Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells.

PURPOSE: Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation. EXPERIMENTAL DESIGN: Using single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics was used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and non-tumor cells within the metastatic microenvironment. RESULTS: Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. CONCLUSIONS: Fibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a pro-metastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the anti-fibrotic TKI nintedanib. Our data shed light on the non-cell autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models.

Vesicular trafficking plays a role in centriole disengagement and duplication.

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair.  During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle.  Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.

Control of mitochondrial homeostasis by endocytic regulatory proteins.

Mitochondria play essential roles in cellular energy processes, including ATP production, control of reactive oxygen species (ROS) and apoptosis. While mitochondrial function is regulated by the dynamics of fusion and fission, mitochondrial homeostasis remains incompletely understood. Recent studies implicate dynamin-2 and dynamin-related protein-1 (Drp1, also known as DNM1L), as GTPases involved in mitochondrial fission. Here, we identify the ATPase and endocytic protein EHD1 as a novel regulator of mitochondrial fission. EHD1 depletion induces a static and elongated network of mitochondria in the cell. However, unlike dynamin-2 and Drp1, whose depletion protects cells from staurosporine-induced mitochondrial fragmentation, EHD1-depleted cells remain sensitive to staurosporine, suggesting a different mechanism for EHD1 function. Recent studies have demonstrated that VPS35 and the retromer complex influence mitochondrial homeostasis either by Mul1-mediated ubiquitylation and degradation of the fusion protein Mfn2, or by removal of inactive Drp1 from the mitochondrial membrane. We demonstrate that EHD1 and its interaction partner rabankyrin-5 interact with the retromer complex to influence mitochondrial dynamics, likely by inducing VPS35-mediated removal of inactive Drp1 from mitochondrial membranes. Our study sheds light on mitochondrial dynamics, expanding a new paradigm of endocytic protein regulation of mitochondrial homeostasis.

The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome.

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous "membrane bridges." Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.

Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1.

Localization of the non-receptor tyrosine kinase Src to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and the movement of Src to the plasma membrane is mediated by endocytic transport. However, the precise pathways and regulatory proteins that are responsible for SRC transport are incompletely understood. Here, we demonstrate that Src partially colocalizes with the endocytic regulatory protein MICAL-L1 (molecule interacting with CasL-like protein 1) in mammalian cells. Furthermore, MICAL-L1 is required for growth-factor- and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner EHD1 (EH domain-containing protein 1) is also required for Src activation and transport. Moreover, the MICAL-L1-mediated recruitment of EHD1 to Src-containing recycling endosomes is required for the release of Src from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mechanism by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases.

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Implicating calpain in tau-mediated toxicity in vivo.

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo.

Convergence of linkage, gene expression and association data demonstrates the influence of the RAR-related orphan receptor alpha (RORA) gene on neovascular AMD: a systems biology based approach.

To identify novel genes and pathways associated with AMD, we performed microarray gene expression and linkage analysis which implicated the candidate gene, retinoic acid receptor-related orphan receptor alpha (RORA, 15q). Subsequent genotyping of 159 RORA single nucleotide polymorphisms (SNPs) in a family-based cohort, followed by replication in an unrelated case-control cohort, demonstrated that SNPs and haplotypes located in intron 1 were significantly associated with neovascular AMD risk in both cohorts. This is the first report demonstrating a possible role for RORA, a receptor for cholesterol, in the pathophysiology of AMD. Moreover, we found a significant interaction between RORA and the ARMS2/HTRA1 locus suggesting a novel pathway underlying AMD pathophysiology.