Macrophages, Nitric Oxide and microRNAs Are Associated with DNA Damage Response Pathway and Senescence in Inflammatory Bowel Disease

Background: Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation. Methodology/Principal Findings: Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn’s disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression. Conclusions: Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.Cancer Research Institute (New York, N.Y.) (Intramural Research Program)National Cancer Institute (U.S.) (Cancer Research Training Award Fellowship)Danish Cancer SocietyDanish National Research FoundationEuropean Commission (projects: Infla-Care, Biomedreg and DDResponse

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis

Induction of APOBEC3 exacerbates DNA replication stress and chromosomal instability in early breast and lung cancer evolution

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast DCIS, and in pre-invasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx pre-invasive to invasive NSCLC lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models, revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in pre-invasive disease, providing fuel for selection early in cancer evolution

Senescence is induced by either macrophages or NO• in primary normal human fibroblasts in culture.

<p>Normal human fibroblasts (WI38 and MRC5) were grown in coculture with murine macrophages (ANA-1), or with the NO• donor spermine NONOate (Sper/NO•). Senescence-associated β-galactosidase activity was used to determine to the percent of senescent fibroblasts divided by the number of total fibroblasts. Results are shown from three experiments, with each experiment done in triplicate. (A) Normal human fibroblasts were cocultured with macrophages, with and without the NO• synthase inhibitor L-NAME (500 µM). Macrophages induced senescence in WI38 and MRC5 cells. Senescence was partially abrogated by L-NAME in WI38 and MRC5 cells. (B) The NO• synthase inhibitor L-NAME reduces diffused NO• in media of cocultures comprised of normal human fibroblasts (WI38 or MRC5) and macrophages (ANA-1). 100 µl of media from three separate cocultures was aliquoted with 100 µL of 5 µM of DAF in 96-well plates. Plates were read for DAF-fluorescence as an indicator of NO•. Addition of the NO• inhibitor L-NAME resulted in decreased levels of NO• in both WI38 (<i>P</i> = 0.008) and MRC5 (<i>P</i> = 0.03) cells. Fluorescence measurements from cocultures were normalized by subtracting the DAF fluorescence measured in media from wells with fibroblasts only. (C) Fibroblasts were dosed with 0.09 µM, 3 µM and 10 µM Sper/NO•, Sper/NO• that was previously incubated in media with sodium hydroxide (vehicle) for 48 hours (exhausted donor), and media alone (negative control) overnight (16 hrs). These concentrations were selected to achieve steady state concentrations of 4.5 nM, 15 nM and 50 nM NO• respectively. 10 µM and 3 µM Sper/NO• induced significant levels of senescence (<i>P</i><0.0001). Exhausted Sper/NO• (negative control) and 0.09 µM Sper/NO• did not induce significant levels of senescence when compared to media alone. Hydrogen peroxide (200 µM; 2 hrs) was used as a positive control.</p

Association of microRNAs with NOS2 and CD68 expression in IBD and microRNAs altered in colon adenomas.

<p>The Venn diagram displays microRNAs that were significantly associated with the mRNA expression of NOS2 (<i>P</i><0.001) and CD68 (<i>P</i><0.05) and those microRNAs that are altered in colon adenomas (<i>P</i><0.001) based on microRNA microarray profiling. MiR-21 was found to be associated in all three comparisons suggesting a potential role for this microRNA in senescence.</p