Department of Biochemistry and Molecular Biology, Buffett Cancer Center, College of Medicine, University of Nebraska Medical Center, 7005 Durham Research Center, 985870 Nebraska Medical Center, Omaha, NE, 68198, USA.
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
Breast Cancer Res. 2022 Jul 14;24(1):48. doi: 10.1186/s13058-022-01548-6.
A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFβ-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer.
We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA.
We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.
已报道促氧化剂酶 NADPH 氧化酶 4（Nox4）是 TGFβ诱导纤维化过程中肌成纤维细胞转化的关键下游效应因子。虽然有少量研究表明源自激活成纤维细胞的 Nox4 具有致癌作用，但直接将这种促氧化剂与支持肿瘤的 CAF 表型联系起来的证据以及所涉及的机制尚缺乏，特别是在乳腺癌中。
我们通过 siRNA 介导的敲低或使用药物抑制剂（GKT137831）靶向靶向乳腺患者衍生的 CAFs 中的 Nox4。我们还使用稳定的 Nox4-/- 同基因小鼠模型来确定植入肿瘤细胞的原发性肿瘤生长和转移。通过共聚焦显微镜分析和自噬标记物（beclin-1 和 LC3B）的表达水平，使用串联荧光标记的 ptfl-LC3 质粒评估 CAF 的自噬通量。Nox4 过表达的 CAFs 的存活依赖于 Nrf2（核因子红细胞 2 相关因子 2）途径。然后，我们确定了 Nox4 过表达的 CAFs 对 Nrf2 介导的适应性应激反应途径的生存依赖性。此外，我们研究了 Birc5 在 CAF 表型（活力和胶原蛋白收缩活性）以及 CAF 标志物 FAP 和αSMA 的表达水平中的参与。
我们发现，基质 Nox4 的缺失和使用 GKT137831 对其活性的药物靶向治疗显著抑制了 E0771 和 4T1 小鼠乳腺癌细胞系在小鼠中的原位肿瘤生长和转移。更重要的是，我们发现与正常乳腺成纤维细胞（RMFs）相比，分离自人类乳腺肿瘤的 CAFs 中 Nox4 表达明显上调。我们对人类乳腺癌肿瘤微阵列进行的 Nox4 转录原位 RNA 杂交分析进一步支持了这种促氧化剂在乳腺癌基质中的作用。此外，我们发现 Nox4 促进 CAFs 中的自噬。此外，我们发现 Nox4 通过激活 Nrf2（氧化应激反应的主要调节剂）促进 CAFs 的存活。我们进一步表明 Birc5 作为 Nrf2 介导的存活表型的下游调节剂参与其中。这些研究表明，通过促进 CAFs 的自噬和存活，Nox4-Nrf2 途径的氧化还原信号在乳腺癌细胞的肿瘤发生和转移中起作用。
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