β-arrestin 2 Is a Prognostic Factor for Survival of Ovarian Cancer Patients Upregulating Cell Proliferation

Abstract

Establishing reliable prognostic factors as well as specific targets for new therapeutic approaches is an urgent requirement in advanced ovarian cancer. For several tumor entities, the ubiquitously spread scaffold protein β-arrestin 2, a multifunctional scaffold protein regulating signal transduction and internalization of activated G protein-coupled receptors (GPCRs), has been considered with rising interest for carcinogenesis. Therefore, we aimed to elucidate the prognostic impact of β-arrestin 2 and its functional role in ovarian cancer. β-arrestin 2 expression was analyzed in a subset of 156 samples of ovarian cancer patients by immunohistochemistry. Cytoplasmic expression levels were correlated with clinical as well as pathological characteristics and with prognosis. The biologic impact of β-arrestin 2 on cell proliferation and survival was evaluated, in vitro. Following transient transfection by increasing concentrations of plasmid encoding β-arrestin 2, different cell lines were evaluated in cell viability and death. β-arrestin 2 was detected in all histological ovarian cancer subtypes with highest intensity in clear cell histology. High β-arrestin 2 expression levels correlated with high-grade serous histology and the expression of the gonadotropin receptors FSHR and LHCGR, as well as the membrane estrogen receptor GPER and hCGβ. Higher cytoplasmic β-arrestin 2 expression was associated with a significantly impaired prognosis (median 29.88 vs. 50.64 months; P = 0.025). Clinical data were confirmed in transfected HEK293 cells, human immortalized granulosa cell line (hGL5) and the ovarian cancer cell line A2780 in vitro, where the induction of β-arrestin 2 cDNA expression enhanced cell viability, while the depletion of the molecule by siRNA resulted in cell death. Reflecting the role of β-arrestin 2 in modulating GPCR-induced proliferative and anti-apoptotic signals, we propose β-arrestin 2 as an important prognostic factor and also as a promising target for new therapeutic approaches in advanced ovarian cancer.

Keywords: G protein-coupled receptor; immunohistochemistry; in vitro analyses; ovarian cancer; survival analysis; β-arrestin 2.

Figure 1

Detection of β-arrestin 2 with immunohistochemistry. (A) Cytoplasmic β-arrestin 2 staining in ovarian cancer with serous, (B) clear cell, (C) endometrioid, and (D) mucinous histology. (E) β-arrestin 2 negative control (F) and positive control in human kidney tissue. Clear cell histology exhibited the strongest cytoplasmic β-arrestin 2 expression (Kruskal-Wallis test; p = 0.01) when compared with different histological subtypes. Arrows demonstrate representatively β-arrestin 2 stained cells. Images representative of three independent experiments.

Figure 2

Detection of β-arrestin 2 with immunohistochemistry in benign ovary and fallopian tube. Detection of β-arrestin 2 with immunohistochemistry in benign ovary (A) and fallopian tube (B). Positive β-arrestin 2 staining was observed in epithelial cells, granulosa cells and theca cells. The ovarian and fallopian tube stroma did not show a β-arrestin 2 staining. Arrows demonstrate representatively β-arrestin 2 stained cells. Images representative of three independent experiments.

Figure 3

Kaplan–Meier estimate of β-arrestin 2. β-arrestin 2 expression (IRS 4–12) was associated with impaired overall survival (50.64 vs. 29.88 months, median; p = 0.025). Censoring events have been marked in the graphs.

Figure 4

Evaluation of cell viability and death under 48-h β-arrestin 2 overexpression. HEK293, hGL5, and A2780 cells were transfected by increasing amount of plasmid encoding β-arrestin 2 and cell viability and death were assessed by MTT and propidium iodide, respectively. Total protein content from 0.5 × 10⁴ 70% confluent log phase cells were loaded. (A) Representative image of β-arrestin 2 detected in cell lysates by Western blotting using a specific rabbit antibody using β-actin as a normalizer. (B) Box and Whiskers plot of MTT data, collected from transfected HEK293 cells overexpressing increasing levels of β-arrestin 2, representing cell viability. (C) hGL5 cell viability evaluated by MTT. (D) β-arrestin 2-dependent cell viability of the A2780 ovarian cancer cell line. (E) Data from the β-arrestin 2-overexpressing HEK293, representing cell death, in propidium iodide-stained samples. (F) β-arrestin 2-overexpressing hGL5 cell death. (G) β-arrestin 2-overexpressing A2780 ovarian cancer cell death. Brightness/contrast of Western blotting pictures were adjusted uniformly in all panels. Asterisks indicate significantly different data distribution than mock-transfected (untransfected) cells (Kruskal-Wallis test and Dunn's correction for multiple comparisons; *P < 0.05; **P < 0.001; ***P < 0.0001; n = 8).

Figure 5

Viability and death of 48-h β-arrestin 2-silenced HEK293, hGL5, and A2780 cells. Samples were transfected by increasing amount of siRNA probes targeting the β-arrestin 2 mRNA and 48-h cell viability and death were assessed by MTT and propidium iodide. Total protein content from 3 × 10⁴ 70% confluent log phase cells were loaded. (A) Box and Whiskers plot representing the HEK293 cell viability under β-arrestin 2 depletion. (B) β-arrestin 2-silenced hGL5 cell viability. (C) β-arrestin 2-silenced A2780 ovarian cancer cell viability. (D) Data from the β-arrestin 2-silenced HEK293, representing cell death evaluated using propidium iodide staining. (E) hGL5 cell death evaluated by propidium iodide. (F) Cell death in β-arrestin 2-silenced A2780 evaluated by propidium iodide. (G) Representative Western blotting images demonstrating β-arrestin 2 depletion and pro-caspase 3 cleavage in HEK293, hGL5, and A2780 cell lysates. β-actin was the normalizer. Brightness/contrast of Western blotting pictures were adjusted uniformly in all panels. Significantly different data distribution than mock-transfected (untransfected) cells were indicated by asterisks. Kruskal-Wallis test and Dunn's correction for multiple comparisons; *P < 0.05; **P < 0.001; ***P < 0.0001; n = 8).