Abstract
Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3‘UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells.
Highlights
Tumor vasculogenic mimicry (VM) is a novel cancer hallmark formerly described in malignant melanoma cells which involves the formation of patterned three dimensional (3D) channels networks by tumor cells [1]
We focused in miR-765 for further functional analysis because: (i) it was severely downregulated after hypoxia (FC < 32.02; p < 0.05), (ii) it was predicted to target a number of genes involved in VM (Figure 3B), and (iii) there is no reports about the functions of miR-765 in ovarian cancer neither in tumor VM
In order to contribute with the understanding of the role of small non-coding RNAs in the molecular mechanisms responsible for VM, here we have uncovered a novel set of miRNAs modulated at the early onset of hypoxia-induced 3D channels-like structures formation, previous to the proper formation of tubules indicative of VM in SKOV3 cells
Summary
Tumor vasculogenic mimicry (VM) is a novel cancer hallmark formerly described in malignant melanoma cells which involves the formation of patterned three dimensional (3D) channels networks by tumor cells [1] These tubular networks resemble embryonic vasculogenesis, and they describe the ability of certain types of aggressive cancer cells to express endotheliumassociated genes [2]. Tumor VM occur de novo without or in combination with blood vessels formation changing our conventional acceptance that classical angiogenesis is the only means by which cancer cells acquire a nutrients supply to nourish tumors Studies supporting these assumptions have demonstrated that in vivo the 3D channels contain plasm, erythrocytes and blood flow with a hemodynamics similar to those occurring in endothelial vessels [3]. The exploration of the multiple roles of VM in cancer hallmarks, especially in drug resistance, would broaden our knowledge and eventually ameliorate the treatment efficacy in cancer
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