Molecular analysis of the human cytoglobin mRNA isoforms.

Multiple functions have been proposed for the ubiquitously expressed vertebrate globin cytoglobin (Cygb), including nitric oxide (NO) metabolism, lipid peroxidation/signalling, superoxide dismutase activity, reactive oxygen/nitrogen species (RONS) scavenging, regulation of blood pressure, antifibrosis, and both tumour suppressor and oncogenic effects. Since alternative splicing can expand the biological roles of a gene, we investigated whether this mechanism contributes to the functional diversity of Cygb. By mining of cDNA data and molecular analysis, we identified five alternative mRNA isoforms for the human CYGB gene (V-1 to V-5). Comprehensive RNA-seq analyses of public datasets from human tissues and cells confirmed that the canonical CYGB V-1 isoform is the primary CYGB transcript in the majority of analysed datasets. Interestingly, we revealed that isoform V-3 represented the predominant CYGB variant in hepatoblastoma (HB) cell lines and in the majority of analysed normal and HB liver tissues. CYGB V-3 mRNA is transcribed from an alternate upstream promoter and hypothetically encodes a N-terminally truncated CYGB protein, which is not recognized by some antibodies used in published studies. Little to no transcriptional evidence was found for the other CYGB isoforms. Comparative transcriptomics and flow cytometry on CYGB+/+ and gene-edited CYGB-/- HepG2 HB cells did not unveil a knockout phenotype and, thus, a potential function for CYGB V-3. Our study reveals that the CYGB gene is transcriptionally more complex than previously described as it expresses alternative mRNA isoforms of unknown function. Additional experimental data are needed to clarify the biological meaning of those alternative CYGB transcripts.

Identification of lncRNAs involved in response to ionizing radiation in fibroblasts of long-term survivors of childhood cancer and cancer-free controls.

Introduction: Long non-coding ribonucleic acids (lncRNAs) are involved in the cellular damage response following exposure to ionizing radiation as applied in radiotherapy. However, the role of lncRNAs in radiation response concerning intrinsic susceptibility to late effects of radiation exposure has not been examined in general or in long-term survivors of childhood cancer with and without potentially radiotherapy-related second primary cancers, in particular. Methods: Primary skin fibroblasts (n=52 each) of long-term childhood cancer survivors with a first primary cancer only (N1), at least one second primary neoplasm (N2+), as well as tumor-free controls (N0) from the KiKme case-control study were matched by sex, age, and additionally by year of diagnosis and entity of the first primary cancer. Fibroblasts were exposed to 0.05 and 2 Gray (Gy) X-rays. Differentially expressed lncRNAs were identified with and without interaction terms for donor group and dose. Weighted co-expression networks of lncRNA and mRNA were constructed using WGCNA. Resulting gene sets (modules) were correlated to the radiation doses and analyzed for biological function. Results: After irradiation with 0.05Gy, few lncRNAs were differentially expressed (N0: AC004801.4; N1: PCCA-DT, AF129075.3, LINC00691, AL158206.1; N2+: LINC02315). In reaction to 2 Gy, the number of differentially expressed lncRNAs was higher (N0: 152, N1: 169, N2+: 146). After 2 Gy, AL109976.1 and AL158206.1 were prominently upregulated in all donor groups. The co-expression analysis identified two modules containing lncRNAs that were associated with 2 Gy (module1: 102 mRNAs and 4 lncRNAs: AL158206.1, AL109976.1, AC092171.5, TYMSOS, associated with p53-mediated reaction to DNA damage; module2: 390 mRNAs, 7 lncRNAs: AC004943.2, AC012073.1, AC026401.3, AC092718.4, MIR31HG, STXBP5-AS1, TMPO-AS1, associated with cell cycle regulation). Discussion: For the first time, we identified the lncRNAs AL158206.1 and AL109976.1 as involved in the radiation response in primary fibroblasts by differential expression analysis. The co-expression analysis revealed a role of these lncRNAs in the DNA damage response and cell cycle regulation post-IR. These transcripts may be targets in cancer therapy against radiosensitivity, as well as provide grounds for the identification of at-risk patients for immediate adverse reactions in healthy tissues. With this work we deliver a broad basis and new leads for the examination of lncRNAs in the radiation response.

Self-administered questionnaire assessing childhood cancer treatments and associated risks for adverse health outcomes - The KiKme study.

Background: Childhood cancer survivors (CCS) are at particularly high risk for therapy-related late sequelae, with secondary primary neoplasms (SPN) being the most detrimental. Since there is no standardized questionnaire for retrospective assessment of associations between prior cancer treatments and late health effects, we developed a self-administered questionnaire and validated it in a cohort of CCS. Methods: CCS of a first primary neoplasm (FPN, N=340) only or with a subsequent SPN (N=101) were asked whether they had received cancer therapies. Self-reports were compared to participants' medical records on cancer therapies from hospitals and clinical studies (N=242). Cohen's Kappa (κ) was used to measure their agreement and logistic regression was used to identify factors influencing the concordance. Associations between exposure to cancer therapies and late health effects (overweight/obesity, diseases of the lipid metabolism and the thyroid gland, cardiovascular diseases, occurrence of SPN) were analyzed in all participants by applying generalized linear mixed models to calculate odds ratios (OR) and 95% confidence intervals (95%CI). Results: For CCS of SPN, a perfect agreement was found between self-reports and medical records for chemotherapy (CT, κ=1.0) while the accordance for radiotherapy (RT) was lower but still substantial (κ=0.8). For the CCS of FPN the accordance was less precise (CT: κ=0.7, RT: κ=0.3). Cancer status, tumors of the central nervous system, sex, age at recruitment, vocational training, follow-up time, and comorbidities had no impact on agreement. CCS with exposure to CT were found to be less often overweight or obese compared to those without CT (OR=0.6 (95%CI 0.39; 0.91)). However, they were found to suffer more likely from thyroid diseases excluding thyroid cancers (OR=9.91 (95%CI 4.0; 24.57)) and hypercholesterolemia (OR=4.45 (95%CI 1.5; 13.23)). All other analyses did not show an association. Conclusion: Our new questionnaire proved reliable for retrospective assessment of exposure to CT and RT in CCS of SPN. For the CCS of FPN, self-reported RT was very imprecise and should not be used for further analyses. We revealed an association between late health outcomes occurring as hypercholesterolemia and thyroid diseases, excluding thyroid cancer, and the use of CT for the treatment of childhood cancer.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice.

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Gene expression variability in long-term survivors of childhood cancer and cancer-free controls in response to ionizing irradiation.

BACKGROUND: Differential expression analysis is usually adjusted for variation. However, most studies that examined the expression variability (EV) have used computations affected by low expression levels and did not examine healthy tissue. This study aims to calculate and characterize an unbiased EV in primary fibroblasts of childhood cancer survivors and cancer-free controls (N0) in response to ionizing radiation. METHODS: Human skin fibroblasts of 52 donors with a first primary neoplasm in childhood (N1), 52 donors with at least one second primary neoplasm (N2 +), as well as 52 N0 were obtained from the KiKme case-control study and exposed to a high (2 Gray) and a low dose (0.05 Gray) of X-rays and sham- irradiation (0 Gray). Genes were then classified as hypo-, non-, or hyper-variable per donor group and radiation treatment, and then examined for over-represented functional signatures. RESULTS: We found 22 genes with considerable EV differences between donor groups, of which 11 genes were associated with response to ionizing radiation, stress, and DNA repair. The largest number of genes exclusive to one donor group and variability classification combination were all detected in N0: hypo-variable genes after 0 Gray (n = 49), 0.05 Gray (n = 41), and 2 Gray (n = 38), as well as hyper-variable genes after any dose (n = 43). While after 2 Gray positive regulation of cell cycle was hypo-variable in N0, (regulation of) fibroblast proliferation was over-represented in hyper-variable genes of N1 and N2+. In N2+, 30 genes were uniquely classified as hyper-variable after the low dose and were associated with the ERK1/ERK2 cascade. For N1, no exclusive gene sets with functions related to the radiation response were detected in our data. CONCLUSION: N2+ showed high degrees of variability in pathways for the cell fate decision after genotoxic insults that may lead to the transfer and multiplication of DNA-damage via proliferation, where apoptosis and removal of the damaged genome would have been appropriate. Such a deficiency could potentially lead to a higher vulnerability towards side effects of exposure to high doses of ionizing radiation, but following low-dose applications employed in diagnostics, as well.

Replication stress drives chromosomal instability in fibroblasts of childhood cancer survivors with second primary neoplasms.

New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.

Late health effects and changes in lifestyle factors after cancer in childhood with and without subsequent second primary cancers - the KiKme case-control study.

Background: Improved treatments for childhood cancer result in a growing number of long-term childhood cancer survivors (CCS). The diagnosis and the prevalence of comorbidities may, however, influence their lifestyle later in life. Nonetheless, little is known about differences in late effects between CCS of a first primary neoplasm (FPN) in childhood and subsequent second primary neoplasms (SPN) and their impact on lifestyle. Therefore, we aim to investigate associations between the occurrence of FPN or SPN and various diseases and lifestyle in the later life of CCS. Methods: CCS of SPN (n=101) or FPN (n=340) and cancer-free controls (n=150) were matched by age and sex, and CCS additionally by year and entity of FPN. All participants completed a self-administered questionnaire on anthropometric and socio-economic factors, medical history, health status, and lifestyle. Mean time between FPN diagnosis and interview was 27.3 years for SPN and 26.2 years for FPN CCS. To confirm results from others and to generate new hypotheses on late effects of childhood cancer as well as CCS´ lifestyles, generalized linear mixed models were applied. Results: CCS were found to suffer more likely from diseases compared to cancer-free controls. In detail, associations with cancer status were observed for hypercholesterinemia and thyroid diseases. Moreover, CCS were more likely to take regular medication compared to controls. A similar association was observed for CCS of SPN compared to CCS of FPN. In contrast to controls, CCS rarely exercise more than 5 hours per week, consumed fewer soft and alcoholic drinks, and were less likely to be current, former, or passive smokers. Additionally, they were less likely overweight or obese. All other exploratory analyses performed on cardiovascular, chronic lung, inflammatory bone, allergic, and infectious diseases, as well as on a calculated health-score revealed no association with tumor status. Conclusion: CCS were more affected by pathologic conditions and may consequently take more medication, particularly among CCS of SPN. The observed higher disease burden is likely related to the received cancer therapy. To reduce the burden of long-term adverse health effects in CCS, improving cancer therapies should therefore be in focus of research in this area.

Transcriptomes of Clusterin- and S100B-transfected neuronal cells elucidate protective mechanisms against hypoxia and oxidative stress in the hooded seal (Cystophora cristata) brain.

BACKGROUND: The hooded seal (Cystophora cristata) exhibits impressive diving skills and can tolerate extended durations of asphyxia, hypoxia and oxidative stress, without suffering from irreversible neuronal damage. Thus, when exposed to hypoxia in vitro, neurons of fresh cortical and hippocampal tissue from hooded seals maintained their membrane potential 4-5 times longer than neurons of mice. We aimed to identify the molecular mechanisms underlying the intrinsic neuronal hypoxia tolerance. Previous comparative transcriptomics of the visual cortex have revealed that S100B and clusterin (apolipoprotein J), two stress proteins that are involved in neurological disorders characterized by hypoxic conditions, have a remarkably high expression in hooded seals compared to ferrets. When overexpressed in murine neuronal cells (HN33), S100B and clusterin had neuroprotective effects when cells were exposed to hypoxia. However, their specific roles in hypoxia have remained largely unknown. METHODS: In order to shed light on potential molecular pathways or interaction partners, we exposed HN33 cells transfected with either S100B, soluble clusterin (sCLU) or nuclear clusterin (nCLU) to normoxia, hypoxia and oxidative stress for 24 h. We then determined cell viability and compared the transcriptomes of transfected cells to control cells. Potential pathways and upstream regulators were identified via Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). RESULTS: HN33 cells transfected with sCLU and S100B demonstrated improved glycolytic capacity and reduced aerobic respiration at normoxic conditions. Additionally, sCLU appeared to enhance pathways for cellular homeostasis to counteract stress-induced aggregation of proteins. S100B-transfected cells sustained lowered energy-intensive synaptic signaling. In response to hypoxia, hypoxia-inducible factor (HIF) pathways were considerably elevated in nCLU- and sCLU-transfected cells. In a previous study, S100B and sCLU decreased the amount of reactive oxygen species and lipid peroxidation in HN33 cells in response to oxidative stress, but in the present study, these functional effects were not mirrored in gene expression changes. CONCLUSIONS: sCLU and S100B overexpression increased neuronal survival by decreasing aerobic metabolism and synaptic signaling in advance to hypoxia and oxidative stress conditions, possibly to reduce energy expenditure and the build-up of deleterious reactive oxygen species (ROS). Thus, a high expression of CLU isoforms and S100B is likely beneficial during hypoxic conditions.

Pro-Apoptotic and Anti-Invasive Properties Underscore the Tumor-Suppressing Impact of Myoglobin on a Subset of Human Breast Cancer Cells.

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

Radiation-response in primary fibroblasts of long-term survivors of childhood cancer with and without second primary neoplasms: the KiKme study.

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 log2 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+.

Knockout of Factor-Inhibiting HIF (Hif1an) in Colon Epithelium Attenuates Chronic Colitis but Does Not Reduce Colorectal Cancer in Mice.

Inflammatory bowel disease such as chronic colitis promotes colorectal cancer, which is a common cause of cancer mortality worldwide. Hypoxia is a characteristic of inflammation as well as of solid tumors and enforces a gene expression response controlled by hypoxia-inducible factors (HIFs). Once established, solid tumors are immunosuppressive to escape their abatement through immune cells. Although HIF activity is known to 1) promote cancer development and 2) drive tumor immune suppression through the secretion of adenosine, both prolyl hydroxylases and an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) negatively regulate HIF. Thus, FIH may act as a tumor suppressor in colorectal cancer development. In this study, we examined the role of colon epithelial FIH in a mouse model of colitis-induced colorectal cancer. We recapitulated colitis-associated colorectal cancer development in mice using the azoxymethane/dextran sodium sulfate model in Vil1-Cre/FIH+f/+f and wild-type siblings. Colon samples were analyzed regarding RNA and protein expression and histology. Vil1-Cre/FIH+f/+f mice showed a less severe colitis progress compared with FIH+f/+f animals and a lower number of infiltrating macrophages in the inflamed tissue. RNA sequencing analyses of colon tissue revealed a lower expression of genes associated with the immune response in Vil1-Cre/FIH+f/+f mice. However, tumor occurrence did not significantly differ between Vil1-Cre/FIH+f/+f and wild-type mice. Thus, FIH knockout in colon epithelial cells did not modulate colorectal cancer development but reduced the inflammatory response in chronic colitis.

Identification of Genetic Predispositions Related to Ionizing Radiation in Primary Human Skin Fibroblasts From Survivors of Childhood and Second Primary Cancer as Well as Cancer-Free Controls: Protocol for the Nested Case-Control Study KiKme.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32395.

Myoglobin Protects Breast Cancer Cells Due to Its ROS and NO Scavenging Properties.

Myoglobin (MB) is an oxygen-binding protein usually found in cardiac myocytes and skeletal muscle fibers. It may function as a temporary storage and transport protein for O2 but could also have scavenging capacity for reactive oxygen and nitrogen species. In addition, MB has recently been identified as a hallmark in luminal breast cancer and was shown to be robustly induced under hypoxia. Cellular responses to hypoxia are regulated by the transcription factor hypoxia-inducible factor (HIF). For exploring the function of MB in breast cancer, we employed the human cell line MDA-MB-468. Cells were grown in monolayer or as 3D multicellular spheroids, which mimic the in vivo avascular tumor architecture and physiology with a heterogeneous cell population of proliferating cells in the rim and non-cycling or necrotic cells in the core region. This central necrosis was increased after MB knockdown, indicating a role for MB in hypoxic tumor regions. In addition, MB knockdown caused higher levels of HIF-1α protein after treatment with NO, which also plays an important role in cancer cell survival. MB knockdown also led to higher reactive oxygen species (ROS) levels in the cells after treatment with H2O2. To further explore the role of MB in cell survival, we performed RNA-Seq after MB knockdown and NO treatment. 1029 differentially expressed genes (DEGs), including 45 potential HIF-1 target genes, were annotated in regulatory pathways that modulate cellular function and maintenance, cell death and survival, and carbohydrate metabolism. Of these target genes, TMEFF1, TREX2, GLUT-1, MKNK-1, and RAB8B were significantly altered. Consistently, a decreased expression of GLUT-1, MKNK-1, and RAB8B after MB knockdown was confirmed by qPCR. All three genes of interest are often up regulated in cancer and correlate with a poor clinical outcome. Thus, our data indicate that myoglobin might influence the survival of breast cancer cells, possibly due to its ROS and NO scavenging properties and could be a valuable target for cancer therapy.

Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.

Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Correction to: Molecular karyotyping and gene expression analysis in childhood cancer patients.

The correct Author names are presented in this paper.

Spontaneous and Radiation-Induced Chromosome Aberrations in Primary Fibroblasts of Patients With Pediatric First and Second Neoplasms.

The purpose of the present study was to investigate whether former childhood cancer patients who developed a subsequent secondary primary neoplasm (SPN) are characterized by elevated spontaneous chromosomal instability or cellular and chromosomal radiation sensitivity as surrogate markers of compromised DNA repair compared to childhood cancer patients with a first primary neoplasm (FPN) only or tumor-free controls. Primary skin fibroblasts were obtained in a nested case-control study including 23 patients with a pediatric FPN, 22 matched patients with a pediatric FPN and an SPN, and 22 matched tumor-free donors. Clonogenic cell survival and cytogenetic aberrations in Giemsa-stained first metaphases were assessed after X-irradiation in G1 or on prematurely condensed chromosomes of cells irradiated and analyzed in G2. Fluorescence in situ hybridization was applied to investigate spontaneous transmissible aberrations in selected donors. No significant difference in clonogenic survival or the average yield of spontaneous or radiation-induced aberrations was found between the study populations. However, two donors with an SPN showed striking spontaneous chromosomal instability occurring as high rates of numerical and structural aberrations or non-clonal and clonal translocations. No correlation was found between radiation sensitivity and a susceptibility to a pediatric FPN or a treatment-associated SPN. Together, the results of this unique case-control study show genomic stability and normal radiation sensitivity in normal somatic cells of donors with an early and high intrinsic or therapy-associated tumor risk. These findings provide valuable information for future studies on the etiology of sporadic childhood cancer and therapy-related SPN as well as for the establishment of predictive biomarkers based on altered DNA repair processes.

The role of myoglobin in epithelial cancers: Insights from transcriptomics.

The muscle­associated respiratory protein myoglobin (MB) is expressed in multiple types of cancer, including breast and prostate tumors. In Kaplan­Meier analyses of the two tumor types, MB positivity is associated with favorable prognoses. Despite its well­characterized function in myocytes, the role of MB in cancer remains unclear. To study the impact of endogenous MB expression, small interfering RNA MB­knockdown cells were engineered using breast, prostate and colon cancer cell lines (MDA­MB468, LNCaP, DLD­1), and their transcriptomes were investigated using RNA­Seq at different oxygen levels. In MB­positive cells, increased expression of glycolytic genes was observed, which was possibly mediated by a higher activity of hypoxia­inducible factor 1α. In addition, the results of the gene set enrichment analysis suggested that MB contributed to fatty acid transport and turnover. MB­positive, wild­type­p53 LNCaP cells also exhibited increased expression of p53 target genes involved in cell cycle checkpoint control and prevention of cell migration. MB­positive cells expressing mutant p53 exhibited upregulation of genes associated with prolonged cancer cell viability and motility. Therefore, it was hypothesized that these transcriptomic differences may result from MB­mediated generation of nitric oxide or reactive oxygen species, thus employing established enzymatic activities of the globin. In summary, the transcriptome comparisons identified potential molecular functions of MB in carcinogenesis by highlighting the interaction of MB with key metabolic and regulatory processes.

Loss of Neuroglobin Expression Alters Cdkn1a/Cdk6-Expression Resulting in Increased Proliferation of Neural Stem Cells.

In the quest to unravel its functional significance, neuroglobin (Ngb), a brain-specific neuroprotective protein, has recently been proposed as an actor in neurodevelopment. As neural stem cells (NSCs) are fundamental during brain development, the present study aimed at investigating the role of Ngb in the growth and proliferation of NSCs by comparing an Ngb-floxed (Ngbfl-)NSC line, equivalent to the wild-type cellular situation, with an in-house created Ngb knockout (NgbKO-)NSC line. NgbKO-NSCs were characterized by an increased growth and proliferation capacity in vitro, supported by RNA sequencing and western blot results reporting the downregulation of Cdkn1a and the upregulation of Cdk6, both enhancing the cell cycle. Based on additional gene ontology enrichment and pathway analyses, we hypothesize that the loss of Ngb affects multiple cellular signaling pathways with the most important being the Akt-Tp53 axis.