Eui Young So

BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction

BackgroundBRAT1 (BRCA1-associated ATM activator 1) interacts with both BRCA1, ATM and DNA-PKcs, and has been implicated in DNA damage responses. However, based on our previous results, it has been shown that BRAT1 may be involved in cell growth and apoptosis, besides DNA damage responses, implying that there are undiscovered functions for BRAT1.MethodsUsing RNA interference against human BRAT1, we generated stable BRAT1 knockdown cancer cell lines of U2OS, Hela, and MDA-MA-231. We tested cell growth properties and in vitro/in vivo tumorigenic potentials of BRAT1 knockdown cells compared to control cells. To test if loss of BRAT1 induces metabolic abnormalities, we examined the rate of glycolysis, ATP production, and PDH activity in both BRAT1 knockdown and control cells. The role of BRAT1 in growth signaling was determined by the activation of Akt/Erk, and SC79, Akt activator was used for validation.ResultsBy taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo. Cell migration was also remarkably lowered when BRAT1 was depleted. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown HeLa cells. Furthermore, both basal and induced activity of Akt and Erk kinases were suppressed in these cells, implicating abnormality in signaling cascades for cellular growth. Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.ConclusionsThese findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

Decreased DNA repair activity in bone marrow due to low expression of DNA damage repair proteins

The bone marrow (BM) is one of the organs that is sensitive to acute exposure of ionizing radiation (IR); however, the mechanism of its high sensitivity to IR remains to be elucidated. BM is differentiated into dendritic cells (DC) with granulocyte macrophage-colony stimulating factor (GM-CSF). Using this in vitro model, we studied whether radiosensitivity is distinctly regulated in undifferentiated and differentiated BM. We discovered that levels of DNA damage repair (DDR) proteins are extremely low in BM, and they are markedly increased upon differentiation to DC. Efficiency of both homologous recombination (HR)- and non-homologous end joining (NHEJ)-mediated repair of DNA double strand breaks (DSBs) is much lower in BM compared with that of DC. Consistent with this, immunofluorescent γH2AX is highly detected in BM after IR. These results indicate that increased radiosensitivity of BM is at least due to low expression of the DNA repair machinery.

Deregulation of DNA Damage Response Pathway by Intercellular Contact

Background: γH2AX (H2AX S139P) is one of the most upstream and important components of the DDR. Results: Intercellular contact increases H2AX by activating the γ-catenin pathway. Conclusion: Cellular microenvironment might determine cellular commitment to apoptosis. Significance: Regulation of the DDR pathway by the cadherin-catenin complex is innovative. Deregulation of the DNA damage response (DDR) pathway could compromise genomic integrity in normal cells and reduce cancer cell sensitivity to anticancer treatments. We found that intercellular contact stabilizes histone H2AX and γH2AX (H2AX phosphorylated on Ser-139) by up-regulating N/E-cadherin and γ-catenin. γ-catenin and its DNA-binding partner LEF-1 indirectly increase levels of H2AX by suppressing the promoter of the RNF8 ubiquitin ligase, which decreases levels of H2AX protein under conditions of low intercellular contact. Hyperphosphorylation of DDR proteins is induced by up-regulated H2AX. Constitutive apoptosis is caused in confluent cells but is not further induced by DNA damage. This is conceivably due to insufficient p53 activation because ChIP assay shows that its DNA binding ability is not induced in those cells. Together, our results illustrate a novel mechanism of the regulation of DDR proteins by the cadherin-catenin pathway.

Mutations in BRAT1 cause autosomal recessive progressive encephalopathy: Report of a Spanish patient.

We describe a 4-year-old male child born to non-consanguineous Spanish parents with progressive encephalopathy (PE), microcephaly, and hypertonia. Whole exome sequencing revealed compound heterozygous BRAT1 mutations [c.1564G > A (p.Glu522Lys) and c.638dup (p.Val214Glyfs*189)]. Homozygous and compound heterozygous BRAT1 mutations have been described in patients with lethal neonatal rigidity and multifocal seizure syndrome (MIM# 614498). The seven previously described patients suffered from uncontrolled seizures, and all of those patients died in their first months of life. BRAT1 acts as a regulator of cellular proliferation and migration and is required for mitochondrial function. The loss of these functions may explain the cerebral atrophy observed in this case of PE. This case highlights the extraordinary potential of next generation technologies for the diagnosis of rare genetic diseases, including PE. Making a prompt diagnosis of PE is important for genetic counseling and disease management.

The screening of a microRNA expression during development of human macrophages and mouse dendritic cells

ABSTRACT There is increasing evidence showing specific roles of microRNA in cell differentiation and cancer progression. Here we examine miRNA profiles during maturation of monocytes and bone marrow-derived dendritic cells (BMDCs) in human and mouse, respectively. We have identified significant changes of various miRNA expression during monocyte and BMDC monocyte development via miRNA microarrays, confirmed by quantitative PCR. Increases in miR155 expression positively correlated with increasing maturity of monocyte and BMDC in both mouse and human microarrays, indicating its importance in development. We describe a requirement of miR155 for MHCII expression during GM-CSF-induced development and LPS-induced maturation of DCs, suggesting reduced immune function of DC when miR155 is absent. Our study suggests that miRNAs might have an important role in differentiation of myeloid cell such as dendritic cells and macrophages.

Tumor suppression by resistant maltodextrin, Fibersol-2

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.

Fibersol-2 induces apoptosis of Apc-deficient colorectal Cancer (SW480) cells and decreases polyp formation in Apc MIN mice.

ABSTRACT The consumption of dietary fibers has been implicated with a lowered risk of human colorectal cancer. Proposed mechanisms involve alterations in the stool consistency, transit time, and formation of short-chain fatty acid by dietary fiber fermentation, and the reorganization of gut microbiota. Here we show that Fibersol-2, a digest-resistant maltodextrin, not only inhibits proliferation of colorectal SW480 cancer cell lines by increasing reactive oxygen species (ROS), but decreases the numbers of the adenoma count in Multiple Intestinal Neoplasia (MIN) mice carrying a mutation in the Adenomatous Polyposis Coli gene by 84 d of age. These observations provide direct evidence that Fibersol-2 intrinsically contains anti-cancer activity, independent of the intestinal metabolism and any potential interactions with the microbiota.

Abstract 5407: The novel roles of BRCA1/ATM-associated BRAT1 in glucose/mitochondrial metabolism and tumor cell growth.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC BRAT1 (BRCA1-associated ATM activator) was identified as a novel DNA-damage response protein. It physically interacts with both BRCA1 and ATM, and is required for ATM-activation under conditions of cell stress. We have recently discovered that it also binds to DNA-PKs, implicating the broad role of BRAT1 in DNA repair as well as in DNA damage response. By taking advantage of BRAT1 knockdown HeLa and U2OS cells, we found that loss of BRAT1 expression significantly decreased cell proliferation and tumorigenecity both in vitro and in vivo. Cell motility is remarkably lowered when BRAT1 is depleted. These results demonstrate novel function of BRAT1 in cell proliferation, migration, and tumorigenesis of cancer cells. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown. These findings suggest novel functions of BRAT1 in regulating glucose metabolism and mitochondrial function. Functional interaction of BRCA1/ATM pathway and glucose/mitochondria metabolism are discussed. Citation Format: Eui Young So, Toru Ouchi. The novel roles of BRCA1/ATM-associated BRAT1 in glucose/mitochondrial metabolism and tumor cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2013-5407

Abstract 3088: Essential roles of ATM in GM-CSF-induced bone marrow differentiation to dendritic cells

The gene mutated in ataxia telangiectasia (ATM) encodes 370-kD protein, belonging to a family of PI-3 protein kinases (PIKKs). ATM has been implicated in cellular responses under conditions of DNA damage that causes DNA lesions, playing crucial roles in activating DNA repair machinery and cell-cycle checkpoints. Recent studies have also illustrated that ATM regulates cellular growth, development, and stem cell renewal. Although mice model has demonstrated that ATM deficiency results in both multiple defects in DNA repair in response to ionizing radiation and severe developmental abnormality in T/B cells development as well as weight-loss, it remains unclear whether ATM is involved in development of other cell lineage. We found that levels of ATM are significantly low in primary bone marrow cells (BMs), and that they are increased when BMs’ are differentiated to dendritic cells (DCs) when stimulated with granulocyte macrophage colony-stimulating factor, GM-CSF. BMs derived from ATM mice (ATM-/- BMs) demonstrate decreased cell proliferation, and their differentiation to DCs is markedly impaired, suggesting that ATM is essential for development of DCs. ATM-deficiency is also associated with a decreased capability of BMs to activate T cells. We also found that an ATM specific inhibitor, KU-55933, abrogated GM-CSF-induced DCs development in BMs cell culture. ATM-deficiency in BMs also resulted in reduced expression of Akt compared with control cells, and inhibited Ser493 phosphorylation of Akt by GM-CSF. Levels of expression and phosphorylation of STAT5 were differentially regulated in ATM-/- BMs, and ectopic expression of STAT5 restored DCs differentiation of ATM-/- BMs. Together, our findings reveal that ATM plays a physiological role in BMs’ differentiation to DCs by regulating Akt/STAT5 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3088. doi:1538-7445.AM2012-3088