Glucocorticoid Regulation of SLIT/ROBO Tumour Suppressor Genes in the Ovarian Surface Epithelium and Ovarian Cancer Cells

Rachel E Dickinson,K Scott Fegan,Stephen G Hillier,W Colin Duncan,Xia Ren,Sumitra Deb

doi:10.1371/journal.pone.0027792

Rachel E Dickinson, K Scott Fegan + Show 4 more

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https://doi.org/10.1371/journal.pone.0027792

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Abstract

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.

Highlights

The secreted Slit glycoprotein and its Robo receptor were originally identified as important axon guidance molecules in the developing Drosophila nervous system [1,2]
RT-PCR analysis confirmed the expression of SLIT2 and ROBO1 in primary cultures of ovarian surface epithelium (OSE) and demonstrated that there was some expression of SLIT3, ROBO2 and ROBO4 in these cells (Fig. 1D)
SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 were expressed in these cells but quantitative analysis showed that they were reduced by 25–82% when compared to primary cultures of normal OSE (Fig. 2A) (P,0.05)

Summary

Introduction

The secreted Slit glycoprotein and its Robo receptor were originally identified as important axon guidance molecules in the developing Drosophila nervous system [1,2]. The SLITs and ROBOs are candidate tumour suppressor genes whose expression is reduced in numerous epithelial tumour cell types, mainly through deletion, loss of heterozygosity and promoter region hypermethylation [9]. This includes cancers derived from reproductive tissues including cervical, prostate and ovarian germ-line tumours [10,11,12,13]. SLIT2 inhibited the invasion of numerous different types of tumour cells including those from the prostate, breast, endometrium and ovary [13,18,19]

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