Alternative NHEJ pathway proteins as components of MYCN oncogenic activity in human neural crest stem cell differentiation: implications for neuroblastoma initiation.

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.

Alternative NHEJ Pathway Components Are Therapeutic Targets in High-Risk Neuroblastoma.

UNLABELLED: In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. IMPLICATIONS: These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets.

Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA.

Caveolin-1 (cav-1) plays a key role in PKC-α and RhoA signaling pathways during acetylcholine (ACh)-induced contraction of colonic smooth muscle cells (CSMC). Aged rat CSMC showed sluggish contractility, concomitant with reduced expression of cav-1 with an associated reduction in activation of PKC-α and RhoA signaling pathway. Real-time monitoring of live human CSMC transfected with yellow fluorescent protein-tagged wild-type caveolin 1 cDNA (YFP-wt-cav-1) cDNA in the present study suggests that cav-1 cycles within and along the membrane in a synchronized, highly organized cytoskeletal path. These studies provide, for the first time, the advantages of real-time monitoring of the dynamic movement of caveolin in living cells. Rapid movement of cav-1 in response to ACh suggests its dynamic role in CSMC contraction. Human CSMC transfected with YFP-ΔTFT-cav-1 dominant negative cDNA show fluorescence in the cytosol of the CSMC and no movement of fluorescent cav-1 in response to ACh mimicking the response shown by aged rat CSMC. Transfection of CSMC from aged rat with YFP-wt-cav-1 cDNA restored the physiological contractile response to ACh as well as the dynamic movement of cav-1 along the organized cytoskeletal path observed in normal adult CSMC. To study the force generation by CSMC, three-dimensional colonic rings were bioengineered. Colonic bioengineered rings from aged CSMC showed reduced force generation compared with colonic bioengineered rings from adult CSMC. Colonic bioengineered rings from aged CSMC transfected with wt-cav-1 cDNA showed force generation similar to colonic bioengineered rings from adult rat CSMC. The data suggest that contraction in CSMC is dependent on cav-1 reorganization dynamics, which restores the physiological contractile response in aged CSMC. We hypothesize that dynamic movement of cav-1 is essential for physiological contractile response of colonic smooth muscle.