The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. The identity and role of cell surface molecules driving complex biological events leading to HCC progression are poorly understood, hence representing major lacunae in HCC therapies. Here, combining SILAC quantitative proteomics and biochemical approaches, we uncover a critical oncogenic role of Agrin, which is overexpressed and secreted in HCC. Agrin enhances cellular proliferation, migration and oncogenic signalling. Mechanistically, Agrin’s extracellular matrix sensor activity provides oncogenic cues to regulate Arp2/3-dependent ruffling, invadopodia formation and epithelial–mesenchymal transition through sustained focal adhesion integrity that drives liver tumorigenesis. Furthermore, Agrin signalling through Lrp4-muscle-specific tyrosine kinase (MuSK) forms a critical oncogenic axis. Importantly, antibodies targeting Agrin reduced oncogenic signalling and tumour growth in vivo. Together, we demonstrate that Agrin is frequently upregulated and important for oncogenic property of HCC, and is an attractive target for antibody therapy.

Kaposi's sarcoma-associated herpesvirus (KSHV), etiologically associated with Kaposi's sarcoma, uses integrins (α3β1, αVβ3, and αVβ5) and associated signaling to enter human dermal microvascular endothelial cells (HMVEC-d), an in vivo target of infection. KSHV infection activated c-Cbl, which induced the selective translocation of KSHV into lipid rafts (LRs) along with the α3β1, αVβ3, and xCT receptors, but not αVβ5. LR-translocated receptors were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to clathrin-mediated entry that was targeted to lysosomes. Because the molecule(s) that integrate signal pathways and productive KSHV macropinocytosis were unknown, we immunoprecipitated KSHV-infected LR fractions with anti-α3β1 antibodies and analyzed them by mass spectrometry. The tyrosine kinase EphrinA2 (EphA2), implicated in many cancers, was identified in this analysis. EphA2 was activated by KSHV. EphA2 was also associated with KSHV and integrins (α3β1 and αVβ3) in LRs early during infection. Preincubation of virus with soluble EphA2, knockdown of EphA2 by shRNAs, or pretreatment of cells with anti-EphA2 monoclonal antibodies or tyrosine kinase inhibitor dasatinib significantly reduced KSHV entry and gene expression. EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. EphA2 shRNA ablated macropinocytosis-associated signaling events, virus internalization, and productive nuclear trafficking of KSHV DNA. Taken together, these studies demonstrate that the EphA2 receptor acts as a master assembly regulator of KSHV-induced signal molecules and KSHV entry in endothelial cells and suggest that the EphA2 receptor is an attractive target for controlling KSHV infection.virus entry | productive endocytosis | receptor tyrosine kinase