Chi Meng, Shi Xiaoding, Huo Xing, Wu Xiaohong, Zhang Pinyi, Wang Guonian
Department of Anesthesiology, Harbin Medical University Cancer Hospital, Harbin 150081, China.
Pain Research Institute of Heilongjiang Academy of Medical Sciences, Harbin 150081, China.
Ann Transl Med. 2020 Apr;8(8):531. doi: 10.21037/atm.2020.04.28.
Dexmedetomidine (DEX), a highly selective α2-adrenergic receptor agonist, has been reported to increase the malignancy of breast cancer cells and stimulate tumor growth in mice. Transmembrane protease serine 2 (TMPRSS2) demonstrates proteolytic activity, resulting in degradation of the extracellular matrix (ECM). This study investigated whether and how TMPRSS2 regulates migration of DEX-treated breast cancer cells.
Breast cancer cell lines MCF-7 and MDA-MB-231 were treated with DEX and scratch assay was performed. Expressions of TMPRSS2, α2-adrenergic receptor, phospho-STAT3, Rab11, and ECM components were assessed using real-time polymerase chain reaction (real-time PCR), Western blotting, and immunofluorescence staining. ELISA and ultracentrifugation were used to quantify secreted exosomal proteins. Knockdown assay was used to inhibit the expression of TMPRSS2 and Rab11.
DEX significantly increased the migration of MCF-7 and MDA-MB-231, which was accompanied by the upregulation and colocalization of TMPRSS2 and α2-adrenergic receptor. Nuclear phospho-STAT3 was increased dramatically following DEX treatment, and TMPRSS2 upregulation was significantly suppressed by the STAT3 inhibitor WP1066. Meanwhile, TMPRSS2 knockdown decreased DEX-induced cellular migration. TMPRSS2 and Rab11 were significantly detected in the media and the isolated exosomes from DEX-treated cells, and their colocalization was also revealed. Rab11 knockdown prevented exosomal TMPRSS2 from increasing in DEX-treated cells. In normal cultured MDA-MB-231, migration was increased by Rab11-positive exosomes isolated from DEX-treated MCF-7. Moreover, transmission electron microscopy showed that Rab11-positive exosomes enriched more components than Rab11-negative exosomes. Additionally, a reduction in ECM components fibronectin, collagen IV, matrix metallopeptidase 16, and Tenascin C was detected after DEX treatment, but was prohibited when TMPRSS2 or Rab11 were knocked down.
This study provides evidence that DEX upregulates TMPRSS2 expression via the activation of α2-adrenergic receptor/STAT3 signaling and promotes TMPRSS2 secretion in exosomes through Rab11, thus resulting in degradation of the ECM, which is responsible for DEX-induced migration of breast cancer cells.
右美托咪定（DEX）是一种高度选择性的α2肾上腺素能受体激动剂，据报道可增加乳腺癌细胞的恶性程度并刺激小鼠肿瘤生长。跨膜蛋白酶丝氨酸2（TMPRSS2）具有蛋白水解活性，可导致细胞外基质（ECM）降解。本研究调查了TMPRSS2是否以及如何调节DEX处理的乳腺癌细胞的迁移。
用DEX处理乳腺癌细胞系MCF-7和MDA-MB-231，并进行划痕试验。使用实时聚合酶链反应（实时PCR）、蛋白质印迹法和免疫荧光染色评估TMPRSS2、α2肾上腺素能受体、磷酸化STAT3、Rab11和ECM成分的表达。采用酶联免疫吸附测定（ELISA）和超速离心法定量分泌的外泌体蛋白。采用敲低试验抑制TMPRSS2和Rab11的表达。
DEX显著增加了MCF-7和MDA-MB-231的迁移，这伴随着TMPRSS2和α2肾上腺素能受体的上调和共定位。DEX处理后，核磷酸化STAT3显著增加，STAT3抑制剂WP1066显著抑制TMPRSS2上调。同时，TMPRSS2敲低减少了DEX诱导的细胞迁移。在DEX处理细胞的培养基和分离的外泌体中显著检测到TMPRSS2和Rab11，并且它们的共定位也被揭示。Rab11敲低阻止了DEX处理细胞中外泌体TMPRSS2的增加。在正常培养的MDA-MB-231中，从DEX处理的MCF-7中分离的Rab11阳性外泌体增加了迁移。此外，透射电子显微镜显示Rab11阳性外泌体比Rab11阴性外泌体富集更多成分。另外，DEX处理后检测到ECM成分纤连蛋白、IV型胶原、基质金属肽酶16和腱生蛋白C减少，但当TMPRSS2或Rab11被敲低时被阻止。
本研究提供的证据表明，DEX通过激活α2肾上腺素能受体/STAT3信号上调TMPRSS2表达，并通过Rab11促进外泌体中TMPRSS2的分泌，从而导致ECM降解，这是DEX诱导乳腺癌细胞迁移的原因。
