The endothelin A receptor and epidermal growth factor receptor signaling converge on β-catenin to promote ovarian cancer metastasis

Aims: Endothelin A receptor (ETAR) and epidermal growth factor receptor (EGFR) cross-talk enhances the metastatic potential of epithelial ovarian cancer (EOC) cells activating different pathways, including β-catenin signalling. Here, we evaluated β-catenin as one of ETAR/EGFR downstream pathway in the invasive behaviour of EOC cells and their therapeutic potential to co-target ETAR and EGFR. Main methods: The phosphorylation status and interactions of different proteins were analysed by immunoblotting and immunoprecipitation. Reporter activity and RT-PCR was used for evaluation of β-catenin transcriptional activity and gene expression. Functional effects were evaluated by gelatin zymography and cell invasion assays. An orthotopic model of metastatic human EOC in mice was used for in vivo studies. Key findings: In EOC cell lines, ET-1 induced Src-dependent EGFR transactivation, causing tyrosine (Y) phosphorylation of β-catenin at the residue Y654, its dissociation from E-cadherin complexes and the accumulation as an active form. This pool of Tyr-β-catenin relocalised to the nucleus promoting its transcriptional activity, and the expression of its target genes, such as MMP-2. At functional level, ET-1 and EGFR circuits enhanced protease activity and cell invasion. All these effects were significantly inhibited by the ETAR antagonist, zibotentan, or EGFR inhibitor, gefitinib, and are completely blocked by co-addition of both drugs. In vivo, zibotentan treatment significantly inhibited metastases, associated with reduced expression and activation of MMPs and active β-catenin, especially when combined with gefitinib. Significance: Altogether these findings provide additional support to the potential use of ETAR and EGFR blockade as a new therapeutic opportunity for EOC treatment. © 2012 Elsevier Inc

Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression

Hypoxia-inducible factor-1α (HIF-1α) mediates the response to hypoxia or other stimuli, such as growth factors, including endothelin-1 (ET-1), to promote malignant progression in numerous tumors. The importance of cofactors that regulate HIF-1α signalling within tumor is not well understood. Here we elucidate that ET-1/ET(A) receptor (ET(A)R)-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signalling. In epithelial ovarian cancer (EOC) cells, ET-1/ET(A)R axis induced vascular-endothelial growth factor (VEGF) expression through HIF-1α nuclear accumulation. In these cells, activation of ET(A)R by ET-1, by mimicking hypoxia, promoted the nuclear interaction between β-arr1 and HIF-1α and the recruitment of p300 acetyltransferase to hypoxia response elements on the target gene promoters, resulting in enhanced histone acetylation, and HIF-1α target gene transcription. Indeed, β-arr1-HIF-1α interaction regulated the enhanced expression and release of downstream targets, such as ET-1 and VEGF, required for tumor cell invasion and pro-angiogenic effects in endothelial cells. These effects were abrogated by β-arr1 or HIF-1α silencing or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. Interestingly, ET(A)R/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit involved in invasive and angiogenic behaviors. In a murine orthotopic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation. Collectively, these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ET(A)R signalling, mediating epigenetic modifications directly involved in the metastatic process, and suggest that targeting ET-1-dependent β-arr1/HIF-1α pathway by using macitentan may impair EOC progression

β-Arrestin 1 is required for endothelin-1-induced NF-κB activation in ovarian cancer cells

Aims In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signalling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion and metastasis. These effects are dependent by the activation of critical signalling pathways, such as MAPK, Akt, and β-catenin, through specific cytosolic and nuclear scaffolding functions of β-arrestin 1 (β-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through β-arr-1 recruitment. Main methods We used cultured HEY EOC cells cultured in the presence or absence of ET-1 and the ETAR antagonist BQ123. The phosphorylation of p65 and Iκ-Bα was evaluated by immunoblotting analysis. The interaction between p65 and β-arr1 was evaluated by immunoprecipitation experiments in nuclear extracts. NF-κB promoter activity was evaluated by transfection with NF-κB-driven luciferase reporter construct. Assessment of the function of β-arr1 was achieved by β-arr1 silencing with shRNA and expression of β-arr1-FLAG expression vector. Key findings In EOC cells, ET-1 promotes the phosphorylation of p65 subunit and the cytoplasmic inhibitor IκB that in turn led to increased NF-κB transcriptional activity. These effects were inhibited by the use of BQ123, as well as by β-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of β-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and β-arr1 indicates a nuclear function of β-arr-1 in ETAR-driven NF-κB transcriptional activity. Significance Altogether these findings reveal a previously unrecognized pathway that depends on β-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer

Endothelin-1 axis fosters YAP-induced chemotherapy escape in ovarian cancer

The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, β-arrestin1 (β-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ETAR/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients

Blocking endothelin-1-receptor/\u3b2-catenin circuit sensitizes to chemotherapy in colorectal cancer

The limited clinical response to conventional chemotherapeutics observed in colorectal cancer (CRC) may be related to the connections between the hyperactivated \u3b2-catenin signaling and other pathways in CRC stem-like cells (CRC-SC). Here, we show the mechanistic link between the endothelin-1 (ET-1)/ET-1 receptor (ET-1R) signaling and \u3b2-catenin pathway through the specific interaction with the signal transducer \u3b2-arrestin1 (\u3b2-arr1), which initiates signaling cascades as part of the signaling complex. Using a panel of patient-derived CRC-SC, we show that these cells secrete ET-1 and express ETAR and \u3b2-arr1, and that the activation of ETAR/\u3b2-arr1 axis promotes the cross-talk with \u3b2-catenin signaling to sustain stemness, epithelial-to-mesenchymal transition (EMT) phenotype and response to chemotherapy. Upon ETAR activation, \u3b2-arr1 acts as a transcription co-activator that binds \u3b2-catenin, thereby promoting nuclear complex with \u3b2-catenin/TFC4 and p300 and histone acetylation, inducing chromatin reorganization on target genes, such as ET-1. The enhanced transcription of ET-1 increases the self-sustained ET-1/\u3b2-catenin network. All these findings provide a strong rationale for targeting ET-1R to hamper downstream \u3b2-catenin/ET-1 autocrine circuit. Interestingly, treatment with macitentan, a dual ETAR and ETBR antagonist, able to interfere with tumor and microenvironment, disrupts the ET-1R/\u3b2-arr1-\u3b2-catenin interaction impairing pathways involved in cell survival, EMT, invasion, and enhancing sensitivity to oxaliplatin (OX) and 5-fluorouracil (5-FU). In CRC-SC xenografts, the combination of macitentan and OX or 5-FU enhances the therapeutic effects of cytotoxic drugs. Together, these results provide mechanistic insight into how ET-1R coopts \u3b2-catenin signaling and offer a novel therapeutic strategy to manage CRC based on the combination of macitentan and chemotherapy that might benefit patients whose tumors show high ETAR and \u3b2-catenin expression

β-arrestin1/YAP/mutant p53 protein complex orchestrates endothelin A receptor-driven metastatization and platinum response in ovarian cancer

The limited clinical response to platinum-based therapeutics observed in high-grade serous ovarian cancer (HG-SOC), characterized by high frequency of recurrence and TP53 mutations, might be related to oncogenic pathway network. The activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling, through the ability of the scaffold protein β-arrestin1 (β-arr1) to interact with a broad repertoire of signaling proteins, integrates several pathways involved in a plethora of tumor functions. Here, we show that the Hippo effector Yes-associated protein (YAP) works as crucial signal transducer to mediate ETAR/β-arr1 signaling during HG-SOC growth, metastasis and platinum response. In patient-derived HG-SOC cells and platinum sensitive and resistant OC cell lines, we demonstrate that ET-1R activation promotes YAP and TAZ de-phosphorylation and to a greater extent in platinum-resistant OC cells. Of note, ET-1R through β-arr1, engaging a physical interaction with the novel partner YAP, triggers its cytoplasmic-nuclear shuttling. In parallel, β-arr1 mediates the ET-1R-induced Trio/RhoA/actin cytoskeleton-dependent YAP nuclear accumulation. Mechanistically, downstream of ETAR signaling, nuclear β-arr1 and YAP bind mutant p53 (mutp53), forming a competent transcriptional complex consisting of β-arr1/YAP/mutp53/TEAD. This leads to the aberrant transcriptional activation of YAP/TEAD target genes, including EDN1 (ET-1 gene), thereby fueling a feed-forward loop that ensures persistent signals sustaining aggressive traits. ET-1R inactivation, by using the dual ET-1R antagonist macitentan, shuts down β-arr1-mediated YAP/mutp53/TEAD transcriptional program, reduces tumor growth, metastatic dissemination and enhances the sensitivity to chemotherapy in the clinical model of patient-derived HG-SOC xenografts (PDX). Of clinical relevance, the combined expression of ETAR/β-arr1/YAP gene signature correlates with a worst prognosis in HG-SOC patients, carrying TP53 mutations. Altogether, these findings identify YAP as a central mediator of ETAR/β-arr1 signaling, thereby providing mechanistic and therapeutic insights to the targeting of the ETAR/β-arr1/YAP/mutp53 complex warranting the repurposing of macitentan for combinatorial treatment of HG-SOC

Clusterin, a gene enriched in intestinal stem cells, is required for L1-mediated colon cancer metastasis

Hyperactive Wnt signaling is a common feature in human colorectal cancer (CRC) cells. A central question is the identification and role of Wnt/β-catenin target genes in CRC and their relationship to genes enriched in colonic stem cells, since Lgr5+ intestinal stem cells were suggested to be the cell of CRC origin. Previously, we identified the neural immunoglobulin-like adhesion receptor L1 as a Wnt/β-catenin target gene localized in cells at the invasive front of CRC tissue and showed that L1 expression in CRC cells confers enhanced motility and liver metastasis. Here, we identified the clusterin (CLU) gene that is also enriched in Lgr5+ intestinal stem cells, as a gene induced during L1-mediated CRC metastasis. The increase in CLU levels by L1 in CRC cells resulted from transactivation of CLU by STAT-1. CLU overexpression in CRC cells enhanced their motility and the reduction in CLU levels in L1 overexpressing cells suppressed the ability of L1 to confer increased tumorigenesis and liver metastasis. Genes induced during L1-mediated CRC cell metastasis and enriched in intestinal stem cells might be important for both CRC progression and colonic epithelium homeostasis

Endothelin-1/endothelin A receptor axis activates RhoA GTPase in epithelial ovarian cancer

AIMS: The endothelin-1 (ET-1)/ET A receptor (ETAR) signaling pathway is critical driver of epithelial ovarian cancer (EOC) progression. Emerging evidences demonstrate that the scaffolding protein β-arrestin-1 (β-arr1) downstream of ETAR guides cell motility, although the signaling pathways by which ETAR activation controls these process are not well understood. Here, we set out to molecularly dissect whether RhoA GTPase activation is a mediator of ET-1 signaling controlling EOC cell migration. MAIN METHODS: We cultured EOC cell lines (HEY, SKOV3, OVCAR, A2780 and 2008) with ET-1 and the ET-1R antagonist macitentan. RhoA expression was evaluated by RT-PCR. Activation of RhoA and ROCK1 was evaluated by pull down and kinase assays, respectively. Cell motility was evaluated by chemotaxis and wound healing assays, in untrasfected cells by using ROCK chemical inhibitors, Y-27632 or Fasudil, or in cells after transfection with dominant negative RhoA construct. The phosphorylation of myosin light chain 2 (MLC2) was evaluated by immunoblotting. Pseudopodia formation was evaluated by a pseudopodia kit assay. KEY FINDINGS: In EOC cells, ET-1 activates RhoA and downstream ROCK1 and MLC2. These effects were inhibited by β-arr1 silencing, suggesting that ET-1/ETAR regulate RhoA signaling through β-arr1. At functional level, the activation of RhoA/ROCK signaling led to enhanced cell migration and pseudopodia formation. The suppressive effect of the ROCK inhibitors, as well as of macitentan, demonstrates that RhoA is involved in ET-1/ETAR-induced cell migration. SIGNIFICANCE: Altogether these findings reveal a new pathway that depends on β-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC