Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.

神经内分泌肽降钙素（calcitonin, CT）及其受体降钙素受体（calcitonin receptor, CTR）在上皮性癌症进展中的作用是一个具有重大临床潜力的新兴研究概念。CT与CTR的表达在前列腺癌（prostate cancers, PCs）中常呈上调状态，非侵袭性前列腺癌细胞中CT-CTR信号轴的激活可诱导其获得侵袭表型。本研究通过酵母双杂交筛选实验证实，CTR可通过其羧基末端的1型PDZ结合基序与闭合小环蛋白-1（Zonula Occludens-1, ZO-1）的PDZ3结构域之间的相互作用，与紧密连接蛋白ZO-1相结合。分别突变CTR的C端PDZ结合基序或ZO-1的PDZ3结构域，均不会影响CTR与其配体的结合能力，亦不会干扰G蛋白介导的信号转导，但可消除CT对紧密连接的稳定性破坏作用，同时阻断裸鼠体内原位移植的前列腺癌细胞形成远处转移的能力，上述结果提示此类PDZ结构域相互作用具有病理相关性。进一步地，我们通过原位连接免疫组化（proximity ligation immunohistochemistry）在前列腺癌组织标本中检测到了CTR与ZO-1的相互作用，且转移性前列腺癌标本中的相互作用数量较非转移性前列腺癌标本高出数倍。本研究首次证实，CTR与ZO-1之间的PDZ介导相互作用，是CT刺激前列腺癌转移所必需的分子机制。鉴于众多受体均含有PDZ结合基序，该研究结果提示PDZ结合基序-衔接蛋白相互作用可能是癌症转移的一种普遍机制。