Table_2_Antidiabetic DPP-4 Inhibitors Reprogram Tumor Microenvironment That Facilitates Murine Breast Cancer Metastasis Through Interaction With Cancer Cells via a ROS–NF-кB–NLRP3 Axis.doc

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS–NRF2–HO-1 axis in nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS–NRF2–HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-κB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2–HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS–NRF2–HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive lymphocytes in metastatic sites, but did not significantly alter cell viability, apoptosis, differentiation, and suppressive activation of 4T1-induced splenic MDSCs. Moreover, we revealed that DPP-4i triggered ROS-NF-κB-dependent NLRP3 inflammasome activation in BC cells, leading to increase in inflammation cytokines such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), IL-1β and IL-33, and MDSCs inductors granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF, which play a crucial role in the remodeling of tumor immune-suppressive microenvironment. Thus, our findings suggest that antidiabetic DPP-4i reprograms tumor microenvironment that facilitates murine BC metastasis by interaction with BC cells via a ROS–NRF2–HO-1–NF-κB–NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic ROS–NRF2–HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the improvement of tumor microenvironment to alleviate DPP-4i-induced BC metastasis.

加深对抗糖尿病药物在乳腺癌（breast cancer, BC）进展中的安全性特征与生物学意义的理解，或可为降低糖尿病合并乳腺癌患者使用抗糖尿病药物时出现的意外不良反应提供新的思路。我们此前的研究发现，两种常用的抗糖尿病二肽基肽酶-4抑制剂（dipeptidyl peptidase-4 inhibitors, DPP-4i）类药物——沙格列汀（Saxagliptin, Sax）与西格列汀（Sitagliptin, Sit），可通过活性氧-核因子E2相关因子2-血红素加氧酶1（ROS–NRF2–HO-1）轴促进非肥胖糖尿病/重症联合免疫缺陷（nonobese diabetic–severe combined immunodeficiency, NOD-SCID）小鼠体内的小鼠乳腺癌4T1细胞转移。然而，在免疫健全状态下，DPP-4i在乳腺癌进展中的潜在作用仍未明确。本研究拓展了此前的实验范围，结果显示沙格列汀与西格列汀同样可在原位移植、同基因背景且免疫健全的BALB/c小鼠体内加速小鼠乳腺癌4T1细胞的转移。机制研究显示，DPP-4i不仅可激活ROS–NRF2–HO-1轴，还可在体内外触发活性氧（reactive oxygen species, ROS）依赖的核因子κB（nuclear factor kappa B, NF-κB）活化及其下游转移相关基因的表达；体外实验中，抑制NF-κB可显著阻断DPP-4i介导的乳腺癌细胞转移。与此同时，抑制NRF2–HO-1轴的活化可减弱DPP-4i介导的NF-κB活化，而NRF2激活剂ALA（ALA）可增强NF-κB活化，这表明ROS–NRF2–HO-1轴在DPP-4i介导的NF-κB活化中发挥关键作用。此外，我们还发现，DPP-4i可增加转移灶内的肿瘤浸润CD45阳性细胞、髓过氧化物酶（myeloperoxidase, MPO）阳性细胞、F4/80阳性细胞、CD4阳性细胞、叉头框P3（Foxp3）阳性细胞以及髓系抑制细胞（myeloid-derived suppressor cells, MDSCs），同时减少CD8阳性淋巴细胞的浸润；但DPP-4i并未显著改变4T1细胞诱导的脾脏髓系抑制细胞的活力、凋亡、分化及抑制性活化状态。进一步研究表明，DPP-4i可在乳腺癌细胞中触发ROS-NF-κB依赖的NLRP3炎症小体活化，进而升高多种炎症细胞因子的水平，包括白细胞介素（interleukin, IL）-6、肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）、血管内皮生长因子（vascular endothelial growth factor, VEGF）、细胞间黏附分子1（intercellular cell adhesion molecule 1, ICAM-1）、血管细胞黏附分子1（vascular cell adhesion molecule 1, VCAM-1）、IL-1β及IL-33；同时升高髓系抑制细胞诱导因子粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）、粒细胞集落刺激因子（G-CSF）及巨噬细胞集落刺激因子（M-CSF）的表达，这些因子在肿瘤免疫抑制微环境的重塑中发挥关键作用。综上，本研究结果表明，抗糖尿病DPP-4i可通过ROS–NRF2–HO-1–NF-κB–NLRP3轴与乳腺癌细胞相互作用，重塑肿瘤微环境，从而促进小鼠乳腺癌的转移。本研究不仅阐明了DPP-4i介导的乳腺癌进展中致癌性ROS–NRF2–HO-1轴的机制，还为通过改善肿瘤微环境以减轻DPP-4i诱导的乳腺癌转移提供了全新的研究思路。