Hypoxia-related Gene Expressions Affecting Prognosis of High-grade Serous Adenocarcinoma of the Ovary

Abstract

Background: Ovarian cancer is the most lethal disease among malignancies of the female genital tract because of a higher rate of diagnosis at advanced-stage disease. Thus, maximal debulking surgery followed by adjuvant chemotherapy is essential to treat ovarian cancer. However, drug resistance of ovarian cancer is expected to be associated with microenvironment, which may hinder the improvement of prognosis and the development of optimal biomarkers for ovarian cancer. Among various types of tumor microenvironment, hypoxia-related genes are known to be associated with carcinogenesis, proliferation and invasion of tumor cells. However, they have been not investigated sufficiently for their roles affecting drug resistance and prognosis of ovarian cancer. Thus, we evaluated expressions of hypoxia-related genes in the most common histologic type, high-grade serous adenocarcinoma of the ovary (HGSO), and investigated the roles of hypoxia-related genes affecting platinum-resistance and survival of HGSO. Methods: We determined IC50 of cisplatin, and then evaluated cell viability after treatment of cisplatin under hypoxia in OV90 cells. Basic mRNA levels of CBP, P300, HIF-1α, HIF-1β, FIH and VHL were evaluated in each five tissues of normal ovary and HGSO by using of quantitative RT-PCR. Then, we investigated protein levels after treatment of cisplatin under hypoxic conditions (normoxia, 5%O2, 3%O2 and 1%O2) in OV90 cells by using Western blot. Furthermore, we estimated cellular proliferation and reactive oxygen species (ROS) production in OV90 cells by N-acetylcysteine (NAC) to determine relationship between hypoxia and ROS generation. Finally, we performed immunohistochemistry (IHC) for the six genes in patients with HGSO (n=149), and investigated prognostic factors affecting platinum-resistance and progression-free survival (PFS) among clinico-pathologic factors and the six hypoxia-related genes. Results: IC50 of cisplatin were 50 μmol/L in OV90 cells, and hypoxic cells were less inhibited than normoxic cells after treatment of IC50 of cisplatin. In HGSO, mRNA levels of CBP, P300, HIF-1α and HIF-1β were lower than those in normal tissues, whereas mRNA levels of FIH and VHL were higher than those in normal tissues. In Western blot, the protein level of HIF-1β was increased, whereas that of VHL was decreased under hypoxic conditions after treatment of cisplatin in OV90 cells. Furthermore, the protein level of HIF-1β was increased, whereas that of VHL was decreased when the concentration of NAC was increased with cisplatin in OV90 cells. On IHC, a low expression of HIF-1β and a high expression of VHL were favorable factors for reducing platinum-resistance in patients with HGSO (Adjusted ORs, 0.657 and 0.381

95% CI, 0.443-0.975 and 0.165-0.878). In addition, a low expression of HIF-1β and a high expression of VHL were related with improved PFS in patients with HGSO (adjusted HRs, 0.657 and 0.381

95% CIs, 0.443-0.975 and 0.165-0.878). Conclusions: A high expression of HIF-1 β and a low expression of VHL may be related with platinum-resistance and poor prognosis in patients with HGSO.