Abstract：
OBJECTIVE: Transactivation of insulin-growth-factor-receptor (IGF-1R) by angiotensin-II-type-1-receptor (AT-1R) was only demonstrated in vascular-smooth-muscle cells and has never been tested in breast-cancer (BC). This investigation addressed the impact of chronic AT-1R blockade by valsartan (Val) on possible concurrent AT-1R/IGF-1R signaling inhibition, regressing BC-tumor-microenvironment (TME) cellular components activation, and hindering BC development.
METHODS: The effect of different Val doses (10, 20, 40 & 80 mg/kg/day for 490 days) was tested on dimethylbenz(a)anthracene (DMBA)-induced progesterone-promoted-BC in rats. The influence on intratumoral/circulating angiotensin-II (ANG-II) levels and AT-1R/Mas-R immunofluorescent-expression were assessed. The potential AT-1R/IGF-1R crosstalk within TME-BC-stem-cells (BCSCs) and cancer-associated-fibroblasts (CAFs) was evaluated by fluorescently marking these cells and locating the immunofluorescently-stained AT-1R/IGF-1R in them using confocal-laser-microscopy and further quantified by flow cytometry. In addition, the molecular alterations following blocking AT-1R were inspected including determining Src; crucial for IGF-1R transactivation by AT-1R, Notch-1; IGF-IR transcriptional-regulator, and PI3K/Akt &IL-6/STAT expression. Further, the suppression of CSCs\' capabilities to maintain pluripotency, stemness features, epithelial-to-mesenchymal-transition (EMT), and angiogenesis was evaluated by assessing NANOG gene, aldehyde-dehydrogenase (ALDH), N-cadherin and vascular-endothelial-growth-factor (VEGF), respectively. Furthermore, the proliferative marker; Ki-67, was detected by immunostaining, and tumors were histologically graded using Elston-Ellis-modified-Scarff-Bloom-Richardson method.
RESULTS: Prophylactic Val significantly reduced tumor size, prolonged latency, reduced tumor histopathologic grade, decreased circulating/intratumoral-ANG-II levels, increased Mas-R, and decreased AT1R expression. AT-1R/IGF-1R were co-expressed with a high correlation coefficient on CAFs/BCSCs. Moreover, Val significantly attenuated IGF-1R transactivation and transcriptional regulation via Src and Notch-1 genes\' downregulation and reduced Src/IGF-IR-associated PI3K/Akt and IL-6/STAT3 signaling. Further, Val significantly decreased intratumoral NANOG, ALDH, N-cadherin, VEGF, and Ki-67 levels.
CONCLUSIONS: Chronic Val administration carries a potential for repurposing as adjuvant or conjunct therapy for patients at high risk for BC.
METHODS: The effect of different Val doses (10, 20, 40 & 80 mg/kg/day for 490 days) was tested on dimethylbenz(a)anthracene (DMBA)-induced progesterone-promoted-BC in rats. The influence on intratumoral/circulating angiotensin-II (ANG-II) levels and AT-1R/Mas-R immunofluorescent-expression were assessed. The potential AT-1R/IGF-1R crosstalk within TME-BC-stem-cells (BCSCs) and cancer-associated-fibroblasts (CAFs) was evaluated by fluorescently marking these cells and locating the immunofluorescently-stained AT-1R/IGF-1R in them using confocal-laser-microscopy and further quantified by flow cytometry. In addition, the molecular alterations following blocking AT-1R were inspected including determining Src; crucial for IGF-1R transactivation by AT-1R, Notch-1; IGF-IR transcriptional-regulator, and PI3K/Akt &IL-6/STAT expression. Further, the suppression of CSCs\' capabilities to maintain pluripotency, stemness features, epithelial-to-mesenchymal-transition (EMT), and angiogenesis was evaluated by assessing NANOG gene, aldehyde-dehydrogenase (ALDH), N-cadherin and vascular-endothelial-growth-factor (VEGF), respectively. Furthermore, the proliferative marker; Ki-67, was detected by immunostaining, and tumors were histologically graded using Elston-Ellis-modified-Scarff-Bloom-Richardson method.
RESULTS: Prophylactic Val significantly reduced tumor size, prolonged latency, reduced tumor histopathologic grade, decreased circulating/intratumoral-ANG-II levels, increased Mas-R, and decreased AT1R expression. AT-1R/IGF-1R were co-expressed with a high correlation coefficient on CAFs/BCSCs. Moreover, Val significantly attenuated IGF-1R transactivation and transcriptional regulation via Src and Notch-1 genes\' downregulation and reduced Src/IGF-IR-associated PI3K/Akt and IL-6/STAT3 signaling. Further, Val significantly decreased intratumoral NANOG, ALDH, N-cadherin, VEGF, and Ki-67 levels.
CONCLUSIONS: Chronic Val administration carries a potential for repurposing as adjuvant or conjunct therapy for patients at high risk for BC.
摘要：
目的：血管紧张素II-1型受体（AT-1R）对胰岛素生长因子受体（IGF-1R）的反式激活仅在血管平滑肌细胞中得到证实，从未在乳腺癌（BC）中进行过测试。这项研究探讨了缬沙坦（Val）慢性AT-1R阻断对可能并发的AT-1R/IGF-1R信号抑制的影响，回归BC-肿瘤-微环境（TME）细胞成分激活，阻碍BC的发展。
方法:在大鼠中测试不同Val剂量(10、20、40和80mg/kg/天,持续490天)对二甲基苯并(a)蒽(DMBA)诱导的孕酮促进的BC的作用。评估了对肿瘤内/循环血管紧张素II（ANG-II）水平和AT-1R/Mas-R免疫荧光表达的影响。通过荧光标记这些细胞并定位免疫荧光染色的AT-1R/IGF-1R来评估TME-BC干细胞（BCSC）和癌症相关成纤维细胞（CAF）内的潜在AT-1R/IGF-1R串扰。此外,检查阻断AT-1R后的分子改变，包括确定Src；对于AT-1R的IGF-1R反式激活至关重要，Notch-1;IGF-IR转录调节因子,和Akt/PI3K&IL-6/STAT表达。Further,抑制CSCs维持多能性的能力，干性特征，上皮-间质转化（EMT），通过评估NANOG基因来评估血管生成，醛脱氢酶（ALDH），N-钙粘蛋白和血管内皮生长因子（VEGF），分别。此外，增殖标志物；Ki-67，通过免疫染色检测，肿瘤采用Elston-Ellis改良Scarff-Bloom-Richardson方法进行组织学分级。
结果：预防性Val可显著缩小肿瘤大小，延长的延迟，降低肿瘤组织病理学分级，循环/肿瘤内ANG-II水平降低，增加Mas-R，AT1R表达降低。AT-1R/IGF-1R在CAF/BCSC上以高相关系数共表达。此外，Val通过Src和Notch-1基因下调显著减弱IGF-1R的反式激活和转录调节，并降低Src/IGF-IR相关Akt/PI3K和IL-6/STAT3信号传导。Further,Val显著降低肿瘤内NANOG，ALDH,N-钙黏着蛋白,VEGF,和Ki-67级别。
结论：慢性Val给药对高危BC患者有可能重新用作辅助或联合治疗。
方法:在大鼠中测试不同Val剂量(10、20、40和80mg/kg/天,持续490天)对二甲基苯并(a)蒽(DMBA)诱导的孕酮促进的BC的作用。评估了对肿瘤内/循环血管紧张素II（ANG-II）水平和AT-1R/Mas-R免疫荧光表达的影响。通过荧光标记这些细胞并定位免疫荧光染色的AT-1R/IGF-1R来评估TME-BC干细胞（BCSC）和癌症相关成纤维细胞（CAF）内的潜在AT-1R/IGF-1R串扰。此外,检查阻断AT-1R后的分子改变，包括确定Src；对于AT-1R的IGF-1R反式激活至关重要，Notch-1;IGF-IR转录调节因子,和Akt/PI3K&IL-6/STAT表达。Further,抑制CSCs维持多能性的能力，干性特征，上皮-间质转化（EMT），通过评估NANOG基因来评估血管生成，醛脱氢酶（ALDH），N-钙粘蛋白和血管内皮生长因子（VEGF），分别。此外，增殖标志物；Ki-67，通过免疫染色检测，肿瘤采用Elston-Ellis改良Scarff-Bloom-Richardson方法进行组织学分级。
结果：预防性Val可显著缩小肿瘤大小，延长的延迟，降低肿瘤组织病理学分级，循环/肿瘤内ANG-II水平降低，增加Mas-R，AT1R表达降低。AT-1R/IGF-1R在CAF/BCSC上以高相关系数共表达。此外，Val通过Src和Notch-1基因下调显著减弱IGF-1R的反式激活和转录调节，并降低Src/IGF-IR相关Akt/PI3K和IL-6/STAT3信号传导。Further,Val显著降低肿瘤内NANOG，ALDH,N-钙黏着蛋白,VEGF,和Ki-67级别。
结论：慢性Val给药对高危BC患者有可能重新用作辅助或联合治疗。
