Molecular Functional Imaging Lab, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India.
Molecular Functional Imaging Lab, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India.
Exp Cell Res. 2020 Nov 15;396(2):112313. doi: 10.1016/j.yexcr.2020.112313. Epub 2020 Sep 28.
Activation of STAT3 via Y705-phosphorylation is well documented across multiple cancer types and thus forms the basis of canonical pathway to judge STAT3 activation. Recently, important roles of two other post translational modification (PTM) sites, i.e. S727-phosphorylation and K685-acetylation, leading to STAT3 activation are reported. However, their critical mode of function in controlling STAT3 dimerization and signaling, independent of canonical activation remains elusive. Therefore, to understand the functional relevance of each STAT3 PTMs in breast cancer (BC), cell models are developed by stable overexpression of PTM-site specific point mutants, i.e. Y705F, S727A or K685R, in a 3'UTR-STAT3 knockdown BC cell background. Results using this model system reveal novel findings showing that phosphorylation at S727 can lead to STAT3 activation independent of phosphoY705. We also demonstrate that loss of pS727 or K685ac significantly affects functional phenotypes such as cell survival and proliferation as well as downstream transcriptional activity (Twist 1, Socs3, c-Myc, Bcl-1 and Mcl-1) of STAT3. Thereafter, by utilizing a BRET biosensor for measuring STAT3 phosphorylation in live cells, a crucial role of pS727 in dictating STAT3 activation and homodimerization formation is uncovered. Further by performing retrospective IHC analysis of total and phospho-forms of STAT3 in a cohort of 76 triple negative breast cancer (TNBC) patient samples, a significant dominant expression of phosphoS727 over phosphoY705 PTM (p < 0.001) is found in STAT3 positive cases. We also focus on validating known STAT3 inhibitor molecules for their action against both pY705 and pS727 activation. This study for the first time demonstrates that an anti-helminth drug compound, Niclosamide, is capable of inactivating both phospho-PTM sites on STAT3 and exhibits excellent anticancer efficacy in preclinical TNBC tumour model.
STAT3 的 Y705 磷酸化激活在多种癌症类型中已有充分的文献记载，因此成为判断 STAT3 激活的经典途径的基础。最近，报道了另外两个翻译后修饰（PTM）位点，即 S727 磷酸化和 K685 乙酰化，对 STAT3 激活的重要作用。然而，它们在控制 STAT3 二聚化和信号转导方面的关键作用，独立于经典激活机制，仍不清楚。因此，为了了解每个 STAT3 PTM 在乳腺癌（BC）中的功能相关性，通过在 3'UTR-STAT3 敲低 BC 细胞背景下稳定过表达 PTM 位点特异性点突变体（即 Y705F、S727A 或 K685R），开发了细胞模型。该模型系统的研究结果揭示了新的发现，表明 S727 的磷酸化可以导致 STAT3 激活，而不依赖于磷酸化 Y705。我们还证明，pS727 或 K685ac 的缺失会显著影响细胞存活和增殖等功能表型，以及 STAT3 的下游转录活性（Twist1、Socs3、c-Myc、Bcl-1 和 Mcl-1）。此后，通过利用活细胞中测量 STAT3 磷酸化的 BRET 生物传感器，揭示了 pS727 在决定 STAT3 激活和同源二聚体形成中的关键作用。进一步通过对 76 例三阴性乳腺癌（TNBC）患者样本中总 STAT3 和磷酸化 STAT3 形式的回顾性 IHC 分析，发现 STAT3 阳性病例中磷酸化 S727 的表达明显高于磷酸化 Y705（p<0.001）。我们还专注于验证已知的 STAT3 抑制剂分子对 pY705 和 pS727 激活的作用。这项研究首次表明，一种抗蠕虫药物化合物尼氯硝唑能够使 STAT3 上的两个磷酸化 PTM 失活，并在 TNBC 肿瘤模型的临床前研究中表现出优异的抗癌疗效。
