Project description:Breast cancer stem cells (BCSCs) have been shown to contribute to tumor growth, metastasis, and recurrence. They are also markedly resistant to conventional cancer treatments, such as chemotherapy and radiation. Recent studies have suggested that hypoxia is one of the prominent micro-environmental factors that increase the self-renewal ability of BCSCs, partially by enhancing CSC phenotypes. Thus, the identification and development of new therapeutic approaches based on targeting the hypoxia-dependent responses in BCSCs is urgent. Through various in vitro studies, we found that hypoxia specifically up-regulates BCSC sphere formation and a subset of CD44+/CD24-/low CSCs. Hypoxia inducible factors 2? (HIF2?) depletion suppressed CSC-like phenotypes and CSC-mediated drug resistance in breast cancer. Furthermore, the stimulatory effects of hypoxia-induced HIF2? on BCSC sphere formation were successfully attenuated by epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) knockdown. Taken together, these data suggest that HIF2? mediates hypoxia-induced cancer growth/metastasis and that EFEMP1 is a downstream effector of hypoxia-induced HIF2? during breast tumorigenesis.
Project description:Hyperactivation of Notch signaling and the cellular hypoxic response are frequently observed in cancers, with increasing reports of connections to tumor initiation and progression. The two signaling mechanisms are known to intersect, but while it is well established that hypoxia regulates Notch signaling, less is known about whether Notch can regulate the cellular hypoxic response. We now report that Notch signaling specifically controls expression of HIF2?, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2? by Notch under normoxic conditions leads to elevated HIF2? protein levels in primary breast cancer cells as well as in human breast cancer, medulloblastoma, and renal cell carcinoma cell lines. The elevated level of HIF2? protein was in certain tumor cell types accompanied by downregulation of HIF1? protein levels, indicating that high Notch signaling may drive a HIF1?-to-HIF2? switch. At the transcriptome level, the presence of HIF2? was required for approximately 21% of all Notch-induced genes: among the 1062 genes that were upregulated by Notch in medulloblastoma cells during normoxia, upregulation was abrogated in 227 genes when HIF2? expression was knocked down by HIF2? siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2?, which may be important for future cancer therapies.
Project description:Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.
Project description:While the functions of hypoxia-inducible factor 1? (HIF1?)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2?/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2? protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2?, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2? knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.
Project description:BACKGROUND:During mammalian cerebral cortex development, different types of projection neurons are produced in a precise temporal order and in stereotypical numbers. The mechanisms regulating timely generation of neocortex projection neurons and ensuring production in sufficient numbers of each neuronal identity are only partially understood. RESULTS:Here, we show that ephrin-B2, a member of the Eph:ephrin cell-to-cell communication pathway, sets the neurogenic tempo in the neocortex. Indeed, conditional mutant embryos for ephrin-B2 exhibit a transient delay in neurogenesis and acute stimulation of Eph signaling by in utero injection of synthetic ephrin-B2 led to a transient increase in neuronal production. Using genetic approaches we show that ephrin-B2 acts on neural progenitors to control their differentiation in a juxtacrine manner. Unexpectedly, we observed that perinatal neuron numbers recovered following both loss and gain of ephrin-B2, highlighting the ability of neural progenitors to adapt their behavior to the state of the system in order to produce stereotypical numbers of neurons. CONCLUSIONS:Altogether, our data uncover a role for ephrin-B2 in embryonic neurogenesis and emphasize the plasticity of neuronal production in the neocortex.
Project description:N4-acetylcytidine (ac4C) is a highly conserved RNA modification and is the first acetylation event described in mRNA. ac4C in mRNA has been demonstrated to be involved in the regulation of mRNA stability, processing and translation, but the exact means by which ac4C works remain unclear. In addition, ac4C is widely distributed within the human transcriptome at physiologically relevant levels and so far only a small fraction of modified sequences have been detected by experiments. In this study, we developed a predictor of ac4C sites in human mRNA named PACES to help mining possible modified motifs. PACES combines two random forest classifiers, position-specific dinucleotide sequence profile and K-nucleotide frequencies. With genomic sequences as input, PACES gives possible modified sequences based on the training model. PACES is freely available at http://www.rnanut.net/paces/.
Project description:Hypoxia-inducible factor (HIF) 1? and HIF2? and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2? protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2? reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2? morphants. The phenotypes induced by HIF2? depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2?. Chromatin immunoprecipitation assay revealed that HIF2? binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2?, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2? morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2? depletion is due in part to the loss of survivin activity.
Project description:Hypoxic-ischemic (HI) encephalopathy is the major cause of mortality and disability in newborns. The neurovascular unit is a major target of acute and chronic brain injury, and therapies that protect simultaneously both neurons and vascular endothelial cells from neonatal HI injury are in demand. Insulin receptors and its key downstream molecule-insulin receptor substrate -1 (IRS-1) are potential neuroprotective targets and expressed both in neuron and endothelial cells. To investigate whether IRS-1 can act similarly in neurons and vascular endothelial cells in protecting neurovascular units and brain form HI injury, we found that neuron-specific IRS-1 transgenic rats showed reduced neurovascular injury and infarct volumes, whereas endothelial-specific IRS-1 transgenic rats showed increased blood-brain barrier (BBB) disruption and exaggerated neurovascular injury after neonatal HI brain injury. Endothelial-specific IRS-1 overexpression increased vascular permeability and disassembled the tight junction protein (zonula occludens-1) complex. Inhibition of mammalian target of rapamycin (mTOR) by rapamycin preserved tight junction proteins and attenuated BBB leakage and neuronal apoptosis after HI in the endothelial-specific IRS-1 transgenic pups. Together, our findings suggested that neuronal and endothelial IRS-1 had opposite effects on the neurovascular integrity and damage after neonatal HI brain injury and that endothelial IRS-1 worsens neurovascular integrity after HI via mTOR-mediated tight junction protein disassembly.
Project description:Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies