PAUF enhances the migration and invasion of human serous ovarian cancer cells in vitro
To determine the endogenous levels of PAUF expression in various human serous ovarian adenocarcinoma cell lines, cells were cultured and a sandwich ELISA of culture supernatants was conducted. Levels of PAUF were highest in OVCAR-5 cells (~19 ng per 1 x 106 cells) while HeyA8, A2780, SK-OV-3, and OVCAR-8 cells showed modest expression (Fig. 1a). PAUF expression in OVCAR-5 was higher when compared with CFPAC-1, the pancreatic cancer cell line previously shown to express PAUF at high levels [19] (Fig. 1b). Next, the potential impact of PAUF on the motility and invasiveness of serous ovarian cancer cell lines was characterized. The three cell lines with a modest PAUF expression-HeyA8, SK-OV-3, and OVCAR-8-were treated with recombinant PAUF (rPAUF). As shown in Fig. 1c-f, migration, invasion, and adhesion activities increased significantly in the treated cells when compared to the control cells (i.e., no PAUF). These results are consistent with our previous findings in pancreatic cancer cell lines [8, 10]. However, the treatment did not show significant changes in the motility and invasiveness of the OVCAR-5 cell line, which expresses PAUF at a high level. Treatment of OVCAR-8 cells with rPAUF activated intracellular signaling pathways involving ERK, Src, and AKT (Fig. 1g and Fig. S1), implying that PAUF may exert similar functions in both ovarian and pancreatic cancer cells [8-10]. These results suggest that PAUF may function as a factor that affects the metastasis in serous ovarian adenocarcinoma.
Reduced PAUF activity diminishes migration, invasion, and proliferation of serous ovarian cancer cells in vitro
We explored the effects of an antibody that neutralizes PAUF function. The anti-PAUF antibody significantly inhibited the migration and invasion of OVCAR-5 cells (Fig. 2). The inhibitory activity of the anti-PAUF antibody was slightly less in cell lines with lower PAUF expression (i.e., HeyA8, SK-OV-3, OVCAR-8). To determine whether cancer-related cellular activities could be attenuated by reducing the level of PAUF, we established a stable PAUF-knockout OVCAR-5 cells line (OVCAR-5_PAUF K/O as described in the Materials and Methods). Successful transduction was confirmed by RT-qPCR and immunoblot analysis (Fig. 3a, b and Fig. S2). OVCAR-5_Mock cells were established as a negative control. As determined by sandwich ELISA, the level of PAUF expression in the OVCAR-5_PAUF K/O cells was reduced by more than 90% compared to the OVCAR-5_Mock cells (Fig. 3c). PAUF K/O cells exhibited significantly lower migratory and invasion capabilities when compared to the control cells (Fig. 3d, e). PAUF K/O cells also showed a decrease in proliferative capacity (Fig. 3f). Treatment of PAUF K/O cells with rPAUF recovered the motility, invasion, and proliferation abilities (Fig. 3g). Therefore, both functional inhibition and expressional knockout of PAUF resulted in lowered cancer cell activities, suggesting that PAUF may be used for a targeted anti-cancer therapy to treat serous ovarian adenocarcinoma.
PAUF-knockout delayed in vivo xenograft tumor growth
In vivo relevance of the in vitro results was determined by evaluating the tumorigenesis and tumor growth rate in ovarian xenograft tumor models. Mice were subcutaneously implanted with OVCAR-5_PAUF K/O or OVCAR-5_Mock cells and tumor growth curves were plotted using tumor volumes measured twice a week for 46 days (Fig. 4). Individual and average tumor growth curves are shown in Fig. 4a, b, respectively. Overall, tumor growth in the OVCAR-5_PAUF K/O group was slower when compared with the OVCAR-5_Mock group. It took a mean of 32 days for tumors in the OVCAR-5_Mock group to reach 500 mm3 compared with 36 days in the OVCAR-5_PAUF K/O group, indicating delayed tumor growth by PAUF-knockout. At the endpoint of the experiment, the tumor growth inhibition (TGI) percentages were calculated. On day 46, the average tumor volumes of OVCAR-5_PAUF K/O and OVCAR-5_ Mock groups were 1,200 mm3 and 1,800 mm3, respectively, correlating to a TGI of 32.92% from PAUF-knockout (Fig. 4c). The largest and smallest volumes of OVCAR-5_PAUF K/O group were 1,300 mm3 and 800 mm3, while those volumes of OVCAR-5_Mock group were 2,000 mm3 and 1,500 mm3. These results strongly suggested that the PAUF expression was involved in ovarian tumor progression in vivo and could be a therapeutic target for human ovarian cancer treatment.
An anti-PAUF antibody demonstrated therapeutic efficacy in an ovarian cancer xenograft mouse model
We tested whether an anti-PAUF antibody is an effective treatment for human serous ovarian cancer. Mice bearing OVCAR-5-derived subcutaneous xenograft tumors were randomly divided into two groups and treated with 10 mg/kg anti-PAUF antibody or control IgG intravenously twice a week (Fig. 5a-d). The tumor growth rate and distribution of tumor volumes between IgG-treated and anti-PAUF antibody-treated groups were analyzed; average tumor growth curves are shown in Fig. 5a. The results indicate that the anti-PAUF antibody tested here is capable of reducing the growth of OVCAR-5 xenograft tumors in a mouse model. As shown in Fig. 5b, on day 23, the average tumor volumes of the anti-PAUF antibody-treated and control IgG-treated groups were 1,400 mm3 and 1,800 mm3, respectively, reflecting a 27% TGI effect of treatment with an anti-PAUF antibody. A Kaplan-Meier analysis indicated that the survival rate of the anti-PAUF antibody-treated group increased 28.6% compared to that of the control IgG-treated group (Fig. 5c). There was no difference in body weight between the two groups (Fig. 5d). To test the combination effect of the antibody with docetaxel, the tumor growth rate and distribution of tumor volumes between IgG, anti-PAUF antibody, docetaxel, and combination-groups were analyzed; average tumor growth curves are shown in Fig. 5e. As shown in Fig. 5f, on day 31 the average tumor volumes of the control IgG, anti-PAUF antibody, docetaxel, and combination treated groups were 2,600 mm3, 2,200 mm3, 1,700 mm3, and 1,600 mm3, respectively. In the current experiment, a Kaplan-Meier analysis indicated that while the anti-PAUF antibody showed a 17% improvement of the median survival rate when compared with control IgG, docetaxel alone showed 24% compared with control IgG. Furthermore, the combination of docetaxel and anti-PAUF antibody exhibited a significantly improved median survival rate of 29% (Fig. 5g). There was no difference in body weight among the test groups (Fig. 5h). Our results clearly demonstrate that the anti-PAUF antibody reduced tumor growth and improved survival in OVCAR-5-derived xenograft tumor models, suggesting that PAUF could be targeted by therapeutic antibodies for the treatment of human ovarian serous carcinoma.