Suppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D3 and its receptor

doi:10.1016/j.jsbmb.2014.11.005

The Journal of Steroid Biochemistry and Molecular Biology

Volume 148, April 2015, Pages 138-147

https://doi.org/10.1016/j.jsbmb.2014.11.005 Get rights and content

Highlights

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1,25D₃ suppresses EOC invasion into the omentum.
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The suppression of EOC invasion by 1,25D₃ is conserved from mice to humans.
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Both epithelial and stromal VDR act as suppressors of EOC invasion.

Abstract

Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D₃ (1,25D₃) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D₃ suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D₃, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D₃ as well as a 1,25D₃-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages.

This article is part of a Special Issue entitled “Vitamin D Workshop”.

Introduction

EOC is the deadliest among all gynecologic cancers and causes more deaths than cervical and uterine cancers combined. Despite the overall advancement in cancer research and clinical development over recent decades, there has been little improvement in EOC mortality rates. The poor prognosis is mainly due to the disease reaching an advanced stage before it is discovered. Primary cytoreductive surgery followed by chemotherapy with cisplatin and paclitaxel is the standard treatment regimen for patients with advanced EOC, yielding high response rates and improving both short and medium-term survivals. However, most patients will eventually relapse and die of their cancer [32]. Intervention strategies that suppress EOC invasion would retain the cancer inside ovaries, allowing simple surgery to suffice as a cure.

Omentum is a sheet-like tissue attached to the greater curvature of the stomach. It is the most common site for EOC metastatic spread [19], [20], [36]. The spread happens rather quickly and, in 80% of EOC patients, the cancer usually has spread to this tissue at the time of diagnosis. The composition and function of omenta are similar between mice and human. They contain primarily adipose tissue and islands of compact immune cells called milky spots, which is a lymphoid organ controlling peritoneal cavity immune response [33]. A recent study has reported that omental adipocytes promote EOC metastasis by providing energy for rapid tumor growth [30]. Nevertheless, the mechanisms underlying EOC invasion into the omentum are largely unknown and key molecular events controlling the process remain to be defined.

1,25D₃ is a fat-soluble seco-steroid hormone best known for its role in calcium and phosphate homeostasis. Effects of 1,25D₃ are mediated through the VDR that belongs to the steroid/thyroid hormone receptor superfamily [9], [39]. In addition, 1,25D₃ and its analogs elicit anti-tumor effects in a wide variety of cancer cell types through the induction of cell death, cell cycle arrest, differentiation, angiogenesis, etc. [3], [6], [7], [11], [12], [37], [38], [40], [41], [45], suggesting that 1,25D₃ holds great promise in cancer intervention. In EOC cells, 1,25D₃ causes cell cycle arrest at the G2/M transition through p53-independent induction of GADD45 [16]. Further studies have identified p27 as the key mediator of 1,25D₃-induced growth arrest in G1/S checkpoint [23] and defined a decrease in hTERT mRNA stability through microRNA as the mechanism underlying 1,25D₃-induced cell death [15], [17]]. However, a role for 1,25D₃ and VDR in EOC invasion and metastasis has not been investigated.

In the present studies, a series of experiments employing in vitro, ex vivo and in vivo EOC tumor models were conducted to assess the possible involvement of 1,25D₃ and VDR in suppressing EOC invasion into the omentum. These studies have revealed a novel role for 1,25D₃ in suppressing EOC invasion through both epithelial and stromal VDR. The findings suggest that VDR-based drug discovery may lead to a new intervention strategy to improve the clinical outcomes of patients with advanced EOC.

Section snippets

Cell culture and reagents

OVCAR3 human ovarian carcinoma cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 medium supplemented with 15% calf serum (CS), 2 mM l-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and 10 μg/ml bovine insulin. SKOV3-Luc cells, human ovarian carcinoma cell line, (Cell Biolabs, San Diego, CA) were maintained in DMEM containing 584 mg/l l-glutamine and 4.5 g/l glucose, supplemented with

Suppression of human and mouse EOC migration and invasion by 1,25D₃ through epithelial VDR and its translation into in vivo suppression of EOC omental invasion in mice

In published microarray studies performed in OVCAR3 cells [44], a group of genes with known functions in regulating invasive behavior of cancer cells, including genes encoding extracellular matrix proteins and chemokines/cytokines, were identified as 1,25D₃ target genes, indicating a role for the hormone in controlling EOC invasion. To test this potential role, OVCAR3 cells were treated with vehicle or 1,25D₃ and the migration and invasion of the treated cells were assessed by monolayer scratch

Discussion

Understanding the mechanisms by which tumor cells become metastatic is of great significance in cancer biology and medicine for the simple reason that cancer mortality is often caused by metastatic rather than by primary tumors. The metastatic process requires that cells gain the ability to migrate and invade adjacent tissues to reach the vasculature and lymphatic system. In this study, we have investigated the potential role of 1,25D₃ and VDR in suppressing EOC invasion into the omentum, the

Conflicts of interest

The authors have no conflicts of interest to report.

Acknowledgements

The authors would like to thank Ms. June Paciga for her assistance in conducting VDR immunohistochemistry, Dr. Takashi Tokono from Sapporo Medical University for providing VDR-shRNA and Dr. Weidong Xu from the University of South Florida for ID8-VEGF cells. We appreciate the technical support and assistance of the Tissue Core Facility, Moffitt Cancer Center. We also thank Mr. Clifford J. Webb for editorial input. The studies were supported by Public Health Service grants CA111334 (to Bai) and

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Further analyses have demonstrated that 1,25D3 induces microRNA target genes to decrease telomerase expression for cell death induction [10,11] and to inhibit leptin and estrogen-induced tumor growth [12], suggesting that 1,25D3 has an important role in cutting down obesity-associated cancer risks in women [13]. More recent studies have shown that ovarian cancer invasion is suppressed by 1,25D3 through the cooperative actions of epithelial and stromal VDR [14]. Overall, these studies project VDR as a new therapeutic target for ovarian cancer intervention.
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ReviewSuppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D3 and its receptor

Highlights

Abstract

Introduction

Section snippets

Cell culture and reagents

Suppression of human and mouse EOC migration and invasion by 1,25D3 through epithelial VDR and its translation into in vivo suppression of EOC omental invasion in mice

Discussion

Conflicts of interest

Acknowledgements

J. Steroid Biochem. Mol. Biol.

J. Steroid Biochem. Mol. Biol.

Biochem. Pharmacol.

Cell

Am. J. Pathol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Ann. Oncol.

Immunity

Mol. Cell. Endocrinol.

Am. J. Pathol.

J. Biol. Chem.

Vitamin D3 analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model

Dis. Colon Rectum

EB1089 inhibits the parathyroid hormone-related protein-enhanced bone metastasis and xenograft growth of human prostate cancer cells

Mol. Cancer Ther.

A calcitriol analogue, EB1089, inhibits the growth of LNCaP tumors in nude mice

Cancer Res.

Vitamin D for the prevention and treatment of pancreatic cancer

World J. Gastroenterol.

Vitamin D status and breast cancer risk

Anticancer Res.

Inhibition of hepatocellular cancer by EB1089: in vitro and in vive study

Anticancer Res.

Effects of 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells

Anticancer Res.

Milky spots as the implantation site for malignant cells in peritoneal dissemination in mice

Cancer Res.

Review
Suppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D₃ and its receptor

Suppression of human and mouse EOC migration and invasion by 1,25D₃ through epithelial VDR and its translation into in vivo suppression of EOC omental invasion in mice

Vitamin D₃ analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model

Effects of 1alpha,25-dihydroxyvitamin D₃ in human colon cancer cells