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Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer (1-5). Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple aKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation (6-10). However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs (4,5), cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional linkbetween IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy., Gain-of-function IDH1/IDH2 mutations occur in ~25% of IHCCs (1,3-5), a liver malignancy that exhibits bile duct differentiation, but have not been identified in hepatocellular carcinomas (HCCs), which exhibit hepatocyte differentiation [...]

Identification of effective targeted therapies for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) remains an unmet medical need. A patient with platinum‐refractory recurrent oral cavity HNSCC underwent comprehensive genomic profiling (CGP) that identified an activating MET mutation (R1004). The patient was treated with the oral MET tyrosine kinase inhibitor crizotinib with rapid response to treatment. Based on this index case, we determined the frequency of MET alterations in 1,637 HNSCC samples, which had been analyzed with hybrid capture‐based CGP performed in the routine course of clinical care. The specimens were sequenced to a median depth of >500× for all coding exons from 182 (version 1, n = 24), 236 (version 2, n = 326), or 315 (version 3, n = 1,287) cancer‐related genes, plus select introns from 14 (version 1), 19 (version 2), or 28 (version 3) genes frequently rearranged in cancer. We identified 13 HNSCC cases (0.79%) with MET alterations (4 point mutation events and 9 focal amplification events). MET‐mutant or amplified tumors represent a small but potentially actionable molecular subset of HNSCC. Key Points: This case report is believed to be the first reported pan‐cancer case of a patient harboring a MET mutation at R1004 demonstrating a clinical response to crizotinib, in addition to the first documented case of head and neck squamous cell carcinoma (HNSCC) with any MET alteration responding to crizotinib.The positive response to MET inhibition in this patient highlights the significance of comprehensive genomic profiling in advanced metastatic HNSCC to identify actionable targetable molecular alterations as current treatment options are limited. Effective therapies for advanced head and neck squamous cell carcinoma (HNSCC) are lacking. This article presents what is believed to be the first reported case of a patient harboring a MET mutation who showed a clinical response to crizotinib, highlighting the benefits of comprehensive genomic profiling in HNSCC. [ABSTRACT FROM AUTHOR]

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration. The microtubules (MTs) provide tracks for long‐range intracellular transport, which requires the assistance of the MT plus‐end tracking proteins (+TIPs). Our study conclusively demonstrates that in response to HGF, the spatially restricted transport of the Met RTK to the cell cortex of migrating cells, occurs via an MT‐directed mechanism. We identified a novel interaction between the +TIP, CLIP‐170, and Met via the adaptor GGA3 and demonstrate that the CLIP‐170‐dependent regulation of Met vesicle transport to MT plus‐ends is required for HGF‐dependent cell migration. [ABSTRACT FROM AUTHOR]

Summary Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach. [ABSTRACT FROM AUTHOR]

The transport and sorting of cell surface receptors into membrane-bound intracellular compartments is crucial for cellular homeostasis. Defects in receptor trafficking are associated with several diseases, including cancer. Recent advances in our understanding of mechanisms that control receptor trafficking have highlighted the involvement of membrane trafficking in cell signaling, as well as in biological processes, including cell migration and invasion. In this review, we summarize current knowledge of how cargos, focusing on receptor tyrosine kinases (RTKs) and integrins, are dynamically transported through the endosomal pathway for recycling, and how this promotes spatially restricted signaling microdomains associated with distinct biological responses. We discuss mechanisms through which dysregulation of membrane trafficking contributes to tumorigenesis and potential therapeutic approaches. [Copyright &y& Elsevier]

Summary: Cells are dependent on correct sorting of activated receptor tyrosine kinases (RTKs) for the outcome of growth factor signaling. Upon activation, RTKs are coupled through the endocytic machinery for degradation or recycled to the cell surface. However, the molecular mechanisms governing RTK recycling are poorly understood. Here, we show that Golgi-localized gamma ear-containing Arf-binding protein 3 (GGA3) interacts selectively with the Met/hepatocyte growth factor RTK when stimulated, to sort it for recycling in association with “gyrating” clathrin. GGA3 loss abrogates Met recycling from a Rab4 endosomal subdomain, resulting in pronounced trafficking of Met toward degradation. Decreased Met recycling attenuates ERK activation and cell migration. Met recycling, sustained ERK activation, and migration require interaction of GGA3 with Arf6 and an unexpected association with the Crk adaptor. The data show that GGA3 defines an active recycling pathway and support a broader role for GGA3-mediated cargo selection in targeting receptors destined for recycling. [Copyright &y& Elsevier]

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4- acetamido-4′-isothiocyabatostilbene-2′,2- disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation. [ABSTRACT FROM AUTHOR]

Down-regulation of receptor tyrosine kinases (RTK) through receptor internalization and degradation is critical for appropriate biological responses. The hepatocyte growth factor RTK (also known as Met) regulates epithelial remodeling, dispersal, and invasion and is deregulated in human cancers. Impaired down-regulation of the Met RTK leads to sustained signaling, cell transformation, and tumorigenesis, hence understanding mechanisms that regulate this process is crucial. Here we report that, following Met activation, the endocytic adaptor protein, Eps15, is recruited to the plasma membrane and becomes both tyrosine-phosphorylated and ubiquitinated. Recruitment of Eps15 requires Met receptor kinase activity and involves two distinct Eps15 domains. Unlike previous reports for the EGF RTK, which requires the EpslS ubiquitin interacting motif, recruitment of Eps15 to Met involves the coiled-coil domain of Eps15 and the signaling adaptor molecule, Grb2, which binds through a proline-rich motif in the third domain of Eps15. Expression of the coiled-coil domain is sufficient to displace the wild-type Eps15 protein complex from Met, resulting in loss of tyrosine phosphorylation of Eps15. Knockdown of Eps15 results in delayed Met degradation, which can be rescued by expression of Eps15 WT but not an Eps15 mutant lacking the coiled-coil domain, identifying a role for this domain in Eps15-mediated Met down-modulation. This study demonstrates a new mechanism of recruitment for Eps15 downstream of the Met receptor, involving the coiled-coil domain of Eps15 as well as interaction of Eps15 with Grb2. This highlights distinct regulation of Eps15 recruitment and the diversity and adaptability of endocytic molecules in promoting RTK trafficking. [ABSTRACT FROM AUTHOR]

Three mouse killer immunoglobulin-like receptors (KIRs), namely, KIR3DL1, KIRL1, and KIRL2, have recently been identified in C56BL/6 (B6) mice. However, only two Kir genes are found in the B6 mouse genome sequence data base. To clarify this discrepancy, we cloned Kir cDNAs from multiple strains of mice. Sequencing of the cDNA clones showed that the Kir3dl1 gene is found in C3H/HeJ and CBA/J but not in B6 mice. Analysis of the single nucleotide polymorphism data base suggested that Kir3dl1 is the C3H/HeJ and CBA/J allele of Kirl1. We generated mAb to the recombinant KIRL1 protein to investigate its expression pattern. The anti-KIRL1 mAb bound to NK1.1+ T cells but only very weakly or at undetectable levels to other lymphocytes including natural killer (NK) cells and conventional T cells. Among NK1.1+ T cells, conventional NK T cells stained with CD1d tetramer did not significantly bind anti-KIRL1 mAb, whereas CD1d-tetramer-negative subset was KIRL1-positive. Furthermore, the expression of KIRL1 is readily detected on NK1.1+ T cells from β2-microglobulin-deficient B6 mice. Thus, KIRL1 is predominantly expressed on CD1d-independent NK1.1+ T cells. [ABSTRACT FROM AUTHOR]