SOX17 expression in ovarian clear cell carcinoma

Abstract

Recent studies have revealed that the Sry-related HMG box gene 17 (SOX17) plays an important role in ovarian carcinogenesis. Unlike other types of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a distinct pathobiological phenotype, often harboring an AT-rich interaction domain 1 A (ARID1A) mutation. In the present study, to determine the SOX17 in OCCC cells, we immunohistochemically examined SOX17 expression in 47 whole-tissue specimens of OCCC. Although not statistically significant, SOX17-high immunoreactivity tended to be related to unfavorable patient outcomes. We also aimed to determine the relationship of SOX17 with ARID1A. Double immunofluorescence staining demonstrated that SOX17 immunoreactivity was not associated with ARID1A immunoreactivity. Immunoblotting revealed that SOX17 was abundantly expressed in cultured OVISE and RMG-V OCCC cells, but not in OVTOKO OCCC cells. Polyubiquitinated bands of SOX17 were observed in MG132 treated OVTOKO, but not in OVISE or RMG-V OCCC cells. Notably, si-RNA-mediated knockdown of a deubiquitinase enzyme, ubiquitin C-terminal hydrolase L1, increased polyubiquitination followed by proteasome degradation of SOX17 in OVISE. These findings indicate that SOX17 is not uniformly and heterogeneously expressed in OCCCs, independent of ARID1A deficiency. Impaired ubiquitin-mediated proteasome degradation may stabilize SOX17 in some OCCC cells.

Keywords: ARID1A; Ovarian clear cell carcinoma; Prognosis; SOX17; Ubiquitination.

Fig. 1

SOX17 immunoreactivity in OCCC cells. SOX17 immunoreactivity was not diffusely but heterogeneously distributed in OCCC cells. Representative staining of diminished SOX17 expression (a). In SOX17-high cases, SOX17 immunoreactivity was focally found in OCCC cells at various intensities (b and c). Intermittent SOX17 immunoreactivity is shown (b) (arrow indicate the SOX17 immunoreactivity). Intratumoral heterogeneous SOX17 immunoreactivity was found in (c) (arrow indicates weak immunoreactivity, while arrowhead indicates strong reactivity). Scale bar, 50 μm

Fig. 2

Representative images of SOX17 immunohistochemical staining in both ARID1A-negative OCCC tissue specimens (a and c) and ARID1A-positive OCCC tissue specimens (b and d). Little or no ARID1A immunoreactivity was found in a, while robust ARID1A immunoreactivity was found in c. Immunohistochemical staining demonstrated that SOX17 immunoreactivity was found in both ARID1A-negative tissue specimens (b) and ARID1A-positive tissue specimens (d). Blue and black arrows indicate SOX17-negative and -positive OCCC cells, respectively. Arrows indicate 100 μm

Fig. 3

Immunoblotting results. This procedure revealed a strong SOX17 protein band (green) in a HGSC cell line, OVSAHO (lane 1). The SOX17 band was also found in all of the examined OCCC cell lines (a) However, SOX17 band was notably smaller in OVTOKO OCCC cells (lane 3) compared to that of the OVISE (lane2) and RMG-V (lane 4) OCCC cells. The internal control, HDAC1, was stained red. SOX17 and HDAC1 bands were extracted as black and white bands (b and c, respectively). The polyubiquitinated SOX17 bands were found in OVTOKO cells (lane 3), but were present at diminished levels in the OVSAHO (lane 1), OVISE (lane 2), and RMG-V cells (lane 4) when they were treated with a proteasome inhibitor, MG132 (d). Input protein loads of cell lysates that were subjected to co-immunoprecipitation (IP) with anti-SOX17, followed by immunoblotting (WB) with anti-ubiquitin is shown as G3PDH bands (e). siRNA-mediated silencing of UCHL1 decreased the intensity of UCHL1 (f) and SOX17 (g) protein bands (lane 1, treated with control siRNA; lane 2, treated with Ambion siRNA ID: 105082) and moreover, increased polyubiquitinated SOX17 proteins (h). Equal input protein loads are shown as G3PDH bands. Similar results were also obtained using another siRNA ID: 105,646