Sub-Sets of Cancer Stem Cells Differ Intrinsically in Their Patterns of Oxygen Metabolism

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Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

Background: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIFprolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.Fil:Acevedo, J.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Centanin, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Dekanty, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Wappner, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Achieving a Preoperative Target HbA1c of < 69 mmol/mol in Elective Vascular and Orthopedic Surgery: A Retrospective Single Center Observational Study

Introduction Diabetes mellitus (DM) is present in 10–15% of the surgical population. It is a known risk factor for adverse postoperative outcomes. UK perioperative guidance recommends optimizing glycemic control preoperatively, aiming for a target glycated hemoglobin (HbA1c) of < 69 mmol/mol. However, real-world compliance with this guidance remains unknown. The aim of our study was to determine how many patients with DM undergoing elective orthopedic and vascular surgery had a preoperative HbA1c of < 69 mmol/mol. We also reviewed the surgical reasons for non-concordance with the recommended preoperative HbA1c target. Methods This was a retrospective observational study of 1000 consecutive patients who had been referred for elective vascular and orthopedic surgery at a large tertiary center. Data were collected on these patients, both those with and without DM, between January 2016 and February 2017. Electronic databases were used to collect information on the patients’ preoperative HbA1c concentration and to determine whether there was a resulting delay in surgery when the preoperative HbA1c target of < 69 mmol/mol was exceeded. Results Of the 1000 patients referred for surgery (500 orthopedic and 500 vascular patients) included in the study, 201 (20%) had diabetes. Among these 201 people with DM, 155 (77%) had a preoperative HbA1c < 69 mmol/mol. Among the 46 people with DM whose HbA1c exceeded the recommended target, 41 were operated on despite the high HbA1c level, and only five had their surgery deferred or canceled due to suboptimal preoperative glycemic control. Conclusions Our data shows that the majority (77% ) of people undergoing elective vascular and orthopedic surgery were able to achieve a target HbA1c of < 69 mmol/mol. The current preoperative guidance is therefore achievable in a real-life setting. However, as is stated in the national guidance, this target should only be used where it is safe to do so and a degree of clinical discretion is necessary

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Anti-Angiogenic Therapy Induces Integrin-Linked Kinase 1 Up-Regulation in a Mouse Model of Glioblastoma

BACKGROUND: In order to improve our understanding of the molecular pathways that mediate tumor proliferation and angiogenesis, and to evaluate the biological response to anti-angiogenic therapy, we analyzed the changes in the protein profile of glioblastoma in response to treatment with recombinant human Platelet Factor 4-DLR mutated protein (PF4-DLR), an inhibitor of angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: U87-derived experimental glioblastomas were grown in the brain of xenografted nude mice, treated with PF4-DLR, and processed for proteomic analysis. More than fifty proteins were differentially expressed in response to PF4-DLR treatment. Among them, integrin-linked kinase 1 (ILK1) signaling pathway was first down-regulated but then up-regulated after treatment for prolonged period. The activity of PF4-DLR can be increased by simultaneously treating mice orthotopically implanted with glioblastomas, with ILK1-specific siRNA. As ILK1 is related to malignant progression and a poor prognosis in various types of tumors, we measured ILK1 expression in human glioblastomas, astrocytomas and oligodendrogliomas, and found that it varied widely; however, a high level of ILK1 expression was correlated to a poor prognosis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that identifying the molecular pathways induced by anti-angiogenic therapies may help the development of combinatorial treatment strategies that increase the therapeutic efficacy of angiogenesis inhibitors by association with specific agents that disrupt signaling in tumor cells

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases

Adenovirus-mediated hypoxia-targeting cytosine deaminase gene therapy enhances radiotherapy in tumour xenografts

Hypoxia is closely associated with the radioresistance of tumours; therefore, targeting hypoxic areas is very important for cancer therapy. The aim of this study is to establish such a targeting strategy by applying a bacterial cytosine deaminase (BCD)/5-fluorocytosine (5-FC) gene therapy system and to examine whether the strategy enhances the efficacy of radiotherapy in a tumour xenograft. The hypoxia-responsive promoter 5HREp, in which five copies of the hypoxia-response element (HRE) enhance transcription from a cytomegalovirus minimal promoter, was employed to induce the expression of BCD under hypoxic conditions. The adenoviral vector Ad/5HREp-BCD, encoding the gene 5HREp-BCD, robustly induced BCD expression under hypoxic conditions and this led to significant cytotoxicity in combination with 5-FC in vitro. Intratumoral Ad/5HREp-BCD administration resulted in the expression of BCD at the border between normoxic and necrotic regions. The BCD/5-FC gene therapy enhanced the therapeutic effects of both single (12.5 Gy) and fractionated (3 Gy × 5 days) radiotherapy with few side effects and significantly increased tumour growth doubling time by up to 2.4-fold (P<0.01) and 2.5-fold (P<0.05), respectively. All of these results suggest that the present BCD/5-FC gene therapy has the ability to specifically target hypoxic tumour cells and significantly improves the control of tumour growth after radiotherapy

Drosophila Genome-Wide RNAi Screen Identifies Multiple Regulators of HIF–Dependent Transcription in Hypoxia

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke

The spatial scale of competition from recruits on an older cohort in Atlantic salmon

Competitive effects of younger cohorts on older ones are frequently assumed to be negligible in species where older, larger individuals dominate in pairwise behavioural interactions. Here, we provide field estimates of such competition by recruits on an older age class in Atlantic salmon (Salmo salar), a species where observational studies have documented strong body size advantages which should favour older individuals in direct interactions. By creating realistic levels of spatial variation in the density of underyearling (YOY) recruits over a 1-km stretch of a stream, and obtaining accurate measurements of individual growth rates of overyearlings (parr) from capture–mark–recapture data on a fine spatial scale, we demonstrate that high YOY density can substantially decrease parr growth. Models integrating multiple spatial scales indicated that parr were influenced by YOY density within 16 m. The preferred model suggested parr daily mass increase to be reduced by 39% when increasing YOY density from 0.0 to 1.0 m−2, which is well within the range of naturally occurring densities. Reduced juvenile growth rates will in general be expected to reduce juvenile survival (via increased length of exposure to freshwater mortality) and increase generation times (via increased age at seaward migrations). Thus, increased recruitment can significantly affect the performance of older cohorts, with important implications for population dynamics. Our results highlight that, even for the wide range of organisms that rely on defendable resources, the direction of competition among age classes cannot be assumed a priori or be inferred from behavioural observations alone