EHD1 is Required for IGF-1R-mediated Oncogenic Signaling in Ewing Sarcoma

Background and Significance: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase(RTK) insulin-like-growth-factor-1-receptor (IGF-1R) is upregulated in 93% of EWS patients with anti-IGF-1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF-1R-driven oncogenesis in EWS could provide avenues for more effective therapy. The RTK levels and compartmentalization at the cell surface determine their access to growth factors, thus dictating the downstream oncogenic signaling. Our lab has demonstrated that EPS15-homology-domain-containing-protein-1 (EHD1) regulates traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues, and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective/Question: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Experimental design and Results: In both dox-inducible EHD1-shRNA knockdown and EHD1-CRISPR-Cas9-knockout (KO) EWS cell line models(TC71, A673, and SKES1), we observed a significant impairment of in vitro oncogenic properties namely, cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation, and the phenotypes were restored upon mouse-EHD1 rescue. Furthermore, by orthotopically implanting TC71 cells in the tibia of nude mice(xenograft model), we demonstrated a significant reduction in tumor size upon EHD1-depletion. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF-1R levels upon EHD1-KD, identifying IGF-1R as a potential target of regulation by EHD1. EHD1-KO reduced surface IGF-1R levels under steady-state and ligand-free conditions in EWS cells. IGF-1R and EHD1 were also found to colocalize intracellularly and co-immunoprecipitate after IGF-1 stimulation. Notably, EHD1-KO impaired the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Mechanistically, EHD1 was shown to regulate traffic of newly synthesized IGF-1R and recycled pools from the Golgi to the cell surface, and in absence of EHD1, intracellular IGF-1R was shunted to the lysosome resulting in degradation. Finally, by dual targeting of EHD1 (genetic depletion) and IGF-1R (small-molecule-inhibitor Linsitinib), we observed an additive effect on inhibition of EWS cell proliferation and migration and upregulation of apoptosis. Conclusions: Our studies indicate a novel regulatory pathway of EHD1 requirement in IGF-1R cell surface display and sustaining IGF-1R-mediated oncogenesis in EWS. This highlights the prospects of therapeutic co-targeting of EHD1 and IGF-1R, thus enhancing IGF-1R targeted therapies in EWS.https://digitalcommons.unmc.edu/chri_forum/1040/thumbnail.jp

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis

Hoogsteen base-pairing revisited: resolving a role in normal biological processes and human diseases

For a long time since the discovery of an alternative type of hydrogen bonding between adenine and thymidine, termed Hoogsteen base-pairing, its biological role remained elusive. Recent experiments provide compelling evidence that Hoogsteen base pairs manifest in a gamut of nuclear processes encompassing gene expression, replication, recombination, and telomere length maintenance. An increasing number of proteins that have been shown to bind, unwind or cleave G-quadruplexes or triplexes with high specificity underscore their biological significance. In humans, the absence of these cellular factors or their dysfunction leads to a wide spectrum of genetic diseases including cancer, neurodegenerative syndromes, and a myriad of other disorders. Thus, development of clinically useful compounds that target G-quadruplexes or triplexes, and interfere with specific cellular processes, provides considerable promise for successful and improved treatment of human diseases

Hoogsteen base-pairing revisited: Resolving a role in normal biological processes and human diseases

For a long time since the discovery of an alternative type of hydrogen bonding between adenine and thymidine, termed Hoogsteen base-pairing, its biological role remained elusive. Recent experiments provide compelling evidence that Hoogsteen base pairs manifest in a gamut of nuclear processes encompassing gene expression, replication, recombination, and telomere length maintenance. An increasing number of proteins that have been shown to bind, unwind or cleave G-quadruplexes or triplexes with high specificity underscore their biological significance. In humans, the absence of these cellular factors or their dysfunction leads to a wide spectrum of genetic diseases including cancer, neurodegenerative syndromes, and a myriad of other disorders. Thus, development of clinically useful compounds that target G-quadruplexes or triplexes, and interfere with specific cellular processes, provides considerable promise for successful and improved treatment of human diseases

The annealing helicase HARP protects stalled replication forks

Mutations in HepA-related protein (HARP) are the only identified causes of Schimke immunoosseous dysplasia (SIOD). HARP has a unique annealing helicase activity in vitro, but the in vivo functional significance remains unknown. Here, we demonstrated that HARP is recruited to stalled replication forks via its direct interaction with Replication protein A (RPA). Cells with HARP depletion displayed increased spontaneous DNA damage and G2/M arrest, suggesting that HARP normally acts to stabilize stalled replication forks. Our data place the annealing helicase activity of HARP at replication forks and propose that SIOD syndrome may be caused by the destabilization of replication forks during cell proliferation

Interaction of Ascorbate and a-tocopherol Enhances Antioxidant Reserve of Erythrocytes during Anemia in Visceral Leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and a-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and a-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and a-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and a-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous a-tocopherol. The results suggest an interacting role of ascorbate and a-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L. © 2000 Elsevier Science Inc. All rights reserved