Goutam Ghosh

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Ku86 represses lethal telomere deletion events in human somatic cells

Nonhomologous end joining (NHEJ), a form of DNA double-strand break (DSB) repair, is conserved from bacteria to humans. One essential NHEJ factor is Ku, which consists of a heterodimer of Ku70 and Ku86. In a plethora of model systems, null mutations for Ku70 or Ku86 present with defects in DNA DSB repair, variable(diversity)joining [V(D)J] recombination, and/or telomere maintenance. The complete loss of Ku from bacteria to mice is, however, compatible with viability. In striking contrast, human patients with mutations of either Ku subunit have never been described. Here, we have used recombinant adeno-associated virus-mediated gene targeting to produce a human somatic cell line that expresses a conditionally null allele of Ku86. The induced loss of Ku86 results in cell death accompanied by massive telomere loss in the form of t-circles. Thus, Ku86 is an essential gene in human somatic cells because of its requirement, not in NHEJ or V(D)J recombination, but in telomere maintenance.

Regulation of Telomere Length and Suppression of Genomic Instability in Human Somatic Cells by Ku86

ABSTRACT Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.

Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and β‐catenin levels

Endostatin is a well‐characterized endogenous inhibitor of angiogenesis that affects cell proliferation and migration by inhibiting integrin and Wnt‐mediated signalling pathways. Here, we show that endothelial cells treated with native and P125A‐endostatin activate autophagy. Because autophagy can either be protective or induce programmed cell death, experiments were carried out to understand the signalling pathways leading to autophagy in endothelial cells. P125A‐endostatin treatment increased the levels of Beclin 1, a crucial molecule in vesicle nucleation and autophagy. The treatment also reduced the levels of Bcl‐2, Bcl‐xL and β‐catenin; however, progressively increasing amounts of Bcl‐2 and Bcl‐xL were found to be complexed with Beclin 1. Increased β‐catenin and Wnt‐mediated signalling reduced Beclin 1 levels and rescued endothelial cells from endostatin‐induced autophagy. Finally, knocking down Beclin 1 levels by RNA interference decreased autophagy and accelerated caspase activation in endostatin‐treated cells. These studies suggest that endothelial cells may initiate autophagy as a survival response to limit the effects of angiogenesis inhibitors. Thus, interfering with autophagy can potentiate the effects of endostatin by promoting a switch to apoptosis.

Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis

Significance Tumor cells adapt to hypoxia by modulating energy production and utilization. Endocytosis is an energy-consuming process that is suppressed during hypoxia. Our studies show that Dynamin 2 (DNM2), a key component of endocytic machinery, is transcriptionally suppressed by HIF-1. Genetic and pharmacological inactivation of DNM2 stabilized HIF-1α and HIF-2α. Furthermore, miR-199a, which is encoded from the opposite strand of DNM2, exerts reciprocal negative regulation upon HIF-1α and HIF-2α. Overexpression of miR-199a decreased HIF-1α and HIF-2α, cell migration, and metastasis. These findings establish a regulatory loop between endocytic pathway and hypoxic response in tumor cells. Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3′-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a–mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

The lethality of Ku86 (XRCC5) loss-of-function mutations in human cells is independent of p53 (TP53).

Abstract Ghosh, G., Li, G., Myung, K. and Hendrickson, E. A. The Lethality of Ku86 (XRCC5) Loss-of-Function Mutations in Human Cells is Independent of p53 (TP53). Radiat. Res. 167, 66–79 (2007). Ku86 is one of the two regulatory subunits of the DNA-PK (DNA-dependent protein kinase) complex that is required for DNA double-strand break repair in mammalian cells. In a previous study, by means of somatic gene targeting, we generated human cell lines deficient in Ku86 (XRCC5). Heterozygous human Ku86 cells exhibited a wide array of haploinsufficient phenotypes, including sensitivity to ionizing radiation, defects in DNA-PK and DNA end-binding activities, elevated levels of p53 (TP53) and γ-H2AX foci, and a defect in cell proliferation with an increase in the frequency of aneuploid cells. Here we demonstrate that the overexpression of a human Ku86 cDNA complemented the deficiencies of these cells to wild-type levels. In contrast, Ku86 overexpression only partially rescued the telomere defects characteristic of Ku86 heterozygous cells and did not rescue their genetic instability. Additionally, in stark contrast to every other species described to date, we had shown earlier that homozygous human Ku86−/− cells are inviable, because they undergo 8 to 10 rounds of cell division before succumbing to apoptosis. The tumor suppressor protein p53 regulates the DNA damage response in mammalian cells and triggers apoptosis in the face of excessive DNA damage. Correspondingly, ablation of p53 expression has repeatedly been shown to significantly ameliorate the pathological effects of loss-of-function mutations for a large number of DNA repair genes. Surprisingly, however, even in a p53-null genetic background, the absence of Ku86 proved lethal. Thus the gene encoding Ku86 (XRCC5) is an essential gene in human somatic cells, and its absence cannot be suppressed by the loss of p53 function. These results suggest that Ku86 performs an essential role in telomere maintenance in human cells.

AAV-P125A-endostatin and paclitaxel treatment increases endoreduplication in endothelial cells and inhibits metastasis of breast cancer

Endostatin potentiates the antimitotic effects of paclitaxel (taxol) on endothelial cells (ECs). P125A-endostatin and taxol-treated ECs showed multipolar spindles and nuclear lobulation, leading to mitotic catastrophe and cell death. Induction of nuclear abnormalities was found to be dependent on β-catenin levels as wnt-mediated overexpression of β-catenin reversed the changes in nuclear morphology. These results prompted us to investigate whether antiangiogenic gene therapy and paclitaxel chemotherapy can synergistically inhibit angiogenesis and tumor growth. We first determined the effect of combination treatment in a transgenic mouse model of breast cancer. Intramuscular injection of recombinant adeno-associated virus type-2 virus induced sustained expression of P125A-endostatin. In vivo studies showed that combination therapy inhibited mammary cancer growth, delayed the onset of multifocal mammary adenocarcinomas, decreased tumor angiogenesis and increased survival in treated mice. In a second model, female athymic mice were orthotopically transplanted with a metastatic human breast cancer cell line. Antiangiogenic gene therapy in combination with paclitaxel inhibited tumor angiogenesis and lung/lymph-node metastasis in this model. These studies demonstrate cooperation between endostatin gene therapy and chemotherapy to inhibit tumor initiation, growth and metastasis.

Abstract 3980: Hypoxia-induced microRNA-424 targets CUL2 to stabilize HIF-α isoforms and promotes angiogenesis

Chronic hypoxia, a hallmark of many tumors, is associated with angiogenesis and tumor progression. Adaptive changes to oxygen availability are critical for cell survival and homeostasis. Cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for a novel mechanism regulating HIF-1α and HIF-2α levels in endothelial cells during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in microvascular EC, HUVEC and EC progenitor cells purified from the blood. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 in EC was regulated by PU.1-dependent transactivation. PU.1 expression was in turn controlled by RUNX-1 and C/EBPα. Knocking down either C/EBPα or RUNX-1 or PU.1 attenuated hypoxia-driven expression of miR-424 and nuclear translocation of HIF-1α. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by specific morpholinos. These results suggest that miR-424 plays an important physiological role in angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2011-3980

Abstract 2020: MiR-210 modulates the hypoxic response in endothelial and ovarian cancer cells

Tumor growth and progression is intricately linked to hypoxia induced adaptive changes. Hypoxia is known to alter chemosensitivity, upregulation of putative cancer initiating stem cells and the microenvironment. The multitude of these changes is brought about by differential gene expression. Hypoxia inducible transcription factor-1 alpha (HIF-α) is a key transducer of altered gene expression under low oxygen or ischemia. In addition to the transcriptional activation of multiple genes, hypoxia also alters miRNA levels in tumor and vascular endothelial cells. miRNAs are small noncoding RNAs ∼22 nucleotides in length representing 1%-2% of the eukaryotic transcriptome. We used microarrays to determine relative changes in 467 miRNAs in endothelial and MA148 ovarian cancer cells exposed to hypoxia. One of the miRNAs, miR-210 was significantly up-regulated under hypoxia by microarray analysis (2-fold, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2020.

Abstract 2070: Mir-199a is downregulated in hypoxia and targets HIF1α in ovarian cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC HIF1 is the main transcriptional regulator of the hypoxic response in mammalian cells and plays an important role in the adaptation of tumor cells to hypoxic microenvironments. While normally degraded under normoxic conditions via oxygen-dependent, ubiquitin-based mechanisms, HIF1α protein, which, with the constitutively expressed HIF1β forms the HIF1 heterodimer, is stabilized in hypoxic tumor cells. Stabilization of HIF1α protein under hypoxia leads to transactivation of a variety of angiogenic and tumorigenic genes including VEGF and matrix metalloproteinase 2. Clinically, high levels of HIF1 in tumors translate to increased patient morbidity and mortality. Our studies show that HIF1α, in addition to being regulated at the protein level, is also directly regulated by microRNAs, small 20-22 base ribonucleotides that specifically bind to the 3’ untranslated regions (UTRs) of target mRNA to inhibit translation or induce degradation. Based on bioinformatics and microRNA-target databases, we determined that HIF1α contains a potential binding site in its 3’ UTR for mir-199a, which, based on our microarray and RT-qPCR data, is significantly downregulated in hypoxic ovarian cancer cells compared to normoxia. Using a luciferase reporter construct in which luciferase expression is under control of the HIF1α 3’ UTR, we showed that overexpression of mir-199a in A2780 or MA148 cells leads to suppression of the luciferase signal. Mutation of the mir-199a binding site in the HIF1α 3’ UTR resulted in no change in luciferase signal with mir-199a overexpression, indicating that mir-199a directly targets the HIF1α 3’ UTR. Furthermore, decreasing mir-199a levels, either by incubating cells in hypoxia or using a morpholino specifically targeting mir-199a, leads to increased levels of HIF1α protein as determined by western blot and increased transcripts of HIF1-activated genes such as VEGF as measured by RT-qPCR. Conversely, overexpression of mir-199a leads to decreased HIF1α protein and VEGF transcript levels. Together, our findings have significant implications in the study of HIF1α regulation in cancers. Mir-199a may have a role in future therapeutic strategies to modulate the tumor microenvironment to limit hypoxia-driven tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2070.