Abstract
BackgroundGranulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS).MethodsThe GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey’s post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05.ResultsControl cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis.ConclusionsThe results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.
Highlights
Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence
Keratin and β-actin expression in KGN cells Immunofluorescent staining of cultured KGN cells revealed an abundance of keratin 8/18 (K8/18) filament expression throughout the cells (Fig. 1, green fluorescence)
Acknowledging that these intermediate filaments help to counteract physiological stressors [28, 39], we show that disrupting K8/18 filament expression in KGN cells reverses resistance to FAS-mediated apoptosis by enhancing FAS expression on the cell surface
Summary
Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Granulosa cell tumors (GCT) represent approximately 5 % of ovarian malignancies, yet constitute the most prevalent sex cord-stromal ovarian neoplasms [1, 2]. Prognosis is favorable when detected early, but recurrent GCT may be life-threatening. The rarity of these tumors and their low potential for malignancy (i.e., slow growth over a prolonged period) has reduced the urgency to study and develop standard treatment regimens for these neoplasms compared to other ovarian cancers. GCT have a high rate of reappearance following resection [2,3,4], and such recurrence often leads to late detection and poor prognosis [5, 6]. GCT have many morphological and biochemical features akin to granulosa cells of mature, preovulatory follicles (e.g., FSH-responsive and steroid hormone production) [11,12,13,14]
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