No abstract

EHD3 [Eps15 homology (EH) domain-containing protein 3] is a protein that resides in tubular and vesicular membrane structures and participates in endocytic recycling, although all its functions are unknown. Since Ehd3 is most abundantly expressed in brain tissues, we examined its role in brain cancer progression. Using immunohistochemistry, we report loss of EHD3 expression in gliomas, including low-grade astrocytomas, suggesting that this is an early event in gliomagenesis. EHD3 expression is also very low in most of glioma cell lines tested. In two cell lines, a bisulfite sequencing method identifies promoter hypermethylation as a mechanism of Ehd3 silencing, and its expression was restored by the demethylating agent 5-Azacytidine. Doxycycline-inducible restoration of EHD3 expression to glioma cells decreases their growth and invasiveness and induces cell cycle arrest and apoptosis. Furthermore, shRNA-mediated Ehd3 silencing increases cell growth. Using a xenograft model, we demonstrate Ehd3 growth inhibitory functions in glioma cells in vivo. We suggest that Ehd3 functions as a tumor suppressor gene and loss of its expression is a very common event in gliomas. This is the first study to highlight the importance of a member of the C-terminal EHD proteins in cancer and to link their functions to the cell cycle and apoptosis.

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.