在乳腺肿瘤微环境（TME）中丰富的肿瘤促进癌相关成纤维细胞（CAFs）维持着转化生长因子-β（TGF-β）-Smad2/3信号通路的激活以及肌成纤维细胞状态，后者是激活成纤维细胞的标志。然而，有关肌成纤维细胞CAF（简称为myCAFs）在TME中的来源以及激活的成纤维细胞表型的表观遗传和代谢改变尚未被充分理解。我们在此研究中发现，肿瘤中存在的myCAFs中的全球组蛋白去乙酰化与乳腺癌患者的预后密切相关。由于TME受到谷氨酰胺（Gln）缺乏的影响，我们培养了人乳腺成纤维细胞（HMFs）在Gln饥饿的培养基中。这些细胞中出现了全球组蛋白去乙酰化和TGF-β-Smad2/3信号通路的激活，其中主要由I类组蛋白去乙酰化酶（HDAC）活性介导。此外，在缺乏Gln的HMFs中，机械/哺乳动物雷帕霉素靶蛋白复合物1（mTORC1）信号传导被抑制，并且Gln补充培养的HMFs中的mTORC1抑制通过雷帕霉素处理提升了TGF-β-Smad2/3信号通路的激活。这些数据表明，mTORC1抑制在Gln饥饿的HMFs中介导TGF-β-Smad2/3信号通路的激活。在培养的人乳腺CAF中，我们还观察到了全球组蛋白去乙酰化、I类HDAC活化以及mTORC1抑制的现象。通过I类HDAC抑制或高剂量Gln补充导致的mTORC1活化显著减弱了这些细胞中的TGF-β-Smad2/3信号通路和肌成纤维细胞状态。这些数据表明，I类HDAC活化和mTORC1抑制对于维持myCAF特征是必需的。综上所述，这些发现表明Gln饥饿通过I类HDAC活性和mTORC1抑制触发了HMFs中的TGF-β信号通路的激活，可能导致myCAF转化。© 2023 The Authors. 由John Wiley & Sons Australia, Ltd发表的《癌症科学》代表日本癌症协会。

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-β (TGF-β)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-β-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-β-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-β-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-β-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-β signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.