C3, a central component of the complement system, is a plasma protein that is synthesized in the liver. We have found that malignant epithelial cells are also able to synthesize and secrete C3. The regulation of expression of the C3 gene in malignant cells is not well understood. We found that C3 is highly expressed in various cancer cell lines (Cancer Cell Line Encyclopedia: CCLE) and cancerous human patient tissues (The Cancer Genome Atlas: TCGA). We then explored which transcription factors were responsible for regulating C3 gene expression in ovarian cancer cells. To determine C3 transcription factors, we performed a gene promoter analysis, which identified a TWIST1-binding consensus motif on the C3 gene promoter. The TWIST proteins (TWIST1 and TWIST2) are well known transcription factors are associated with more advanced, invasive and metastatic lesions. There is significant evidence suggesting that TWIST1 promotes tumor progression.
To investigate whether TWIST1 is a transcription factor of C3 in human ovarian cancer cells, we performed a chromatin immunoprecipitation (ChIP) analysis and confirmed TWIST1 binding at the C3 promoter (Figure 1A,,1B and 1c). The C3 promoter binding affinity is 5.83-fold higher in Twist antibody used pull down compared to the IgG control (n=3, p=0.001, t-test, Figure 1C). To investigate the functional effect of TWIST1 binding to C3 promoter, we performed a Luciferase reporter gene analysis, showing that a mutant TWIST1 binding site on the C3 promoter decreases luciferase reporter gene activity (control vs. C3 vs. mC3=1 vs. 3.3 vs.1.8, n=3, p=0.01, t-test, Figure 1Dand 1E).
Next, we manipulated expression of the Twist1 gene in ovarian cancer cells and monitored its effect on C3 gene expression. Small interfering RNAs (siRNA) against Twist1 drastically reduced C3 expression (n=3, p=0.01, t-test, Figure 1F); on the other hand, transducing these cells with lentivirus containing Twist1 increased C3 expression (n=3, p=0.01, t-test, Figure 1G). Therefore, we conclude that there is a positive correlation between expression of C3 and TWIST1 in ovarian cancer cells.
Taken together, these data provide evidence that a novel transcription factor of C3, TWIST1, upregulates C3 expression in malignant cancer cells, leading to an increase in cell proliferative potential.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.