Hypoxia mediated expression of stem cell markers in VHL-associated hemangioblastomas

https://doi.org/10.1016/j.bbrc.2013.07.028Get rights and content

Highlights

  • Hemangioblastomas are observed in patients with mutations in the VHL gene.

  • This leads to constitutive activation of the hypoxia-inducible-factor (HIF) pathway.

  • Activated HIF pathway induces stem cell markers in hemangioblastomas.

  • Inhibitors of the HIF pathway block the expression of stem cell markers.

  • Specific inhibitors of the HIF pathway can be used to treat hemangioblastomas.

Abstract

Hemangioblastomas of the retina, central nervous system, and kidney are observed in patients with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Mutations in the VHL lead to constitutive activation of hypoxia-inducible-factor (HIF) pathway. HIF-mediated expression of pro-angiogenic genes causes extensive pathological neovascularization in hemangioblastomas. A number of studies have shown coexistence of pro-angiogenic and stem cell markers in ‘tumorlet-like stromal cells’ in the retinal and optic nerve hemangioblastomas, leading to suggestions that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors. Since recent studies have shown that the HIF pathway also plays a role in the maintenance/de-differentiation of normal and cancerous stem cells, we evaluated the role of the HIF pathway in the expression of stem cell markers in VHL−/− renal cell carcinoma cells under normoxia or VHL+/+ retinal pigment epithelial cells under hypoxia. Here we show that the expression of stem cell markers in hemangioblastomas is due to activation of the HIF pathway. Further, we show that honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources, blocks the expression of stem cell markers. Our results show the mechanism for the cytological origin of neoplastic stromal cells in hemangioblastomas, and suggest that inhibition of the HIF pathway is an attractive strategy for the treatment of hemangioblastomas.

Introduction

Hemangioblastomas (also known as the blood vessel tumor or VHL disease) are multisystem tumor syndromes, where mutations in the VHL protein (pVHL) lead to constitutive activation of the HIF pathway. HIF is a member of PAS family of basic helix-loop-helix heterodimeric transcription factors, consisting of α subunits (HIF-1α, -2α, and -3α) and HIF-1β/ARNT subunit. Normally, hydroxylation of the conserved proline residues (Pro402 and Pro564 in human HIF-1α) by the oxygen-dependent HIF prolyl hydroxylases/dioxygenases (PHD1–3) under normoxic conditions allows the binding of HIF-α isoforms to the wild-type (wt)-pVHL. This binding of hydroxylated HIF-α to pVHL, the substrate recognition subunit of an E3 ubiquitin ligase, allows rapid proteosomal degradation of HIF-α under normoxia [1], [2]. However, in patients with mutated pVHL, even though HIF-α gets hydroxylated at the proline residues under normoxia, it is not bound by mutated pVHL, allowing it to escape proteosomal degradation. Subsequently, HIF-α translocates to the nucleus, dimerizes with the HIF-1β subunit, and recruits transcriptional co-activators. The active HIF-α/β heterodimer binds to a core DNA sequence (G/ACGTG) in the hypoxia-response-element (HRE) present in the promoters of target genes, causing overexpression of hypoxia response genes, which includes a number of pro-angiogenic factors [e.g. vascular endothelial growth factor (VEGF), erythropoietin (EPO), platelet-derived growth factor (PDGF), etc.]. As a result, hemangioblastomas of retina, central nervous system (CNS), and kidney are highly vascular in nature [3].

In addition to these vascular cells, hemangioblastomas are composed of ‘stromal cells’. However, the cytological origin of these neoplastic stromal cells remains unknown. Number of studies have shown coexistence of pro-angiogenic and stem cell markers in tumorlet-like stromal cells in the retinal and optic nerve hemangioblastomas [4], [5], leading to the suggestion that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors [4], [5], [6], [7]. Failure to specify the histological origin of stromal cells in hemangioblastomas has precluded the development of nonsurgical therapies for these multisystem blood vessel tumor syndromes.

All current therapies for hemangioblastomas have significant limitations and side-effects; while anti-VEGF therapies (e.g. Macugen, Lucentis, etc.) had minimal detectable beneficial effects [8], [9]. This lack of efficacy of anti-VEGF therapies is possibly due to overexpression of other pro-angiogenic factors (e.g. EPO, PDGF, etc.) in hemangioblastomas. The possible future approaches to successfully control hemangioblastomas may rely on blocking some master modulator, such as the HIF pathway. Thus, characterization of novel HIF inhibitors may have a considerable therapeutic impact on the treatment of hemangioblastomas. We have recently shown that honokiol, a biphenolic phytochemical extracted from the Magnolia genus (which has been used for thousands of years in the traditional Japanese and Chinese medicine) is a potent inhibitor of the HIF pathway as well as hypoxia-induced expression of histone lysine demethylases in a number of cancer and retinal pigment epithelial cell lines [10], thus providing an evidence-based scientific explanation of honokiol’s therapeutic benefits. Further, screening a library of compounds that are in clinical practice/trials using a reporter assay identified cardiac glycosides (e.g. digoxin, ouabain, and proscillaridin A) and anthracycline chemotherapeutic agents (e.g. doxorubicin and daunorubicin) as potent inhibitors of the HIF pathway [11], [12]. Administration of digoxin or doxorubicin in xenograft mouse models inhibited the transcription of endogenous HIF-dependent genes, increased latency, and decreased tumor growth [11], [12]. Here, we investigate the role of the HIF pathway in the expression of stem cell markers in hemangioblastomas and suggest that inhibition of the HIF pathway is an attractive target for the treatment of hemangioblastomas.

Section snippets

Chemicals and reagents

All chemicals were supplied by the Sigma–Aldrich Chemical Co. (Saint Louis, MO) unless otherwise stated, and were of analytical grade or higher. Honokiol was purchased from the Stanford Chemicals (Irvine, CA), while digoxin and doxorubicin were ordered from Carbosynth LLC (San Diego, CA).

Cell culture

Human retinal pigment epithelial cell lines (D407 and ARPE19) and renal cell carcinoma derived cell lines (RCC4, RCC4–T314, PRC3, and WT8) were used for these studies. RCC4 and PRC3 cells lack a functional VHL

HIF-mediated expression of pro-angiogenic genes in renal cell carcinoma and retinal pigment epithelial cell lines

The human RCC4 cell line lacks wt-pVHL and hence has a constitutively active HIF pathway. RCC4–T314 cells were derived from the RCC4 cells by stably transfecting wt-VHL, and thus, these cells do not activate the HIF pathway under normoxia. Under hypoxia, HIF-α isoforms are stabilized in RCC4–T314 cells leading to activation of the HIF pathway [15]; While, 786-O cells, another renal cell carcinoma cell line that is also VHL−/−, expresses only HIF-2α [16], [17]. Unlike VHL−/− RCC4 cells [15],

Acknowledgments

This research has been funded by UMKC internal support in the form of start-up to M.M. The RCC4, RCC4–T314, PRC3, and WT8 cell lines were generously provided by Dr. M. Celeste Simon (University of Pennsylvania Cancer Center), while D407 cell line was generously gifted by Dr. Richard Hunt (University of South Carolina). The authors would like to thank Dr. Kun Cheng for allowing us to use the real-time qPCR instrument.

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