S100P上调AGTR1实现自我激活从而促进绒毛膜癌的生长和转移

Choriocarcinoma is a common pregnancy associated tumor, characterized by its easily metastasis. We have been committed to investigating S100P function in infertility and gynaecological cancers since. By cDNA chip, we found S100P raised the expression level of cell-cycle and cell-motility related genes, indicated S100P might promote tumor cell growth and motility. Immunohistochemistry results showed that, S100P was only expressed in syncytiotrophoblast not cytotrophoblast in normal placental villi, comparing to its expression both in syncytiotrophoblast and cytotrophoblast of choriocarcinoma tissues. Overexpression of S100P in choriocarcinoma cells can promote proliferation and migration by the activation of MAPK pathway, enhancing ERK and P38 phosphorylation. Besides, S100P upregulated AGTR1 expression both in mRNA and protein level. S100P is a calcium ion binding protein. And AGTR1 is well known for its capability of calcium ion releasing activity. Furthermore, blocking the AGTR1 signal by its antagonist in S100P overexpression cells reversed the invasion increment by S100P. Those results suggested that AGTR1 may be involved in S100P signal activation, and S100P and AGTR1 were likely to form a positive-feedback regulation loop in choriocarcinoma cells. In this study, we will investigate whether S100P promotes the growth and motility of choriocarcinoma by self-activating through upregulating the expression of AGTR1.

绒毛膜癌是常见的妊娠相关肿瘤，具有极易发生转移的特点。本课题组一直致力于S100P在妇科肿瘤的研究。通过cDNA芯片，我们发现S100P上调细胞周期和移动相关的基因（包括AGTR1）表达，提示S100P可能与肿瘤的生长和转移有关。免疫组化发现，S100P在正常绒毛组织中只表达于合体滋养细胞，而在绒毛膜癌中却同时表达于合体滋养细胞和细胞滋养细胞。过表达S100P可促进绒癌细胞增殖与迁移，上调ERK和P38磷酸化水平，激活MAPK通路；此外S100P上调AGTR1 mRNA和蛋白的表达；据报道，S100P是钙结合蛋白，而AGTR1具有激活钙离子释放的功能；同时我们发现，对过表达S100P细胞施用AGTR1拮抗剂可导致S100P促迁移能力被逆转，提示S100P可能通过AGTR1调控细胞的迁移。本课题拟研究，S100P是否通过调控AGTR1的表达实现自我激活从而促进绒毛膜癌的生长和转移。

绒毛膜癌在欧美发病率极为罕见，但在我国发病率较高，为每2415次妊娠中有1次，即0.41％。由于绒毛膜癌细胞高度增生并侵犯子宫肌层和血管，并经常发生阴道或肺等远处转移，尽管对患者广泛开展化学疗法，绒毛膜癌及恶性葡萄胎的死亡率仍有20％左右。我们通过对正常绒毛、葡萄胎、绒毛膜癌原发灶和肺部转移灶的免疫组化发现，S100P在正常绒毛组织中只表达于合体滋养细胞，而在绒毛膜癌中却同时表达于合体滋养细胞和细胞滋养细胞。TCGA和GTEx大数据分析，我们发现S100P在乳腺癌、宫颈癌和内膜癌的癌组织表达水平均高于正常组织，提示S100P在肿瘤细胞高表达具有普遍性。细胞体内外功能分析发现，过表达S100P可提高绒毛膜癌细胞的增殖、克隆形成、迁移、裸鼠皮下成瘤和裸鼠转移瘤形成的能力，而干扰S100P可逆转这些生物学特征。并且过表达S100P促进绒癌细胞EMT转化，导致上皮细胞标志物E-cadherin表达下调，间质细胞Vimentin和beta-catenin表达水平上调。过表达S100P的JAR细胞P38-MAPK和ERK-MAPK的磷酸化水平显著高于对照细胞，而AKT磷酸化水平没显著差异，提示S100P激活细胞P38和ERK的MAPK信号通路。P38蛋白抑制剂SB203580处理后，S100P促进的增殖能力被SB203580显著抑制；而ERK蛋白抑制剂PD98059处理没有相应作用。提示S100P可能通过激活P38 MAPK信号通路影响绒毛膜癌细胞增殖。通过芯片筛查，我们发现与细胞增殖和转移相关的基因表达上调，包括AGTR1，后续验证过表达S100P诱导AGTR1升高；同时p53和p21表达下调。CoIP实验发现S100P与p53相互结合，并且S100P上调的AGTR1在P53的作用下，上调趋势被显著抑制，提示P53可能是S100P发挥功能的关键中间蛋白。我们的研究为进一步理解绒毛膜癌生长和转移可能的作用机制奠定了理论基础。