Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase.

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1, a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells.

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.

The MYCN oncogene and differentiation in neuroblastoma.

Childhood neuroblastoma exhibits a heterogeneous clinical behavior ranging from low-risk tumors with the ability to spontaneously differentiate and regress, to high-risk tumors causing the highest number of cancer related deaths in infants. Amplification of the MYCN oncogene is one of the few prediction markers for adverse outcome. This gene encodes the MYCN transcriptional regulator predominantly expressed in the developing peripheral neural crest. MYCN is vital for proliferation, migration and stem cell homeostasis while decreased levels are associated with terminal neuronal differentiation. Interestingly, high-risk tumors without MYCN amplification frequently display increased c-MYC expression and/or activation of MYC signaling pathways. On the other hand, downregulation of MYCN leads to decreased proliferation and differentiation, emphasizing the importance of MYC signaling in neuroblastoma biology. Furthermore, expression of the neurotrophin receptor TrkA is associated with good prognosis, the ability to differentiate and spontaneous regression while expression of the related TrkB receptor is correlated with bad prognosis and MYCN amplification. Here we discuss the role of MYCN in neuroblastoma with a special focus on the contribution of elevated MYCN signaling for an aggressive and undifferentiated phenotype as well as the potential of using MYCN as a therapeutic target.

BARD1 participates with BRCA1 in homology-directed repair of chromosome breaks.

The BRCA1 tumor suppressor has been implicated in the maintenance of chromosomal stability through homology-directed repair of DNA double-strand breaks. Much of the BRCA1 in cells forms a heterodimeric complex with a structurally related protein BARD1. We report that expression of truncated mouse or human BARD1 peptides capable of interacting with Brca1 results in a homologous-repair deficiency. Repair is mildly reduced in Brca1 wild-type cells and severely reduced in cells that harbor a Brca1 splice product deleted for exon 11. Nuclear localization of the Brca1 or BARD1 peptides is not compromised, implying that the repair deficiency is caused by a more direct effect on repair. The tumor suppressor activity of BRCA1 may require the participation of BARD1 to maintain chromosome integrity through the homologous-repair pathway.

Regulation of Nuclear Hormone Receptors by MYCN-Driven miRNAs Impacts Neural Differentiation and Survival in Neuroblastoma Patients.

MYCN amplification and MYC signaling are associated with high-risk neuroblastoma with poor prognosis. Treating these tumors remains challenging, although therapeutic approaches stimulating differentiation have generated considerable interest. We have previously shown that the MYCN-regulated miR-17∼92 cluster inhibits neuroblastoma differentiation by repressing estrogen receptor alpha. Here, we demonstrate that this microRNA (miRNA) cluster selectively targets several members of the nuclear hormone receptor (NHR) superfamily, and we present a unique NHR signature associated with the survival of neuroblastoma patients. We found that suppressing glucocorticoid receptor (GR) expression in MYCN-driven patient and mouse tumors was associated with an undifferentiated phenotype and decreased survival. Importantly, MYCN inhibition and subsequent reactivation of GR signaling promotes neural differentiation and reduces tumor burden. Our findings reveal a key role for the miR-17∼92-regulated NHRs in neuroblastoma biology, thereby providing a potential differentiation approach for treating neuroblastoma patients.

First application of liquid-metal-jet sources for small-animal imaging: high-resolution CT and phase-contrast tumor demarcation.

PURPOSE: Small-animal studies require images with high spatial resolution and high contrast due to the small scale of the structures. X-ray imaging systems for small animals are often limited by the microfocus source. Here, the authors investigate the applicability of liquid-metal-jet x-ray sources for such high-resolution small-animal imaging, both in tomography based on absorption and in soft-tissue tumor imaging based on in-line phase contrast. METHODS: The experimental arrangement consists of a liquid-metal-jet x-ray source, the small-animal object on a rotating stage, and an imaging detector. The source-to-object and object-to-detector distances are adjusted for the preferred contrast mechanism. Two different liquid-metal-jet sources are used, one circulating a Ga∕In∕Sn alloy and the other an In∕Ga alloy for higher penetration through thick tissue. Both sources are operated at 40-50 W electron-beam power with ∼7 µm x-ray spots, providing high spatial resolution in absorption imaging and high spatial coherence for the phase-contrast imaging. RESULTS: High-resolution absorption imaging is demonstrated on mice with CT, showing 50 µm bone details in the reconstructed slices. High-resolution phase-contrast soft-tissue imaging shows clear demarcation of mm-sized tumors at much lower dose than is required in absorption. CONCLUSIONS: This is the first application of liquid-metal-jet x-ray sources for whole-body small-animal x-ray imaging. In absorption, the method allows high-resolution tomographic skeletal imaging with potential for significantly shorter exposure times due to the power scalability of liquid-metal-jet sources. In phase contrast, the authors use a simple in-line arrangement to show distinct tumor demarcation of few-mm-sized tumors. This is, to their knowledge, the first small-animal tumor visualization with a laboratory phase-contrast system.

RAD51 can inhibit PDGF-B-induced gliomagenesis and genomic instability.

Faithful replication and DNA repair are vital for maintenance of genome integrity. RAD51 is a central protein in homologous recombination repair and during replication, when it protects and restarts stalled replication forks. Aberrant RAD51 expression occurs in glioma, and high expression has been shown to correlate with prolonged survival. Furthermore, genes involved in DNA damage response (DDR) are mutated or deleted in human glioblastomas, corroborating the importance of proper DNA repair to suppress gliomagenesis. We have analyzed DDR and genomic instability in PDGF-B-induced gliomas and investigated the role of RAD51 in glioma development. We show that PDGF-B-induced gliomas display genomic instability and that co-expression of RAD51 can suppress PDGF-B-induced tumorigenesis and prolong survival. Expression of RAD51 inhibited proliferation and genomic instability of tumor cells independent of Arf status. Our results demonstrate that the RAD51 pathway can prevent glioma initiation and maintain genome integrity of induced tumors, suggesting reactivation of the RAD51 pathway as a potential therapeutic avenue.

C/EBP is an essential component of PDGFRA transcription in MG-63 cells.

Interleukin-1beta (IL-1beta) is a potent inhibitor of platelet-derived growth factor alpha receptor (PDGFRalpha) expression in MG-63 cells. Its effect is mediated at the transcriptional level, but the transcription factors involved in this process are unknown. In the current study, we found that IL-1beta could inhibit the PDGFRalpha gene promoter activity, and this effect was strongly correlated with increased binding of CCAAT/enhancer-binding protein (C/EBP) to the responsive promoter region. In addition, forced expression of C/EBPbeta could mimic the IL-1beta effect on the promoter activity, but subsequent mutation analysis of the C/EBP binding sites indicated that direct C/EBP binding to the promoter is not required for the IL-1beta response. However, our data clearly demonstrated that the C/EBP binding site at position-162 relative to the transcriptional start site is essential for high basal level PDGFRalpha promoter activity.