RESUMEN
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.
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Carcinoma , Mioglobina , Animales , Ratones , Mioglobina/genética , Biopsia , Modelos Animales de Enfermedad , Hipoxia/genética , Ratones NoqueadosRESUMEN
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.
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Ácidos Grasos , Metabolismo de los Lípidos , Animales , Extractos Celulares , Células Epiteliales/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , Mioglobina/metabolismo , Oxígeno/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
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.
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Neoplasias de la Mama , Mioglobina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclinas/metabolismo , Doxorrubicina/farmacología , Transición Epitelial-Mesenquimal , Femenino , Humanos , Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
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.
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Neoplasias Asociadas a Colitis/patología , Colitis/patología , Neoplasias Colorrectales/patología , Mucosa Intestinal/patología , Oxigenasas de Función Mixta/metabolismo , Adenosina/metabolismo , Animales , Azoximetano/toxicidad , Hipoxia de la Célula/fisiología , Colitis/inducido químicamente , Colitis/genética , Neoplasias Asociadas a Colitis/genética , Colon/patología , Neoplasias Colorrectales/genética , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Células Epiteliales/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxigenasas de Función Mixta/genética , Prolil Hidroxilasas/metabolismo , Transducción de Señal/fisiología , Escape del Tumor/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
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.
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Neoplasias de la Mama/patología , Mioglobina/fisiología , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Depuradores de Radicales Libres/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Mioglobina/genética , Mioglobina/metabolismo , Sustancias Protectoras/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the "browning" BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.
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Gotas Lipídicas/metabolismo , Mitocondrias/metabolismo , Mioglobina/genética , Oxígeno/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Músculo Esquelético/metabolismo , Mioglobina/metabolismo , PPAR alfa/genética , Palmitatos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Proteínas/genética , Termogénesis/genética , Proteína Desacopladora 1/genéticaRESUMEN
Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis-the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)-were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of â¼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.
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Ascomicetos/genética , Genoma Fúngico , Líquenes/genética , Metagenoma , Simbiosis , Ascomicetos/crecimiento & desarrollo , Líquenes/crecimiento & desarrollo , FilogeniaRESUMEN
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.
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Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Mioglobina/genética , Neoplasias de la Próstata/genética , Carcinogénesis/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , TranscriptomaRESUMEN
The blind subterranean mole rat Spalax shows a remarkable tolerance to hypoxia, cancer-resistance and longevity. Unravelling the genomic basis of these adaptations will be important for biomedical applications. RNA-Seq gene expression data were obtained from normoxic and hypoxic Spalax and rat liver tissue. Hypoxic Spalax broadly downregulates genes from major liver function pathways. This energy-saving response is likely a crucial adaptation to low oxygen levels. In contrast, the hypoxia-sensitive rat shows massive upregulation of energy metabolism genes. Candidate genes with plausible connections to the mole rat's phenotype, such as important key genes related to hypoxia-tolerance, DNA damage repair, tumourigenesis and ageing, are substantially higher expressed in Spalax than in rat. Comparative liver transcriptomics highlights the importance of molecular adaptations at the gene regulatory level in Spalax and pinpoints a variety of starting points for subsequent functional studies.
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Hipoxia/metabolismo , Ratas Topo/genética , Ratas Topo/fisiología , Adaptación Fisiológica/genética , Envejecimiento/genética , Animales , Reparación del ADN , Tolerancia a Medicamentos/fisiología , Metabolismo Energético/fisiología , Hipoxia/fisiopatología , Tolerancia Inmunológica/fisiología , Hígado/metabolismo , Longevidad/genética , Longevidad/fisiología , Ratas , Análisis de Secuencia de ARN , Spalax/genética , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient's HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Genes MHC Clase I , Melanoma/genética , Melanoma/inmunología , Mutación , Linfocitos T/inmunología , Linfocitos T/metabolismo , Alelos , Animales , Presentación de Antígeno , Línea Celular Tumoral , Biología Computacional/métodos , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Melanoma/patología , Ratones , Metástasis de la Neoplasia , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , TranscriptomaRESUMEN
BACKGROUND/AIM: Ectopic myoglobin (MB) expression, mediated by alternative and hypoxia-inducible transcription, has recently been demonstrated in several epithelial tumours. This study aimed to examine the expression of MB in hormone-independent head and neck squamous cell carcinomas (HNSCCs). PATIENTS AND METHODS: Using imunohistochemistry, ectopic MB expression was analyzed on tissue microarrays (TMAs) of 524 patients with localized and locally advanced primary and recurrent HNSCC who had undergone surgical treatment with curative intent. Associations of MB expression with survival and clinicopathological parameters were analyzed. RESULTS: MB expression was found in 45.8% of HNSCC patients being significantly lower in normal adjacent tissue (NAT) compared to primary and recurrent tumours (p<0.001) and significantly associated with a favourable overall survival (OS) in HNSCC [p=0.037, hazard ratio (HR)=0.72, 95% confidence interval (CI)=0.53-0.98]. Furthermore, MB expression negatively correlated with human papillomavirus (HPV) status (p=0.013). CONCLUSION: MB is differentially expressed in HNSCC and correlates with a better OS.
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Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Mioglobina/metabolismo , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
Neuroglobin (Ngb) is a respiratory protein that is almost exclusively expressed in the vertebrate nervous system. Despite many years of research, the exact function and even the expression sites of Ngb are still a matter of debate. However, to investigate hypotheses surrounding the potential roles of Ngb, a detailed knowledge of its major and minor expression sites is indispensable. We have therefore evaluated Ngb expression by extensive bioinformatic analysis using publicly available transcriptome data (RNA-Seq). During mammalian brain development, we observed low embryonic expression of Ngb mRNA and an increase after birth, arguing against a role of Ngb in fetal hypoxia tolerance. In adult mouse brain, we found highest Ngb mRNA levels in the hypothalamus, where expression was up to 100-fold stronger than in cerebral cortex, cerebellum or hippocampus, as confirmed by qRT-PCR and Western blotting. High Ngb expression in the hypothalamus was found conserved in humans and other mammals. Thus, Ngb mRNA is expressed at a basal level in many mammalian brain regions, but shows distinctive regional peaks. RNA-Seq analysis further revealed only low levels of Ngb mRNA in retina and testes and no signal in standard tumor cell lines, thus raising questions concerning previous studies and functional hypotheses. In conclusion, this broad-scale expression study may point to distinct Ngb functions for high- and low-expressing cells and tissues and argues against a single, generic role of Ngb as an oxygen supplier or as an endogenous protectant in all nerve cells.
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Corteza Cerebral/metabolismo , Globinas/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Cerebelo/metabolismo , Mamíferos , Ratones , Neuroglobina , ARN Mensajero/metabolismoRESUMEN
Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.
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Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/genética , Proteínas de Neoplasias/inmunología , Humanos , Melanoma/patología , Metástasis de la NeoplasiaRESUMEN
Myoglobin (MB) is not only strongly expressed in myocytes, but also at much lower levels in different cancer entities. 40% of breast tumors are MB-positive, with the globin being co-expressed with markers of tumor hypoxia in a proportion of cases. In breast cancer, MB expression is associated with a positive hormone receptor status and patient prognosis. In prostate cancer, another hormone-dependent cancer type, 53% of tumors were recently shown to express MB. Especially in more aggressive prostate cancer specimen MB expression also correlates with increased patient survival rates. Both findings might be due to tumor-suppressing properties of MB in cancer cells. In contrast to muscle, MB transcription in breast and prostate cancer mainly depends on a novel, alternative promoter site. We show here that its associated transcripts can be upregulated by hypoxia and downregulated by estrogens and androgens in MCF7 breast and LNCaP prostate cancer cells, respectively. Bioinformatic data mining of epigenetic histone marks and experimental verification reveal a hitherto uncharacterized transcriptional network that drives the regulation of the MB gene in cancer cells. We identified candidate hormone-receptor binding elements that may interact with the cancer-associated MB promoter to decrease its activity in breast and prostate cancer cells. Additionally, a regulatory element, 250 kb downstream of the promoter, acts as a hypoxia-inducible site within the transcriptional machinery. Understanding the distinct regulation of MB in tumors will improve unraveling the respiratory protein's function in the cancer context and may provide new starting points for developing therapeutic strategies.
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Neoplasias de la Mama/genética , Redes Reguladoras de Genes , Mioglobina/genética , Mioglobina/metabolismo , Neoplasias de la Próstata/genética , Sitios de Unión , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Cromatina/metabolismo , Biología Computacional , Minería de Datos , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Reguladores , Genes Reporteros , Humanos , Masculino , Pronóstico , Neoplasias de la Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Recent studies identified unexpected expression and transcriptional complexity of the hemoprotein myoglobin (MB) in human breast cancer but its role in prostate cancer is still unclear. Expression of MB was immunohistochemically analyzed in three independent cohorts of radical prostatectomy specimens (n = 409, n = 625, and n = 237). MB expression data were correlated with clinicopathological parameters and molecular parameters of androgen and hypoxia signaling. Expression levels of novel tumor-associated MB transcript variants and the VEGF gene as a hypoxia marker were analyzed using qRT-PCR. Fifty-three percent of the prostate cancer cases were MB positive and significantly correlated with androgen receptor (AR) expression (p < 0.001). The positive correlation with CAIX (p < 0.001) and FASN (p = 0.008) as well as the paralleled increased expression of the tumor-associated MB transcript variants and VEGF suggest that hypoxia participates in MB expression regulation. Analogous to breast cancer, MB expression in prostate cancer is associated with steroid hormone signaling and markers of hypoxia. Further studies must elucidate the novel functional roles of MB in human carcinomas, which probably extend beyond its classic intramuscular function in oxygen storage.
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Mioglobina/biosíntesis , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/biosíntesis , Anciano , Biomarcadores de Tumor/análisis , Hipoxia de la Célula/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
The blind mole rat (BMR), Spalax galili, is an excellent model for studying mammalian adaptation to life underground and medical applications. The BMR spends its entire life underground, protecting itself from predators and climatic fluctuations while challenging it with multiple stressors such as darkness, hypoxia, hypercapnia, energetics and high pathonecity. Here we sequence and analyse the BMR genome and transcriptome, highlighting the possible genomic adaptive responses to the underground stressors. Our results show high rates of RNA/DNA editing, reduced chromosome rearrangements, an over-representation of short interspersed elements (SINEs) probably linked to hypoxia tolerance, degeneration of vision and progression of photoperiodic perception, tolerance to hypercapnia and hypoxia and resistance to cancer. The remarkable traits of the BMR, together with its genomic and transcriptomic information, enhance our understanding of adaptation to extreme environments and will enable the utilization of BMR models for biomedical research in the fight against cancer, stroke and cardiovascular diseases.
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Adaptación Fisiológica/genética , Evolución Molecular , Genoma , Hipercapnia , Hipoxia , Spalax/genética , Estrés Fisiológico , Transcriptoma/genética , Animales , Oscuridad , Perfilación de la Expresión Génica , Edición de ARN/genética , Elementos de Nucleótido Esparcido CortoRESUMEN
Recently, the ectopic expression of myoglobin (MB) was reported in human epithelial cancer cell lines and breast tumor tissues, where MB expression increased with hypoxia. The better prognosis of MB-positive breast cancer patients suggested that the globin exerts a tumor-suppressive role, possibly by impairing mitochondrial activity in hypoxic breast carcinoma cells. To better understand MB gene regulation in cancer, we systematically investigated the architecture of the human MB gene, its transcripts and promoters. In silico analysis of transcriptome data from normal human tissues and cancer cell lines, followed by RACE-PCR verification, revealed seven novel exons in the MB gene region, most of which are untranslated exons located 5'-upstream of the coding DNA sequence (CDS). Sixteen novel alternatively spliced MB transcripts were detected, most of which predominantly occur in tumor tissue or cell lines. Quantitative RT-PCR analyses of MB expression in surgical breast cancer specimen confirmed the preferential usage of a hitherto unknown, tumor-associated MB promoter, which was functionally validated by luciferase reporter gene assays. In line with clinical observations of MB up-regulation in avascular breast tumors, the novel cancer-associated MB splice variants exhibited increased expression in tumor cells subjected to experimental hypoxia. The novel gene regulatory mechanisms unveiled in this study support the idea of a non-canonical role of MB during carcinogenesis.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula , Mioglobina/genética , Adenocarcinoma/genética , Empalme Alternativo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Exones , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Mitocondrias/metabolismo , Mioglobina/metabolismo , Filogenia , Regiones Promotoras Genéticas , TranscriptomaRESUMEN
Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O(2) uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O(2) consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O(2). Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.