Novel Interaction Between ECD and EIF4A1 Indicates ECD Regulates Eukaryotic Translation

ECD, Ecdysoneless protein, is evolutionarily conserved. It was identified as a human homologue of Drosophila ecdysoneless, interacting with HPV16 E6 in a yeast two-hybrid assay. ECD has been proven to be essential for cell cycle progression from G1 to S phase, mitigating endoplasmic reticulum (ER) stress, and embryogenesis. ECD KO mice are embryonic lethal as it halts the cell cycle at G1. ECD interacts with p53 and Rb. ECD also associates with pre-mRNA splicing factor PRPF8. ECD is over-expressed in breast, pancreatic, gastric, and Human Papilloma-driven cancers and is correlated with shorter patient survival. ECD interacts with co-chaperone complex R2TP involved in protein assembly and folding. ECD over-expression has been proven to increase oncogenesis regulated by c-MYC in a recent mice model. Biochemical analyses showed ECD to have a role in mRNA splicing and nucleus to cytoplasm export. The purpose was to determine if there is an interaction between ECD and EIF4A1 and determine ECD’s effect on global translation using HEK293T (human embryonic kidney) and SUM159 (breast cancer) cell lines. Overall, the interaction between ECD and EIF4A1 supports a novel mechanism by which ECD protein regulates eukaryotic mRNA translation. This mechanism may contribute to the resistance of cancer cells over-expressing ECD to the translation-inhibitory effect of endoplasmic reticulum stress prevalent in tumors.https://digitalcommons.unmc.edu/surp2022/1031/thumbnail.jp

EHD2 Promotes Store-Operated Calcium Entry (SOCE) and Cellular Migration in Ovarian Cancer Cells

Ovarian cancer (OC) ranks as the 5th most common cause of cancer deaths of women, reflecting late diagnoses and lack of targeted therapies. EHD2, a member of the Eps15 homology (EH) domain containing (EHD) proteins family, regulates cell surface expression of Orai1, the mediator of store-operated calcium entry (SOCE) in breast cancer. Disrupting the EHD2-Orai1 axis in OC could provide novel targeted therapies against metastatic disease.https://digitalcommons.unmc.edu/surp2023/1002/thumbnail.jp

Comparative Immunohistochemical Analysis of EHD1 Expression in Adjacent, Metastatic, and Normal Thyroid Tissue

The discovery of prognostic biomarkers plays a crucial role in enhancing the treatment and care of individuals with differentiated thyroid cancer (DTC) who are at risk of disease progression. A significant breakthrough came with earlier research, which revealed higher levels of the EHD1 protein in papillary DTC when compared to the surrounding healthy tissue. This exciting finding served as the driving force behind the initiation of a more extensive investigation aimed at validating EHD1 as a potential biomarker and exploring its connection with clinical outcomes. By unraveling the potential implications of EHD1 in DTC cases, this study holds the promise of advancing our understanding and approach to managing this type of cancer effectively.https://digitalcommons.unmc.edu/surp2023/1001/thumbnail.jp

Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy.

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling

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

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism

Loss of the nuclear pool of ubiquitin ligase CHIP/STUB1 in breast cancer unleashes the MZF1-cathepsin pro-oncogenic program

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two-thirds of ErbB2+ and triple-negative breast cancers and in one-third of ER+ breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2+ and triple-negative breast cancer cell lines. Ectopic CHIP expression in ErbB2+ lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up-or down-regulated by CHIP. We characterized Myeloid Zinc Finger 1 (MZF1) as a CHIP target given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2+ and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2+ breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells

Summary: Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity

Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

Summary: Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5Hi ISCs with transient expansion of the Lgr5Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation

EHD1-dependent traffic of IGF-1 receptor to the cell surface is essential for Ewing sarcoma tumorigenesis and metastasis

Abstract Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting