乳腺癌细胞中协同作用以促进其最大转移适应性的第一个证据。