Role of hypoxia and PARP1 in the regulation of vasculogenic mimicry

Abstract

Melanomas represent a variety of neoplastic diseases that derive from the malignant transformation of melanocytic cells. Cutaneous and uveal melanoma are the most common presentations of this cancer type. Unfortunately, current therapies have proved insufficient in the management of melanoma, highlighting the need for further research. Solid tumors usually present a state of low oxygenation known as hypoxia. Hypoxic microenvironments trigger adaptive responses that contribute to the malignization of tumors. For instance, the hypoxia response plays a key role in tumor neovascularization. Angiogenesis consists in the sprouting of new blood vessels from pre-existing vasculature and it is the best known form of tumor neovascularization. However, alternative neovascularization mechanisms, such as vasculogenic mimicry, have been gaining attention in recent years. Vasculogenic mimicry describes the ability of highly aggressive tumor cells to acquire endothelial traits and develop vessel-like structures capable of carrying blood without the participation of endothelial cells. It is correlated with metastasis, shorter survival and poor prognosis in cancer patients. Unfortunately, there are currently no available drugs to target vasculogenic mimicry in clinical settings, and its treatment remains a major biomedical challenge. Poly-(ADP-ribose) polymerase (PARP) 1 catalyzes the synthesis and transfer of poly-(ADP-ribose) to target proteins. PARP inhibitors cause synthetic lethality in cells with genetic deficiencies in the homologous recombination DNA repair pathway, inducing the selective elimination of cancer cells with these defects. As a result, four PARP inhibitors have been approved so far for their clinical use against tumors displaying characteristics of “BRCAness”. Furthermore, PARP1 has been implicated in multiple physiological and pathological processes. For instance, PARP1 can promote the stability and function of hypoxia-inducible factors, which raises PARP1 as an attractive druggable target for the control of hypoxiaderived tumor processes, like vasculogenic mimicry. The purpose of the present thesis was to explore the consequences of PARP inhibition in the context of melanoma vasculogenic mimicry, which could expand the therapeutic benefit of PARP inhibitors while potentially targeting a malignant trait which so far remains untreatable. Our study shows that PARP inhibition and hypoxia can modulate the expression of hundreds of genes during melanoma tube formation on matrigel. The profound impact of PARP inhibition was partly due to the modulation of long non-coding RNA, a class of RNA transcripts that do not encode any protein products but can regulate gene expression in a variety of ways. PARP inhibition during hypoxia significantly modulated the expression of numerous genes implicated in vascular biology, resulting in the global modulation of a number of vasculature-related signaling pathways and seemingly enhancing the endothelial-like characteristics of melanoma cells. Consistent with previous results, PARP1 chemical inhibition or genetic knockdown could reduce the phosphorylation of vascular endothelial cadherin on tyrosine 658 in normoxia and hypoxia. Previous publications have proved the crucial role of this cadherin and this specific phosphorylation in vasculogenic mimicry. Moreover, this phosphorylation is reportedly correlated with increased endothelial vessel permeability, which can favor metastatic spread. Our study also found that PARP inhibition can upregulate the expression and secretion of the plateletderived growth factor β in vasculogenic melanoma cells. This cytokine is involved in the recruitment of pericytes to blood vessels. In endothelial vessels, low permeability and high pericyte coverage are markers of vascular normalization, which is considered a sign of good prognosis in tumors. In addition, melanoma tubular networks on matrigel seemed to undergo a structural normalization after treatment with PARP inhibitors. Finally, studies with human uveal melanoma xenografts showed that treatment with PARP inhibitor olaparib improved pericyte coverage specifically of vasculogenic mimicry pseudovessels in vivo, which was concomitant with a decrease in metastasis in tumors with the ability to develop vasculogenic mimicry. In conclusion, the results of this thesis indicate that PARP inhibitors may reduce the metastatic spread of melanoma tumors capable of vasculogenic mimicry by normalizing vasculogenic mimicry pseudovessels.