Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

Background: Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results: This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. Conclusion: This study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene

The pyrenoidal linker protein EPYC1 phase separates with hybrid Arabidopsis–Chlamydomonas Rubisco through interactions with the algal Rubisco small subunit

Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein–protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoidlike aggregation for Rubisco derived from a higher plant

Hypoxia Regulates BMP4 Expression in the Murine Spleen during the Recovery from Acute Anemia

Bone marrow erythropoiesis is primarily homeostatic, producing new erythrocytes at a constant rate. However at times of acute anemia, new erythrocytes must be rapidly produced much faster than bone marrow steady state erythropoiesis. At these times stress erythropoiesis predominates. Stress erythropoiesis occurs in the fetal liver during embryogenesis and in the adult spleen and liver. In adult mice, stress erythropoiesis utilizes a specialized population of stress erythroid progenitors that are resident in the spleen. In response to acute anemia, these progenitors rapidly expand and differentiate in response to three signals, BMP4, SCF and hypoxia. In absence of acute anemic stress, two of these signals, BMP4 and hypoxia, are not present and the pathway is not active. The initiating event in the activation of this pathway is the up-regulation of BMP4 expression in the spleen.In this paper we analyze the regulation of BMP4 expression in the spleen by hypoxia. Using stromal cell lines, we establish a role for hypoxia transcription factor HIFs (Hypoxia Inducible Factors) in the transcription of BMP4. We identified putative Hypoxia Responsive Elements (HREs) in the BMP4 gene using bioinformatics. Analysis of these elements showed that in vivo, Hif2alpha binds two cis regulatory sites in the BMP4 gene, which regulate BMP4 expression during the recovery from acute anemia.These data show that hypoxia plays a key role in initiating the BMP4 dependent stress erythropoiesis pathway by regulating BMP4 expression

The ancestral role of ATP hydrolysis in type II topoisomerases: prevention of DNA double-strand breaks

Type II DNA topoisomerases (topos) catalyse changes in DNA topology by passing one double-stranded DNA segment through another. This reaction is essential to processes such as replication and transcription, but carries with it the inherent danger of permanent double-strand break (DSB) formation. All type II topos hydrolyse ATP during their reactions; however, only DNA gyrase is able to harness the free energy of hydrolysis to drive DNA supercoiling, an energetically unfavourable process. A long-standing puzzle has been to understand why the majority of type II enzymes consume ATP to support reactions that do not require a net energy input. While certain type II topos are known to ‘simplify’ distributions of DNA topoisomers below thermodynamic equilibrium levels, the energy required for this process is very low, suggesting that this behaviour is not the principal reason for ATP hydrolysis. Instead, we propose that the energy of ATP hydrolysis is needed to control the separation of protein–protein interfaces and prevent the accidental formation of potentially mutagenic or cytotoxic DSBs. This interpretation has parallels with the actions of a variety of molecular machines that catalyse the conformational rearrangement of biological macromolecules

Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer

Hypoxia and angiogenesis are important factors in breast cancer progression. Little is known of hypoxia and angiogenesis in lymph node metastases of breast cancer. The aim of this study was to quantify hypoxia, by hypoxia-induced marker expression levels, and angiogenesis, by endothelial cell proliferation, comparing primary breast tumours and axillary lymph node metastases. Tissue sections of the primary tumour and a lymph node metastasis of 60 patients with breast cancer were immunohistochemically stained for the hypoxia-markers carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1α (Hif-1α) and DEC-1 and for CD34/Ki-67. Endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. On haematoxylin–eosin stain, the growth pattern and the presence of a fibrotic focus were assessed. Hypoxia-marker expression, ECP% and TCP% in primary tumours and in lymph node metastases were correlated to each other and to clinico-pathological variables. Median ECP% and TCP% in primary tumours and lymph node metastases were comparable (primary tumours: ECP%=4.02, TCP%=19.54; lymph node metastases: ECP%=5.47, TCP%=21.26). ECP% correlated with TCP% (primary tumours: r=0.63, P<0.001; lymph node metastases: r=0.76, P<0.001). CA9 and Hif-1α expression were correlated (primary tumours P=0.005; lymph node metastases P<0.001). In primary tumours, CA9 and Hif-1α expression were correlated with DEC-1 expression (P=0.05), presence of a fibrotic focus (P<0.007) and mixed/expansive growth pattern (P<0.001). Primary tumours and lymph node metastases with CA9 or Hif-1α expression had a higher ECP% and TCP% (P<0.003); in primary tumours, mixed/expansive growth pattern and fibrotic focus were characterised by higher ECP% (P=0.03). Furthermore, between primary tumours and lymph node metastases a correlation was found for ECP%, TCP%, CA9 and Hif-1α expression (ECP% r=0.51, P<0.001; TCP r=0.77, P<0.001; CA9 and Hif-1α P<0.001). Our data demonstrate that the growth of breast cancer lymph node metastases is angiogenesis dependent and that angiogenesis and hypoxia in the primary tumour predict angiogenesis and hypoxia in the lymph node metastases. Together with previous findings in breast cancer liver metastases, which grow in 96% of cases angiogenesis independently, these data suggest that both the intrinsic growth characteristics and angiogenic potential of breast cancer cells and the site-specific tumour microenvironment determine angiogenesis and hypoxia in breast cancer

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [&lt;1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Hif1a inactivation rescues photoreceptor degeneration induced by a chronic hypoxia-like stress

Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients