The role of KIF14 in patient-derived primary cultures of high-grade serous ovarian cancer cells

Abstract

Objective: Previously, it has been shown that KIF14 mRNA is overexpressed in ovarian cancer (OvCa), regardless of histological subtype. KIF14 levels are independently predictive of poor outcome and increased rates of recurrence in serous OvCa patients. Furthermore, it has been shown that KIF14 also controls the in vivo tumorigenicity of OvCa cell lines. In this study, we evaluate the potential of KIF14 as a therapeutic target through selective inhibition of KIF14 in primary high-grade serous patient-derived OvCa cells.

Methods: To assess the dependence of primary serous OvCa cultures on KIF14, protein levels in 11 prospective high grade serous ovarian cancer samples were increased (KIF14 overexpression by transfection) or decreased (anti-KIF14 shRNA) in vitro, and proliferative capacity, anchorage independence and xenograft growth were assessed.

Results: Seven of eleven samples demonstrated increased/decreased in vitro proliferation in response to KIF14 overexpression/knockdown, respectively. When examining in vitro tumorigenicity (colony formation) and in vivo growth (subcutaneous xenografts) in response to KIF14 manipulation, none of the samples demonstrated growth in soft agar (11 samples), or xenograft growth (4 samples).

Conclusions: Although primary high-grade serous OvCa cells may depend on KIF14 for in vitro proliferation we were unable to demonstrate a role for KIF14 on tumorigenicity or develop an in vivo model for assessment. We have, however developed an effective in vitro method to evaluate the effect of target gene manipulation on the proliferative capacity of primary OvCa cultures.

Figure 1

KIF14 mRNA expression in response to short-term in vitro culture conditions. Eleven primary OvCa samples (high-grade serous histology) were measured for KIF14 expression in the primary tissue (dark blue) and in short-term culture (passage 3, light blue). Previously characterized KIF14 ^HIGH and KIF14 ^LOW primary OvCa samples (5) were included as controls. Error bars represent standard deviation of 3 independent measurements for each sample.

Figure 2

KIF14 knockdown in primary OvCa samples. Eleven primary OvCa samples were transduced with lentivirus expressing anti-KIF14 shRNA (LV-816) (green), and mRNA expression measured 21 days (passage 5) post-transduction in comparison to a scrambled shRNA control (control; blue). KIF14 expression in LV-816 cells normalized to control cell expression (set as 1). Error bars represent standard deviation of three independent experiments.

Figure 3

The effect of KIF14 overexpression/knockdown on primary OvCa cultures. Cell viability readings in response to KIF14 overexpression (KIF14, dark blue) or KIF14 knockdown (LV-816, light blue) relative to their respective controls (pcDNA (KIF14), dark red; SCR (LV-816), light red). *P <0.05. Error bars represent standard deviation of 3 independent experiments.

Figure 4

KIF14 overexpression in primary OvCa samples. Eleven primary OvCa samples were transiently transfected with KIF14-EGFP (red), and mRNA expression measured 21 days (passage 5) post-transfection in comparison to the empty vector control (pcDNA; blue). KIF14 expression in KIF14-EGFP cells normalized to control cell expression (set as 1). Error bars represent standard deviation of three independent experiments.

Figure 5

Colony assays. A Representative primary OvCa short-term cultured sample (6972) grown in soft agar either untreated (top), expressing control vector (middle) or overexpressing KIF14 (bottom panels). Images taken 2 and 30 days post seeding. B SKOV3 cells expressing either control vectors (pcDNA or SCR), overexpressing KIF14 (SKOV3_KIF14) or knocked-down in KIF14 (SKOV3_LV-816). Images taken 14 days post seeding. Magnification, 20X.