Regulation of extracellular matrix degradation and metastatic spread by IQGAP1 through endothelin-1 receptor signaling in ovarian cancer through in

The invasive phenotype of serous ovarian cancer (SOC) cells is linked to the formation of actin-based protrusions, invadopodia, operating extracellular matrix (ECM) degradation and metastatic spread. Growth factor receptors might cause engagement of integrin-related proteins, like the polarity protein IQ-domain GTPase-activating protein 1 (IQGAP1), to F-actin core needed for invadopodia functions. Here, we investigated whether IQGAP1 forms a “signalosome” with endothelin-1 (ET-1)/β-arrestin1 (β-arr1) network, as signal-integrating module for adhesion components, cytoskeletal remodelling and ECM degradation. In SOC cells, ET-1 receptor (ET-1R) activation, besides altering IQGAP1 expression and localization, coordinates the binding of IQGAP1 with β-arr1, representing an “hotspot” for ET-1R-induced invasive signalling. We demonstrated that the molecular interaction of IQGAP1 with β-arr1 affects relocalization of focal adhesion components, as vinculin, and cytoskeleton dynamics, through the regulation of invadopodia-related pathways. In particular, ET-1R deactivates Rac1 thereby promoting RhoA/C activation for the correct functions of invasive structures. Silencing of either IQGAP1 or β-arr1, or blocking ET-1R activation with a dual antagonist, prevents matrix metalloproteinase (MMP) activity, invadopodial function, transendothelial migration and cell invasion. In vivo, targeting ET-1R/β-arr1 signalling controls the process of SOC metastasis, associated with reduced levels of IQGAP1, as well as other invadopodia effectors, such as vinculin, phospho-cortactin and membrane type 1-MMP. High expression of ET A R/  -arr1/IQGAP1 positively correlates with poor prognosis, validating the clinical implication of this signature in early prognosis of SOC. These data establish the ET-1R-driven β-arr1/IQGAP1 interaction as a prerequisite for the dynamic integration of pathways in fostering invadopodia and metastatic process in human SOC. and proteolytic signalling platform in ET-1R-driven invadopodia and ECM degradation in ovarian cancer. 1. IQGAP1 interacts with β-arr1 in ET-1-stimulated SOC cells; 2. Engagement of IQGAP1 by β-arr1 favours the tight control of RhoA/C activity over Rac1, thereby coordinating invadopodia function and ECM degradation; 3. Interruption of ET-1R/IQGAP1/β-arr1 network impairs invasive and metastatic behaviour; 4. Concomitant high expression of EDNRA/ARRB1/IQGAP1 is a negative prognostic factor in ovarian cancer patients. role of IQGAP1 potentially independent predictor of highly aggressive tumors our reveal a new mechanistic link between IQGAP1 and the progression of SOC, demonstrating that IQGAP1 is a new interactor of β-arr1 downstream of ET-1R signalling in shaping cytoskeleton remodelling, and invadopodia-dependent ECM degradation. It is conceivable that the interaction between IQGAP1 and β-arr1 might expand the potential of cross-talk between signalling cascades, highlighting an additional complexity in the molecular mechanism by which ET-1R regulates invadopodia. According with the role of IQGAP1 in delivering accumulation of MT1-MMP at invadopodia dots we propose that ET-1-dependent invadopodia maturation in SOC cells relies on the local coordination of effectors, such as RhoC and Rac1, triggered by the interaction of IQGAP1 with β-arr1. Since IQGAP1 possesses an inactive RasGAP domain lacking the ability to direct act as a GEF or GAP, we provide evidence that IQGAP1/β-arr1 could act as small GTPase scaffolding platform, as RacGAP1, to promote GTP hydrolysis and consequently Rac1 inhibition and concomitant RhoA,C activation. Given that different inputs can promote selective Rho GTPase activation and inhibition, it is likely that ET-1R-guided β-arr1 interactions determine the convergence and activation/inhibition of specific signals for invadopodia, such as Rho GTPases, by recruiting GAP, as RacGAP1, where IQGAP1 helps to define the discrete locations and/or time. The coordination of cytoskeleton remodelling from ET-1R-driven IQGAP1/β-arr1 interaction For WB analysis, cells were lysed as previously described and resolved on Mini-PROTEAN TGX gels (Biorad Laboratories). Immunoblotting was performed using the following primary antibodies (Abs): anti-IQGAP1 (cat no. ab86064, Abcam), anti-β-arr1 (cat ab32099, Abcam), anti-Tubulin (cat no. Santa anti-HSP70 (cat no. ab2787, Abcam), anti-GFP (cat no. 83525, Immunological Science), anti-AU5 (cat no. ab24576, Abcam), anti-RacGAP1 (cat no. ab180802, Abcam), anti-MT1-MMP (cat no. Abcam), anti-cortactin (cat no. Cell Signalling), anti-phospho-cortactin Tyr 421 (cat no. 45696, Cell Signalling), anti-vinculin (cat no. Cruz Biotechnology). Primary antibodies were revealed using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Biorad Laboratories). immunoprecipitation (IP), precleared whole-cell lysates were incubated with anti-IQGAP1 (cat no. ab86064) and anti-β-arr1 (cat no. ab32099) Abs (Abcam) or the correspondent IgG control Ab (Thermo Fisher Scientific) and protein G agarose beads (Thermo Fisher Scientific) at 4°C overnight. For detection of co-immunoprecipitated β-arr1, si-IQGAP1 and si-β-arr1 transfected SKOV3 cells stimulated with ET-1 and/or MAC inputs were analysed WB for the indicated proteins. Histograms, the mean±SD from measurements of RhoA and C activity normalized to RhoA,B,C shown as fold over si-SCR; were obtained GST-Pak1-PBD beads transfected HEY inputs three different experiments of densitometric measurements of Rac1 activity normalized to Rac1 expression and as fold over si-SCR; one-way ANOVA test. HEY cells with ET-1 were immunoprecipitated with anti-β-arr1 indicated proteins. mean±SD three independent experimentsof the average band intensity of IQGAP1 or RacGAP1 in β-arr1 IP normalized to unstimulated cells shownasfold of Ctr. Statistics were obtained one-way ANOVA Rac1-GTP si-RacGAP1 transfected HEY cells stimulated with ET-1 for 5 min. Pull down samples and inputs were analysed by WB for the indicated proteins. Histograms, the mean±SD of from three different experiments densitometric measurements of Rac1 activity maturation and increased MMP secretion/activation with a consequent increase of cell invasion.


Introduction
Among the different histological subtypes of ovarian cancer, ~90% include epithelial cancers and the most common diagnosed is serous ovarian cancer (SOC) [1]. Although in the last decade minimal improvement in mortality has been observed, 5-year overall survival is 25% for patients with stage III and stage IV cancer, highlighting the need to further explore molecular mechanisms underpinning invasive and metastatic behaviour and its relationship with the extent of disease [2]. Aggressive traits might be associated with the formation of invasive protrusions, invadopodia, characterized by the presence of a core of F-actin, associated with actin regulators (e.g., cortactin, cofilin), adaptor proteins (e.g., paxillin, TSK5), and matrix metalloproteases (MMP) (e.g., MMP-2, MMP-9, membrane type 1-MMP), which are surrounded by an adhesion ring containing adhesive components [3]. Invadopodia are activated in response to microenvironmental factors, such as extracellular matrix (ECM) stiffness or hypoxia, and growth factor receptor activation [4]. It has been previously demonstrated that integrins and adhesive components are essential to induce the formation of the invadopodial structures and that the dynamic changes of downstream signalling pathways are a prerequisite for a tight

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4 [16]. In SOC, the endothelin-1 (ET-1) axis, including the peptide ligand ET-1 and two Gprotein coupled receptors (GPCR) (ET A R and ET B R), is recognized as key regulator of tumor progression [17]. In SOC cells, autocrine activation of ET-1 receptors (ET-1R) is functionally involved in regulating cell-cell adhesion and communication, linked to epithelial to mesenchymal transition (EMT) and stem-cell like features, which dynamically regulate invasive and metastatic potential of these cells [17][18][19]. Concordantly, functional activation of ET B R enhances protumorigenic activity both promoting the evasion of the immune response, and tumor angiogenesis/lymphangiogenesis [17]. One interesting feature of ET-1R activation is the dynamic recruitment of the protein β-arrestin (β-arr), serving as ligandregulated adapter protein for selected intracellular proteins and the generation of cytosolic and nuclear "signalsomes" [20][21][22][23][24][25][26][27]. In particular, we identify β-arr1 as an integrator of ET-1driven cytoskeleton and signalling network regulating invadopodia in SOC cells. Indeed, in response to ET-1, β-arr1 acts as a linker of different proteins, coupling RhoC GTPase activation, cytoskeleton architecture changes and formation/activation of invadopodia [28,29].
Previous studies demonstratedthat IQGAP1 is an important player in coordinating the balance between actin polymerization and MT1-MMP secretion in invadopodia, by linking adhesion with the exocytic delivery of MT1-MMP [9,13]. Considering the interplay between IQGAP1 and upstream invadopodia signalling pathways, as Rho GTPases, focal adhesion components, and Arp2/3-N-WASP interacting complexes [30][31][32][33], in this study we delineate a network between β-arr1 and IQGAP1, as acto-adhesive and proteolytic signalling platform in ET-1R-driven invadopodia and ECM degradation in ovarian cancer.

ET-1 upregulates IQGAP1 expression in SOC cells.
To study the potential involvement of IQGAP1 and -arr1 interaction in ET-1R-driven invasive signalling, we analysed their expression in a panel of SOC cell lines, showing that IQGAP1 is expressed in 100% of these cells at both mRNA and protein levels (Fig. 1A, B), in parallel with -arr1, and confirmed by immunofluorescence (IF) analysis (Fig. 1C). To test the possibility that ET-1 axis could affect the expression of IQGAP1 in these cells, we analysed its expression upon ET-1 stimulation. Fig. 1D and E show a two fold increase of IQGAP1 expression, at mRNA and protein levels, with a maximum after 24 hours of ET-1 addition, indicating that IQGAP1 expression is regulated by ET-1 in SOC cells.

ET-1R activation promotes interaction of IQGAP1 with β-arr1 to regulate Rho
GTPases activity.

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A C C E P T E D M A N U S C R I P T 6 dependent manner, which are not observed after macitentan treatment (Fig. 3A). In addition, proximity ligation assays (PLA) between endogenous IQGAP1 and β-arr1 demonstrate that, in ET-1-dependent manner, these proteins interact each other and are in close proximity in both HEY and SKOV3 cells, establishing for the first time IQGAP1 as novel interactor of β-arr1 in ET-1-stimulated SOC cells ( Fig. 3B and Supplementary Fig.   1C and D). Moreover, by performing PLA experiments and counterstaining with specific markers of Golgi (GM130) and early endosome (Rab5), we found that none of these markers were found to label the IQGAP1/β-arr1 complexes (Supplementary Fig. 1C and D). On the other hand, we evaluated the effect of ET-1 on IQGAP1 localization in relation to F-actin. While plasma membrane localization of IQGAP1 is observed in control cells, ET-1 addition promotes the localization of IQGAP1 in areas of intense F-actin polymerization in dot-like structures, which is inhibited by macitentan treatment (Fig. 4), suggesting a putative involvement of IQGAP1 in the process of dynamic remodelling of cytoskeleton promoted by ET-1.
Then, we assessed the contribution of IQGAP1 as partner of β-arr1 in regulating dynamic activation of Rho GTPases. In SKOV3 and HEY cells, 2-fold-increase in RhoA activity and 2-and 3-fold increase in RhoC activity, respectively, is evident after ET-1 stimulation, which is inhibited by macitentan treatment, or either IQGAP1 or β-arr1 silencing ( Fig. 5A and Supplementary Fig. 2A). Moreover, overexpression of IQGAP1-GFP promotes their activation, which is further potentiated by the addition of ET-1 ( Supplementary Fig. 2B).
Addition of ET-1, in parallel to enhance RhoA/RhoC activation, decreases the activity of Rac1 of about 40%, while deletion of either IQGAP1 and β-arr1 restores its activity ( Fig.   5B and Supplementary Fig. 3A,B), demonstrating that β-arr1/IQGAP1 complex is required for the fine regulation of ET-1-induced RhoA,C and Rac1 signalling in SOC cells. Previous

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A C C E P T E D M A N U S C R I P T 7 works reported RacGAP1 as a binding partner of IQGAP1 mediating Rac1 deactivation [44]. To test the possibility that RacGAP1 could act in our system as a complex with β-arr1 and IQGAP1, we performed IP experiments in HEY and SKOV3 cells and report a 3-and 2-fold enhanced association between RacGAP1, IQGAP1 and β-arr1 after ET-1 stimulus, respectively, which is impaired by macitentan treatment (Fig. 5C and Supplementary Fig.   3C). CSLM analysis shows that in ET-1-dependent manner RacGAP1 is recruited to β-arr1 ( Supplementary Fig. 3D). Moreover, RacGAP1 silencing significantly impairs ET-1dependent Rac1 deactivation and RhoA/C activity (Fig. 5D, E). These data demonstrate that ET-1 promotes the interaction of RacGAP1 with IQGAP1/β-arr1, leading to suppression of Rac1 activity and activation of RhoA/C.

ET-1R axis regulates invadopodia activity through IQGAP1/β-arr1 complex.
Since input from adhesion signalling is a critical regulatory event to invadopodia formation and IQGAP1 is an integrator of the cytoskeleton and cell adhesion machinery [6,7], we Since enhanced secretion and activity of MMPs is a key feature of the ET-1-induced invasive phenotype of SOC cells [45], we performed gelatin zymography analyses.
Then, we assessed the effect of ET-1-driven β-arr1/IQGAP1 network on SOC degradative and invasive capability in a 3D cell culture system using collagen matrix-embedded tumor spheroids derived from SOC cells, in which tumor cells are organized into a 3D structure mimicking a tumor micro-region or a micro-metastasis. In spheroid cultures from SKOV3 cells, ET-1 addition results in enhanced invasion into the surrounding matrix and radial outgrowth compared to control cells, which is impaired by silencing of IQGAP1 or β-arr1,

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A C C E P T E D M A N U S C R I P T 9 as well as by macitentan or Ilomastat treatment ( Fig 7B). In addition, 3D spheroid cells,
To mimic aspects of SOC metastatic dissemination, we tested the effects of ET-1R blockade with macitentan in metastatic behaviour and molecular effectors of invadopodia by performing intraperitoneal (i.p.) injection. Mice in the control group show evident dissemination pattern of SKOV3 cells on the peritoneal surfaces, intestines, omentum, small bowel, liver, and spleen and ovaries. The average of i.p. nodules is significantly reduced in macitentan-treated mice, associated with awell-tolerated toxicity profile.
Consistent with results obtained in vitro, in i.p. nodules from macitentan-treated mice, the inhibition of IQGAP1, vinculin, MT1-MMP expression, as well as phosphorylation of

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cortactin is observed ( Fig. 8B and C). These results indicate that the therapeutic efficacy of macitentan to control ET-1R/β-arr1-driven SOC progression relies also on its ability to reduce critical regulators of invadopodia, such IQGAP1.
To determine the clinical relevance of mRNA expression of IQGAP1alone and in combination with ET A R (EDNRA) and β-arr1 (ARBB1), we investigated the prognostic significance in ovarian cancer patients using the KM plotter [47]. As shown in Fig. 8D (Fig. 8D), supporting the possibility that EDNRA/IQGAP1/ARRB1 could be used as prognostic biomarkers in SOC.

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11

Discussion
We have identified a novel mechanism that coordinates invasive pathways downstream of ET-1R, in which the scaffolding protein IQGAP1 integrates signal output from this GPCR to invadopodia function in SOC cells by linking β-arr1 (Fig. 8E). This conclusion is based on the following major findings: 1. IQGAP1 interacts with β-arr1 in ET-1-stimulated SOC cells; 2. Engagement of IQGAP1 by β-arr1 favours the tight control of RhoA/C activity over Rac1, thereby coordinating invadopodia function and ECM degradation; 3. Interruption of ET-1R/IQGAP1/β-arr1 network impairs invasive and metastatic behaviour; 4. Concomitant high expression of EDNRA/ARRB1/IQGAP1 is a negative prognostic factor in ovarian cancer patients.
Invadopodia are very dynamic membrane protrusions, whose function is to degrade components of ECM, enabling invasion and metastasis [3,4,46,47]. To make a functionally active invadopodia, local concentration of ligands for motogenic receptors, such as GPCR, acts as input for invadopodia assembly and maturation. Among wellrecognized drivers of invadopodia, the ET-1R signalling is a crucial inducer, in which β-arr1 is instrumental for bringing and assembling cytoskeleton and signalling modules that direct the degradative activity at invadopodia [28,29]. Indeed, ET-1R/β-arr1 core is necessary to assemble elements for invadopodia formation, as cortactin and TSK5, as well as regulating invadopodia maturation [28,29]. However, a deep understanding of how protein complexes, including effectors of invasive signalling, are assembled into a functional unit to enhance specific signalling pathways after ET-1R cue is not reached. Emerging data indicate that the interactions between scaffold proteins might determine the formation of unique signalosome to provide spatio-temporal organization of events in actin cytoskeleton signalling and function, and in this context IQGAP1 is considered a master signalosome for the plethora of IQGAP1-interacting proteins [15,16].

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12 According with previous data demonstrating the involvement of IQGAP1 in the progression and spread of ovarian cancer, and the role of IQGAP1 as potentially independent molecular predictor of highly aggressive tumors [12], our findings reveal a new mechanistic link between IQGAP1 and the progression of SOC, demonstrating that IQGAP1 is a new interactor of β-arr1 downstream of ET-1R signalling in shaping cytoskeleton remodelling, and invadopodia-dependent ECM degradation. It is conceivable that the interaction between IQGAP1 and β-arr1 might expand the potential of cross-talk between signalling cascades, highlighting an additional complexity in the molecular mechanism by which ET- In order to highlight new effective biomarkers for the early prognosis of SOC progression, we previously demonstrated that overexpression of ET A R in SOC patients is associated with chemoresistance, EMT marker expression, and poor prognosis [18,19], indicating biological relevance of ET-1R as prognostic factor. The findings that high expression levels of EDNRA/ARRB1/IQGAP1 positively correlate with poor prognosis, further validate the clinical implication of these predictive markers in early prognosis of metastatic ovarian cancer patients.  plasmid, respectively. The plasmid pEGFP-IQGAP1 was a gift from David Sacks (Addgene plasmid #30112) and the full-length β-arr1-AU5 plasmid was kindly provided by Professor

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Richard D Ye (Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL, USA).

Western Blotting (WB) and immunoprecipitation (IP)
For WB analysis, cells were lysed as previously described [29] and resolved on Mini-PROTEAN TGX gels (Biorad Laboratories). Immunoblotting was performed using the  The PCR products were analysed by electrophoresis on 1% agarose gel and visualized by using ChemiDoc Imaging System and ImageLab Software (Biorad Laboratories).

Gelatin zimography
Conditioned media of untreated or treated cells were collected and analysed as previously reported [29].

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20 overnight. From the cell-pellet, recombinant protein was purified using the MagneGST Protein Purification System (Promega), according to the manufacturer's instructions.
For GST-pull down assay, cell lysate was incubated with GST-β-arr1-beads for 3 hours at 4°C. After washes with PBS 1X, proteins were eluted in Leammli 2X by heating to 95°C for 5 min. Proteins were analysed by WB and detection of IQGAP1 or GST-β-arr1 was performed using IQGAP1 and GST (cat. no. SC-138 Santa Cruz) Abs.

3D-invasion assay
3D invasion assay was performed using Cultrex 3-D Spheroid Cell Invasion Assay (#3500-096-K) according to the manufacturer's instructions. SKOV3 cells spheroids were generated by plating 3000 cells for 48 hours in 3D Culture Qualified 96 Well Plate. Then, spheroids were embedded into invasion matrix. After 1 hour at 37°C, serum-free media with or without ET-1 and/or MAC and/or Ilomastat was added to spheroids wells. Plates were incubated for 72 hours and all spheroids were photographed using a phase-contrast microscope at X4 magnification. Quantification of invasion area and cumulative sprout length was performed using Image J Software.

Transendothelial migration assay
For the trans-endothelial migration, HUVEC were seeded (1×10 5 cells) in 8.0 μm pore sized membranes BD BioCoat growth factor reduced Matrigel Invasion Chamber (BD Biosciences) and left to form a monolayer for 24 hrs at 37°C. Cells were analysed as previously described [29].

In vivo assay
To mimic some aspects of SOC seeding on the peritoneal surfaces, representing typical metastatic sites observed in patients with an advanced stage of disease, female athymic (nu+/nu+) mice, 4-6 weeks of age (Charles River Laboratories, Milan, Italy) were injected

Kaplan-Meyer analysis
The Kaplan-Meier plotter (http://kmplot.com/analysis/) [49] was used to investigate the correlation between combined expression of IQGAP1, ARRB1 and EDNRA mRNA levels Subsequently, PFS for the two groups were compared with a Kaplan-Meier survival plot on the webpage (http://kmplot.com/analysis/index.php?p = service&cancer = ovar). The ovarian cancer patients were followed up to 5 years. Hazard ratio (HR), 95% confidence intervals, and log rank P were presented on the main plots. P value of < 0.05 was considered to be statistically significant.

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22 Statistical analyses were performed with the GraphPad Prism (GraphPad Software) and the values represent mean ±SD obtained with not less than three independent experiments with similar results. The statistical significance of the differences was determined by the Student t-test for the comparisons between two groups; for more than two groups, the 1-way ANOVA analysis of variance was used.
For in vivo studies, a priori power analysis was performed using the G*Power statistical analysis program [50,51]. By assuming two tails with normal distribution between two groups, calculations revealed that a sample size of n=8 would be sufficient to provide 95% power with 0.05 %-error probability and an effect size of 2.   and cultured with/without ET-1 for 60 min, were immunoprecipitated with anti-AU5 Ab. IP and inputs were subjected to WB for IQGAP1-GFP or β-arr1-AU5 expression. Data

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31 represent the mean±SD from three independent experimentsof the average band intensity of GFP-IQGAP1 in AU5-β-arr1 IP normalized to unstimulated cells (Ctr) and shown as fold of Ctr. Statistics were obtained using the Student's t-test.