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Metalloproteinase Meprin Α Regulates Migration and Invasion of Human Hepatocarcinoma Cells and Is a Mediator of the Oncoprotein Reptin

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Metalloproteinase Meprin Α Regulates Migration and Invasion of Human Hepatocarcinoma Cells and Is a Mediator of the Oncoprotein Reptin www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 5), pp: 7839-7851 Research Paper Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin Osman Breig1, Maïlyn Yates1, Véronique Neaud1, Gabrielle Couchy2, Aude Grigoletto1, Carlo Lucchesi3, Johannes Prox4, Jessica Zucman-Rossi2, Christoph Becker-Pauly4, Jean Rosenbaum1 1University Bordeaux, INSERM, U1053, BordeAux Research in Translational Oncology, BaRITOn, Bordeaux, France 2Inserm, U1162, Génomique Fonctionnelle des Tumeurs Solides, Université Paris Diderot, Université Paris Descartes, Université Paris 13, Paris, France 3SIRIC BRIO, Bordeaux, France 4Unit for Degradomics of the Protease Web, University of Kiel, Germany Correspondence to: Jean Rosenbaum, email: [email protected] Keywords: RUVBL2, proteolysis, prognosis Received: August 23, 2016 Accepted: November 30, 2016 Published: December 16, 2016 ABSTRACT Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects. MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers. Silencing Reptin decreased Mep1A expression and activity, without affecting meprin β. Mep1A, but not meprin β, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted. Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC. INTRODUCTION responsible for HCC cells migration and invasion have been identified, this knowledge has not so far impacted Hepatocellular carcinoma (HCC) remains one of the handling of patients. There is thus a great need for the deadliest cancers worldwide. The prognosis of HCC discovering new actionable targets. is dictated both by the status of the host liver, very often In the last few years, we have discovered the cirrhotic, and by the tumor extension. HCC is characterized overexpression in HCC of Reptin, or RUVBL2, a by a very frequent intra-hepatic invasion, notably of portal member of the AAA+ ATPase family [2]. Reptin, and its veins, and by microvascular invasion, which carries a very homolog and partner protein, Pontin/RUVBL1 (also found poor prognosis [1]. Although a number of mechanisms overexpressed in HCC [3]) are part of several protein www.impactjournals.com/oncotarget 7839 Oncotarget complexes that have pleiotropic functions (for review, meprin α proteolytic activity in the conditioned medium see [4]). Briefly, they are involved in the remodeling of was decreased upon Reptin silencing. As a control, chromatin, the regulation of gene transcription through we confirmed that the substrate cleaving activity was various mechanisms, and they also act as chaperones abrogated by the meprin inhibitor actinonin (Figure 1F). for proteins of the Phosphatidylinositol Kinase-like Kinases family such as mTOR, ATM or DNA-PKcs. We Meprin α is overexpressed in human HCC and found that both proteins were required for the growth its expression correlates with that of Reptin and viability of HCC cells [2, 3, 5] and that silencing Reptin within established HCC xenografts induced tumor Expression of meprin α, meprin β and Reptin regression, thus establishing Reptin as a potential target mRNAs was assessed using RT-PCR on a previously in HCC [6]. There is also evidence showing that Reptin described series of 242 HCC from the French Biological silencing reduces invasion of cancer cells from kidney Resource Center on HCC [22]. Meprin α mRNA level [7] and prostate [8]. The downstream mediators of Reptin was significantly higher in HCC samples as compared to underlying its oncogenic effects remain however to be non-tumor liver (2.04 fold, p < 0.0001; Figure 2A). On found. the other hand, meprin β expression was not increased, Meprin α is a secreted metalloproteinase that can and was even significantly decreased (p < 0.0001) when cleave a potentially large number of protein substrates, comparing HCC to non-tumor liver (Figure 2A). many of them being highly relevant for cancer [9]. Immunohistochemistry experiments in 3 HCC Those include extracellular matrix and related proteins tumors confirmed in all cases an overexpression of Meprin (pro-collagen I, fibronectin, SPARC, osteopontin), α in HCC as compared to the non-tumor liver in the same cytokines (pro-IL1β, IL6), growth factors (VEGF-A, patient. Furthermore, these experiments demonstrated CTGF), membrane proteins (occludin), and other unambiguously that Meprin α overexpression takes place proteinases (MMP1, ADAM10) [10]. Meprin α had so far in tumor cells and not stromal cells (Figure 2B). received little attention in cancer, with limited evidence Analysis of HCC subgroups revealed that meprin showing that it could stimulate cancer cell proliferation α expression was especially high in transcriptomic [11], migration [12] and invasion [13]. While this work groups G1, G2 and G3 (Figure 2C). These groups are was in progress, however, OuYang et al. showed that it characterized by a higher rate of cell proliferation, could induce HCC cell migration and invasion [14]. and accordingly meprin α expression was significantly Interestingly, there are already available good meprin correlated with that of Ki67 mRNA (Spearman r = 0.41, α inhibitors like actinonin [15] that can be used in vivo p < 0.0001). Groups G1-G3 are also enriched in patients [16–18], and more specific inhibitors are currently under with TP53 mutations [23] and we found indeed that development [19], raising the hope that meprin α may meprin α expression was significantly higher in become a clinically useful target. patients with TP53 mutation, and conversely lower in Knowing that Reptin regulates the expression those with CTNNB1 mutations, that are enriched in of many genes [20, 21], we hypothesized that it G4-G6 patients (Figure 2D). There was no significant might regulate meprin α. Thus, in this study, we have difference in meprin α expression whether the cause investigated the regulation of the expression of meprin α of liver disease was excess alcohol consumption, B or by Reptin in HCC, the effects of meprin α on HCC cell C viral hepatitis, hemochromatosis or metabolic liver phenotype, and whether meprin α mediated the oncogenic disease (not shown). effects of Reptin. Additionally, several data pointed to a significant correlation of high meprin α expression with a poor RESULTS prognosis. First, meprin α expression was significantly higher in tumors from Edmonson grades II-IV, as Reptin silencing decreases the expression of compared to grades I-II (Figure 2E). We previously found meprin α that a score based on the expression of the 5 genes HN1, RAN, RAMP3, KRT19, and TAF9, was associated with Reptin expression was efficiently silenced in the survival of HCC patients following surgery [22]. We HuH7 cells using two different siRNAs, or with a found here that meprin α expression was significantly doxycycline-inducible shRNA, as described previously higher in tumors from patients with high 5-gene prognosis [2, 6]. This resulted in a significantly decreased expression score (Figure 2E). Finally, we performed a Kaplan-Meier of meprin α mRNA, whereas the expression of meprin β analysis by first dichotomizing the patients as meprin was not decreased (Figure 1A–1C). Similar results were low or high according to the median of expression in all obtained in another HCC cell line (Hep 3B (Supplementary samples. This analysis showed that the overall survival of Figure S1A). Furthermore, meprin α, but not meprin β patients was significantly worse in patients with a high protein expression was also reduced as shown by Western expression of meprin α, whereas meprin β expression blot (Figures 1D–1E, Supplementary Figure S1D). Finally, carried no prognostic value (Figure 2F). www.impactjournals.com/oncotarget 7840 Oncotarget Very interestingly, and in line with our results strategies. First, we constructed HuH7 and Hep3B cell showing the regulation of meprin α expression by Reptin, lines constitutively overexpressing meprin α with a V5 we found that meprin α expression was significantly C-terminal tag. Because the C-terminus of meprin α is and positively correlated with that of Reptin in HCC removed during the maturation and secretion process, (Spearman r = 0.23, p = 0.0003), whereas no correlation the tag is no more present in the secreted protein and was found for meprin β (Spearman r = 0.083, p = 0.20). does not interfere with the proteolytic activity. As shown on Figure 3D, the V5-tagged protein was found in Generation of tools for loss and gain of function large amounts in cell extracts where it can be detected of meprin α with both a meprin α and a V5 antibody. On the other hand, the overexpressed protein is detectable only with For loss of function, we designed two siRNAs the meprin α antibody in the supernatant of the cells. targeting meprin α. Both siRNAs efficiently depleted Immunofluorescence with both anti-meprin α and anti-V5 meprin α mRNA and protein (Figure 3A–3C).
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