Obesity results from chronic energy surplus and excess lipid storage in white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) efficiently burns lipids through adaptive thermogenesis. ...Studying mouse models, we show that cyclooxygenase (COX)-2, a rate-limiting enzyme in prostaglandin (PG) synthesis, is a downstream effector of β-adrenergic signaling in WAT and is required for the induction of BAT in WAT depots. PG shifted the differentiation of defined mesenchymal progenitors toward a brown adipocyte phenotype. Overexpression of COX-2 in WAT induced de novo BAT recruitment in WAT, increased systemic energy expenditure, and protected mice against high-fat diet-induced obesity. Thus, COX-2 appears integral to de novo BAT recruitment, which suggests that the PG pathway regulates systemic energy homeostasis.
Abstract
Background
The etiology and most risk factors for a sporadic first primary neoplasm in childhood or subsequent second primary neoplasms are still unknown. One established causal factor for ...therapy-associated second primary neoplasms is the exposure to ionizing radiation during radiation therapy as a mainstay of cancer treatment. Second primary neoplasms occur in 8% of all cancer survivors within 30 years after the first diagnosis in Germany, but the underlying factors for intrinsic susceptibilities have not yet been clarified. Thus, the purpose of this nested case–control study was the investigation and comparison of gene expression and affected pathways in primary fibroblasts of childhood cancer survivors with a first primary neoplasm only or with at least one subsequent second primary neoplasm, and controls without neoplasms after exposure to a low and a high dose of ionizing radiation.
Methods
Primary fibroblasts were obtained from skin biopsies from 52 adult donors with a first primary neoplasm in childhood (N1), 52 with at least one additional primary neoplasm (N2+), as well as 52 without cancer (N0) from the KiKme study. Cultured fibroblasts were exposed to a high 2 Gray (Gy) and a low dose (0.05 Gy) of X-rays. Messenger ribonucleic acid was extracted 4 h after exposure and Illumina-sequenced. Differentially expressed genes (DEGs) were computed using
limma
for R, selected at a false discovery rate level of 0.05, and further analyzed for pathway enrichment (right-tailed Fisher’s Exact Test) and (in-) activation (z ≥|2|) using
Ingenuity Pathway Analysis
.
Results
After 0.05 Gy, least DEGs were found in N0 (n = 236), compared to N1 (n = 653) and N2+ (n = 694). The top DEGs with regard to the adjusted
p
-value were upregulated in fibroblasts across all donor groups (
SESN1
,
MDM2
,
CDKN1A
,
TIGAR
,
BTG2
,
BLOC1S2
,
PPM1D
,
PHLDB3
,
FBXO22
,
AEN
,
TRIAP1
, and
POLH)
. Here, we observed activation of
p53 Signaling
in N0 and to a lesser extent in N1, but not in N2+. Only in N0, DNA (excision-) repair (involved genes:
CDKN1A
,
PPM1D
, and
DDB2
) was predicted to be a downstream function, while molecular networks in N2+ were associated with cancer, as well as injury and abnormalities (among others, downregulation of
MSH6
,
CCNE2
, and
CHUK
). After 2 Gy, the number of DEGs was similar in fibroblasts of all donor groups and genes with the highest absolute log
2
fold-change were upregulated throughout (
CDKN1A, TIGAR, HSPA4L
,
MDM2
,
BLOC1SD2
,
PPM1D
,
SESN1
,
BTG2
,
FBXO22
,
PCNA
, and
TRIAP1
). Here, the
p53 Signaling
-
Pathway was activated in fibroblasts of all donor groups. The
Mitotic Roles of Polo Like Kinase
-
Pathway was inactivated in N1 and N2+.
Molecular Mechanisms of Cancer
were affected in fibroblasts of all donor groups.
P53
was predicted to be an upstream regulator in fibroblasts of all donor groups and
E2F1
in N1 and N2+. Results of the downstream analysis were
senescence
in N0 and N2+,
transformation of cells
in N0, and no significant effects in N1. Seven genes were differentially expressed in reaction to 2 Gy dependent on the donor group (
LINC00601
,
COBLL1
,
SESN2
,
BIN3
,
TNFRSF10A
,
EEF1AKNMT
, and
BTG2
).
Conclusion
Our results show dose-dependent differences in the radiation response between N1/N2+ and N0. While mechanisms against genotoxic stress were activated to the same extent after a high dose in all groups, the radiation response was impaired after a low dose in N1/N2+, suggesting an increased risk for adverse effects including carcinogenesis, particularly in N2+.
Exposure to ionizing radiation induces complex stress responses in cells, which can lead to adverse health effects such as cancer. Although a variety of studies investigated gene expression and ...affected pathways in human fibroblasts after exposure to ionizing radiation, the understanding of underlying mechanisms and biological effects is still incomplete due to different experimental settings and small sample sizes. Therefore, this study aims to identify the time point with the highest number of differentially expressed genes and corresponding pathways in primary human fibroblasts after irradiation at two preselected time points.
Fibroblasts from skin biopsies of 15 cell donors were exposed to a high (2Gy) and a low (0.05Gy) dose of X-rays. RNA was extracted and sequenced 2 h and 4 h after exposure. Differentially expressed genes with an adjusted p-value < 0.05 were flagged and used for pathway analyses including prediction of upstream and downstream effects. Principal component analyses were used to examine the effect of two different sequencing runs on quality metrics and variation in expression and alignment and for explorative analysis of the radiation dose and time point of analysis.
More genes were differentially expressed 4 h after exposure to low and high doses of radiation than after 2 h. In experiments with high dose irradiation and RNA sequencing after 4 h, inactivation of the FAT10 cancer signaling pathway and activation of gluconeogenesis I, glycolysis I, and prostanoid biosynthesis was observed taking p-value (< 0.05) and (in) activating z-score (≥2.00 or ≤ - 2.00) into account. Two hours after high dose irradiation, inactivation of small cell lung cancer signaling was observed. For low dose irradiation experiments, we did not detect any significant (p < 0.05 and z-score ≥ 2.00 or ≤ - 2.00) activated or inactivated pathways for both time points.
Compared to 2 h after irradiation, a higher number of differentially expressed genes were found 4 h after exposure to low and high dose ionizing radiation. Differences in gene expression were related to signal transduction pathways of the DNA damage response after 2 h and to metabolic pathways, that might implicate cellular senescence, after 4 h. The time point 4 h will be used to conduct further irradiation experiments in a larger sample.
Therapy for a first primary neoplasm (FPN) in childhood with high doses of ionizing radiation is an established risk factor for second primary neoplasms (SPN). An association between exposure to low ...doses and childhood cancer is also suggested; however, results are inconsistent. As only subgroups of children with FPNs develop SPNs, an interaction between radiation, genetic, and other risk factors is presumed to influence cancer development.
Therefore, the population-based, nested case-control study KiKme aims to identify differences in genetic predisposition and radiation response between childhood cancer survivors with and without SPNs as well as cancer-free controls.
We conducted a population-based, nested case-control study KiKme. Besides questionnaire information, skin biopsies and saliva samples are available. By measuring individual reactions to different exposures to radiation (eg, 0.05 and 2 Gray) in normal somatic cells of the same person, our design enables us to create several exposure scenarios for the same person simultaneously and measure several different molecular markers (eg, DNA, messenger RNA, long noncoding RNA, copy number variation).
Since 2013, 101 of 247 invited SPN patients, 340 of 1729 invited FPN patients, and 150 of 246 invited cancer-free controls were recruited and matched by age and sex. Childhood cancer patients were additionally matched by tumor morphology, year of diagnosis, and age at diagnosis. Participants reported on lifestyle, socioeconomical, and anthropometric factors, as well as on medical radiation history, health, and family history of diseases (n=556). Primary human fibroblasts from skin biopsies of the participants were cultivated (n=499) and cryopreserved (n=3886). DNA was extracted from fibroblasts (n=488) and saliva (n=510).
This molecular-epidemiological study is the first to combine observational epidemiological research with standardized experimental components in primary human skin fibroblasts to identify genetic predispositions related to ionizing radiation in childhood and SPNs. In the future, fibroblasts of the participants will be used for standardized irradiation experiments, which will inform analysis of the case-control study and vice versa. Differences between participants will be identified using several molecular markers. With its innovative combination of experimental and observational components, this new study will provide valuable data to forward research on radiation-related risk factors in childhood cancer and SPNs.
DERR1-10.2196/32395.
The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitroassay to study nuclear export mediated by ...the C-terminal shuttle domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin β-related export receptor CRM1 competes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich ...NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).
Abstract
‘Synthetic lethality’ offers an attractive therapeutic strategy for the development of cancer-specific cytotoxic agents. We therefore decided to exploit the genetic differences that exist ...between the tumor cell and the normal cell by searching for drugs that display ‘synthetic lethal’ interactions with the cancer-associated LKB1 mutation that occurs in about 30% of human lung adenocarcinomas.
Two genes are said to be ‘synthetic lethal’, if disruption of either individual gene is compatible with viability, but if loss of both causes death. It follows that a drug that is synthetic lethal to LKB1 should kill only the mutated LKB1 negative cells and spare normal cells.
A cell-based high-content screen for cell death was carried out with 48,000 small molecule compounds on an isogenic cell line pair that differs in its status of LKB1 (wild type versus mutant). We identified Simvastatin, an inhibitor of HMG-CoA reductase and widely used cholesterol-lowering drug, as one of the main hits. Other statins in the screening set, such as Lovastatin and Compactin, displayed similar activity.
Follow-up experiments confirmed synthetic lethal interactions between Simvastatin and LKB1-deficiency. Importantly, addition of mevalonate, the product of the HMG-CoA reduction rescued and siRNAs targeting HMG-CoA reductase further enhanced cell death induced by Simvastatin in LKB1-negative cells. Furthermore, Simvastatin specifically inhibited LKB1-negative tumor growth in a xenograft model in nude mice. The synthetic lethal relationship between Simvastatin and LKB1-deficiency indicates statins as potential cancer therapeutics for LKB1-negative tumors.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A167.
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