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Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America, underscoring the need for the development of new therapeutic strategies for the management of this disease. Although many drugs are pre-clinically tested every year, only a few are selected to be evaluated in clinical trials, and only a small number of these are successfully incorporated into standard care. Inaccuracies with the initial in vitro drug testing may be responsible for some of these failures. Drug testing is often performed using 2D monolayer cultures or 3D spheroid models. Here, we investigate the impact that these different in vitro models have on the carboplatin response of four EOC cell lines, and in particular how different 3D models (polydimethylsiloxane-based microfluidic chips and ultra low attachment plates) influence drug sensitivity within the same cell line. Our results show that carboplatin responses were observed in both the 3D spheroid models tested using apoptosis/cell death markers by flow cytometry. Contrary to previously reported observations, these were not associated with a significant decrease in spheroid size. For the majority of the EOC cell lines (3 out of 4) a similar carboplatin response was observed when comparing both spheroid methods. Interestingly, two cell lines classified as resistant to carboplatin in 2D cultures became sensitive in the 3D models, and one sensitive cell line in 2D culture showed resistance in 3D spheroids. Our results highlight the challenges of choosing the appropriate pre-clinical models for drug testing.

PURPOSE:Epithelial ovarian cancer has the highest mortality rate of all gynecological malignancies. We have shown that high RAN expression strongly correlates with high-grade and poor patient survival in epithelial ovarian cancer. However, as RAN is a small GTPase involved in two main biological functions, nucleo-cytoplasmic transport and mitosis, it is still unknown which of these functions associate with poor prognosis. METHODS:To examine the biomarker value of RAN network components in serous epithelial ovarian cancer, protein expression of six specific RAN partners was analyzed by immunohistochemistry using a tissue microarray representing 143 patients associated with clinical parameters. The RAN GDP/GTP cycle was evaluated by the expression of RANBP1 and RCC1, the mitotic function by TPX2 and IMPβ, and the nucleo-cytoplasmic trafficking function by XPO7, XPOT and IMPβ. RESULTS:Based on Kaplan-Meier analyses, RAN, cytoplasmic XPO7 and TPX2 were significantly associated with poor overall patient survival, and RAN and TPX2 were associated with lower disease free survival in patients with high-grade serous carcinoma. Cox regression analysis revealed that RAN and TPX2 expression were independent prognostic factors for both overall and disease free survival, and that cytoplasmic XPO7 expression was a prognostic factor for overall patient survival. CONCLUSIONS:In this systematic study, we show that RAN and two protein partners involved in its nucleo-cytoplasmic and mitotic functions (XPO7 and TPX2, respectively) can be used as biomarkers to stratify patients based on prognosis. In particular, we reported for the first time the clinical relevance of the exportin XPO7 and showed that TPX2 expression had the strongest prognostic value. These findings suggest that protein partners in each of RAN's functions can discriminate between different outcomes in high-grade serous epithelial ovarian cancer patients. Furthermore, these proteins point to cellular processes that may ultimately be targeted to improve the survival in serous epithelial ovarian cancer.

BACKGROUND:Epithelial ovarian cancer (EOC) is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 3p in serous EOC pathogenesis. Our objective was to evaluate the expression of HYAL1, located at chromosome 3p21.3, in these EOC subtypes, and to investigate its correlation with the expression of steroid hormone receptors. METHODOLOGY/PRINCIPAL FINDINGS:We determined the mRNA expression of HYAL1, estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in EOC tumor samples and cell lines using quantitative RT-PCR. We also examined the expression of these genes in a publicly available microarray dataset. HYAL-1 enzyme activity was measured in EOC cell lines and in plasma samples from patients. We found that HYAL1 mRNA expression was elevated in clear cell and mucinous EOC tissue samples, but not in serous and endometrioid samples, normal ovaries or benign tumors. Similar results were obtained by two different techniques and with tissue sample cohorts from two independent institutions. Concordantly, HYAL1 mRNA levels and enzymatic activity were elevated only in EOC cell lines derived from clear cell and mucinous subtypes. We also showed that HYAL1 mRNA was inversely correlated to that of ERα specifically in clear cell and mucinous EOCs. Additionally, ectopic expression of ERα in a clear cell EOC cell line (ER- and PR-negative) induced 50% reduction of HYAL1 mRNA expression, supporting a role of ERα in HYAL1 gene regulation. Significantly, HYAL-1 activity was also high in the plasma of patients with these EOC subtypes. CONCLUSIONS/SIGNIFICANCE:This is the first report showing high HYAL-1 levels in EOC and demonstrating HYAL1 gene repression by ERα. Our results identify Hyaluronidase-1 as a potential target/biomarker for clear cell and mucinous EOCs and especially in tumors with low ERα levels.