The α-arrestin domain containing protein-3 (ARRDC3) is a tumor suppressor in triple-negative breast carcinoma (TNBC), a highly metastatic subtype of breast cancer which lacks targeted therapies. Thus, understanding the mechanisms and targets of ARRDC3 in TNBC is important. ARRDC3 regulates trafficking of protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis. Loss of ARRDC3 causes overexpression of PAR1 and aberrant signaling. Moreover, dysregulation of GPCR-induced Hippo signaling is associated with breast cancer progression. However, the mechanisms responsible for Hippo dysregulation remain unknown. Here, we report that the Hippo pathway transcriptional co-activator TAZ is the major effector of GPCR signaling and is required for TNBC migration and invasion. Additionally, ARRDC3 suppresses PAR1-induced Hippo signaling via sequestration of TAZ, which occurs independent of ARRDC3-regulated PAR1 trafficking. The ARRDC3 C-terminal PPXY motifs and TAZ WW domain are critical for this interaction and required for suppression of TNBC migration and lung metastasis in vivo. These studies are the first to demonstrate a role for ARRDC3 in regulating GPCR-induced TAZ activity in TNBC and reveal multi-faceted tumor suppressor functions of ARRDC3.

BackgroundThe oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.MethodsIn our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex.ResultsLoss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype.ConclusionsThe small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment.Electronic supplementary materialThe online version of this article (10.1186/s12885-019-5454-2) contains supplementary material, which is available to authorized users.

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Aberrant G protein‐coupled receptor (GPCR) signaling drives cancer initiation and progression, but the mechanism underlying dysregulated GPCR signaling is unknown and represents a major gap in knowledge. Protease‐activated receptor‐1 (PAR1) is proteolytically activated and requires lysosomal degradation for signal termination. PAR1 overexpression correlates with high tumor grade and poor prognosis in breast cancer patients. Increased PAR1 expression results from altered receptor trafficking, which causes persistent signaling and increased invasiveness. We recently discovered that loss of the tumor suppressor α‐arrestin domain‐containing protein‐3 (ARRDC3) is responsible for dysregulated PAR1 trafficking, persistent signaling and breast cancer invasion. This work establishes for the first time a link between ARRDC3 and GPCR dysfunction in cancer. However, it is not known how ARRDC3 alters the dynamics of PAR1 signaling via receptor trafficking or if other determinants are important. We found that ARRDC3 regulates PAR1‐induced non‐canonical endosomal p38α MAPK signaling, but not PAR1‐rapid initiation of Gα12/13‐RhoA signaling from the plasma membrane. Interestingly, ARRDC3 interacts with NEDD4 ubiquitin E3 ligases via C‐terminal PPXY motifs, which is required for agonist‐induced PAR1 degradation, but not essential for regulation of p38α MAPK signaling. However, deletion of the C‐terminus tail abrogated both processes, suggesting that an additional determinant within the C‐terminustail distinct from the PPxY motifs is also important for mediating ARRDC3 function. In addition, PAR1‐stimulated invasion is dependent on p38a MAPK and regulated by ARRDC3, which requires the C‐terminal domain but is only partially suppressed by ARRDC3 PPXY mutant. We further discovered a novel phosphorylated Ser/Thr cluster in the C‐terminus that is critical for ARRDC3 function including modulation of ARRDC3 homo‐oligomerization and basal conformation. Meta‐analysis of breast cancer patients indicates that ARRDC3 and genes involved in PAR1‐activated p38α MAPK are associated with poor prognosis in the aggressive subtype of breast cancer patients. Currently, we are determining the molecular mechanisms by which phospho‐Ser/Thr cluster effects ARRDC3 conformation and function. In conclusion, this work has provided critical insight that improves our understanding of ARRDC3 function in spatiotemporal regulation of GPCRs signaling in invasive breast carcinoma, which may provide new therapeutic options for treating metastatic breast cancer. Support or Funding Information NIH/NIGMS R35 GM127121

G protein‐coupled receptors (GPCRs) are the largest family of cell surface receptors and are known to play key roles in human health and disease, such as cancer. However, while they have proven to be highly druggable, GPCRs are widely underutilized as drug targets in cancer. In cancer, although GPCR signaling is known to be altered, GPCRs themselves are rarely mutated, and therefore it is important that we better understand how GPCR signaling is regulated. Protease‐activated receptor‐1 (PAR1), the GPCR for thrombin, has been shown to be a key driver of invasive breast cancer progression. We recently demonstrated that PAR1 is regulated by arrestin domain‐containing protein‐3 (ARRDC3), a member of the newly discovered family of α‐arrestins. Humans express both β‐ and α‐arrestins, and while these families share very little sequence homology, they are predicted to be structurally similar, and therefore may be subject to similar regulatory mechanisms. However, while GPCR regulation of β‐arrestin function has been well characterized, it remains entirely unknown how GPCRs might regulate α‐arrestins, such as ARRDC3. Here, by using a variety of biochemical and microscopy‐based techniques, we show that PAR1 may regulate ARRDC3 through modulation of post‐translational modifications, including ubiquitination, and that identified ARRDC3 regulatory domains may play a role in its subcellular localization. We hypothesize that PAR1‐mediated regulation of ARRDC3 is critical for normal ARRDC3 function. We will use a variety of approaches to elucidate the role of ARRDC3 regulatory mechanisms in its function in regulating PAR1 signaling and trafficking and breast cancer progression. The results of these studies will advance our understanding of how PAR1 signaling is regulated and may lead to identification of novel targets for therapeutic development to treat breast cancer.