Sp1-CD147 Positive Feedback Loop Promotes The Invasion Ability Of Ovarian Cancer

Ovarian cancer(OC) is one of the common malignant tumors of female reproductive system, ranked the third[1-3] after the cervical cancer and endometrial cancer, and the epithelial ovarian cancer accounted for 50%~70% in primary ovarian tumor. In the epithelial ovarian cancer, the malignant type accounted for 85%~90%[4]. Because of the lack of specific symptoms and effective means of screening in the early stage, 70% of patients were found to be in advanced.The treatment of ovarian cancer is very difficult and the recurrence rate is high so as to the 5 year survival rate is less than 50%[5].CD147, also known as EMMPRIN or Basigin, is a highly glycosylated transmembrane protein that belongs to the immunoglobulin superfamily[6-9]. CD147 promotes cancer cell migration, invasion and metastasis by enhancing the activity of matrix metalloproteinases(MMPs) in digesting the components of the extracellular matrix(ECM) or regulating the expression of VEGF and impressing apoptosis[10-12]. High-expression of CD147 in ovarian cancer is closely related to a poor prognosis [13]. As an important tumor marker, It is important to reveal its regulation mechanism so as to make every effort to help diagnosis and treatment in ovarian cancer.Kong et [14]al found that transcription factor Sp1 can directly bind to the promoter of CD147 and effectively activate its transcription to increase CD147 expression. In addition, they also found that promoter hypomethylation up-regulated CD147 expression through increasing Sp1 binding and made a poor prognosis in hepatocellular cancer.Researches indicated that Sp1 phosphorylation[15] has an important impact on gene transcription. The phosphorylation of Sp1 can increase its binding ability to DNA, or increase the protein stability of itself or can influence its transcriptional activity. Tan et al found that Angiotensin Ⅱwas found to be able to activate PKCζ, with the subsequent phosphorylation of Sp1 in the zinc finger domain(Thr668, Ser670, Thr681), and Sp1 phosphorylation in the zinc finger domain increased its binding capacity to the platelet derived growth factor-D promoter[16]. Blocking the JNK signaling pathway can impress the phosphorylation of Sp1 in the Thr278 and Thr739 residues, which cause the the hematoxylin of Sp1, accelerating its degradation[17, 18]. Because the transcriptional activity of Sp1 is influenced by the rate of Sp1 protein synthesis, nuclear translocation, DNA-binding affinity, interaction with other proteins and the protein stability, it is difficult to judge the effects of Sp1 phosphorylation on the transcriptional activity of CD147.This subject has four purposes: The first is to find what effect the phosphorylation of Sp1 expression makes on CD147 gene expression; The second is to reveal its specific regulatory mechanism[19]; The third is to test the interaction between Sp1 phosphorylation and CD147 gene in the invasive ability of ovarian cancer; The last is to find the correlation between Sp1 expression and CD147 in ovarian cancer cells and tissues[20].The first part: Sp1 phosphorylation was involved in the regulation of CD147 gene expression. We mutated six Ser/Thr residues(including Thr355, Thr453, Thr668, Ser670, Thr681, Thr739) [21, 22]of Sp1 to alanine. The Quick Change Mutagenesis kit was used according to the manufacturer’s instructions. After transient transfection of the mutant and CD147 promoter, we found Sp1 mutations in the Thr453 and Thr739 two residues can significantly reduce the up- regulation on the expression of CD147 affected by Sp1 uesd by dual-luciferase assay report[23]. This conclusion was also verified in the subsequent quantitative real-time PCR and Western blot experiments.The second part: The mechanism of Sp1 phosphorylation regulated the expression of CD147 gene. In order to explore the specific mechanism of phosphorylation of Sp1 was involved in the regulation of CD147 gene expression, we used the PI3K/AKT and MAPK/ERK phosphorylation pathway specific inhibitors of LY294002, rapamycin and PD98059 to pretreate the ovarian cancer cells[24], then we found Sp1 phosphorylation level decreased in Thr453 and Thr739 two residues, but the total protein had no obvious change. In addition, we also found that the protein levels of CD147 also decreased, suggesting that Sp1 phosphorylation can be activated through PI3K/AKT/m TOR and MAPK/ERK pathways. As the level of Sp1 phosphorylation was inhibited, the expression levels of CD147 also decreased, suggesting that Sp1 phosphorylation can activate the transcription of CD147 gene. And when we up-regulated or down-regulated the expression of CD147, we found p70S6 k and p ERK and p AKT were decreased, and then Sp1 phosphorylation level were also decreased in Thr453 and Thr739 two residues, but the total protein showed no obvious change. From the first part of the experiment, we learned that Sp1 phosphorylationcan in the Thr453 and Thr739 two residues can increase the transcription activation on CD147 gene expression, and up-regulation of CD147 expression may increase the Sp1 phosphorylation levels on Thr453 and Thr739 two residues through the activation of PI3K/AKT/m TOR and MAPK/ERK pathway[25]. Such a positive feedback loop may be one of the reasons for the high-expression of CD147 in ovarian cancer.The third part: the influence of interaction between the expression of Sp1 phosphorylation and CD147 gene on invasion ability of ovarian cancer. Using the Sp1(at the Thr453 and Thr739 residues) phosphorylation antibodies and CD147 monoclonal antibody alone or in combination to block the ovarian carcinoma cell line HO-8910 with high potential metastatic ability. Transwell experiment is used to find that invasion ability of ovarian cancer cells was decreased, suggesting that the Sp1-CD147 feedback loop can increase the invasion ability of ovarian cancer.The fourth part: The correlation between the expression of Sp1 phosphorylation and CD147 gene in ovarian carcinoma cells and tissues. We used Western blot to detect the p-Sp1(Thr453), p-Sp1(Thr739) and CD147 expression in ovarian cancer cell lines SKOV3, HO-8910 and HO-8910 PM. The results showed in ovarian cancer cells the expression of Sp1 phosphorylation level had a positive correlation with CD147. Immunohistochemical experiments was used to detect the expression and distribution of p-Sp1(Thr453), p-Sp1(Thr739) and CD147 in the 53 cases of ovarian cancer specimens. The results showed the protein of Sp1 phosphorylation was mainly expressed in ovarian cancer cell nuclear membrane and the nucleus, whereas CD147 is mainly expressed in the cell membrane and cytoplasm.Statistical results showed that the expression of CD147 was 12 cases(strong, 22.64%); 15 cases(moderate, 28.30%); 6 cases(weak, 11.32%); 20 cases(negative, 37.74%); phospho-Sp1(T453) was 24 cases(strong, 45.28%); 13 cases(moderate, 24.53%); 7 cases(weak, 13.21%); 9 cases(negative, 16.98%) and phospho-Sp1(T739) expression was 16 cases(strong, 30.19%), 14 cases(moderate, 26.42%), 10 cases(weak, 18.87%); 13 cases(negative, 24.53%). The positive expression of CD147 accounted for 62.26%, while phospho-Sp1(T453) and phospho-Sp1(T739) positive expression rates were 83.02% and 75.47%. Spearman’rho correlation analysis showed that p-Sp1(Thr453), p-Sp1(Thr739) were weakly correlated with the expression of CD147(r T453=0.477, P<0.01; r T739=0.461, P<0.01).Based on the above results, we clarified the interaction of the transcription factor Sp1 phosphorylation and expression of CD147 gene, and found that Sp1-CD147 positive feedback loop can promote invasion ability of ovarian cancer cells. These results will help us to well understand the mechanism of high expression of CD147 gene in ovarian cancer. It may promte the treatment plan of ovarian cancer through blocking the Sp1-CD147 positive feedback loop.