The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells.

BackgroundIdentification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.ResultsWe demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.ConclusionsThese studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance

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

Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans

BACKGROUND: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. RESULTS: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. CONCLUSION: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved

Role of mammalian Ecdysoneless in cell cycle regulation.

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway

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

Mutant Cbl proteins as oncogenic drivers in myeloproliferative disorders

Casitas B-lineage lymphoma (Cbl) family proteins are evolutionarily-conserved attenuators of protein tyrosine kinase (PTK) signaling. Biochemical analyses over the past two decades have firmly established that the negative regulatory functions of Cbl proteins are mediated through their ability to facilitate ubiquitination and thus promote degradation of PTKs. As aberrant activation of PTKs is frequently associated with oncogenesis, it has long been postulated that loss of normal Cbl functions may lead to unregulated activation of PTKs and cellular transformation. In the last few years, mutations in the CBL gene have been identified in a subset of human patients with myeloid malignancies. Here we discuss insights gained from the analyses of Cbl mutants both in human patients and in animal models and propose potential mechanisms of oncogenesis through this pathway

An essential role of human Ada3 in p53 acetylation.

The p53 tumor suppressor protein functions as a critical component of genotoxic stress response by regulating the expression of effector gene products that control the fate of a cell following DNA damage. Unstressed cells maintain p53 at low levels through regulated degradation, and p53 levels and activity are rapidly elevated upon genotoxic stress. Biochemical mechanisms that control the levels and activity of p53 are therefore of great interest. We and others have recently identified hAda3 (human homologue of yeast alteration/deficiency in activation 3) as a p53-interacting protein and enhancer of p53 activity. Here, we show that endogenous levels of p53 and Ada3 interact with each other, and by using inducible overexpression and short hairpin RNA-mediated knockdown strategies we demonstrate that hAda3 stabilizes p53 protein by promoting its acetylation. Use of a p53 mutant with mutations of known p300/CREB-binding protein acetylation sites demonstrated that hAda3-dependent acetylation is required for increase in p53 stability and target gene induction. Importantly, we demonstrate that endogenous hAda3 is essential for DNA damage-induced acetylation and stabilization of p53 as well as p53 target gene induction. Overall, our results establish hAda3, a component of coactivator complexes that include histone acetyltransferase p300/CREB-binding protein, as a critical mediator of acetylation-dependent stabilization and activation of p53 upon genotoxic stress in mammalian cells

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC

Background: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized.Methods: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients.Results: Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 andc-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients’ outcome was independent of tumor grade, stage and size, and ER status.Conclusion: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients