bims-pideca 2021-01-24 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2021–01–24
nineteen papers selected by
Ralitsa Radostinova Madsen, University College London

Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity.
Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes.
mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.
Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.
Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice.
Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24.
PTEN inhibitor bpV(HOpic) confers protection against ionizing radiation.
Insulin resistance and exaggerated insulin sensitivity triggered by single-gene mutations in the insulin signaling pathway.
Systematic alteration of in vitro metabolic environments reveals empirical growth relationships in cancer cell phenotypes.
Mdm2 phosphorylation by Akt regulates the p53 response to oxidative stress to promote cell proliferation and tumorigenesis.
Unlike dietary restriction, rapamycin fails to extend lifespan and reduce transcription stress in progeroid DNA repair-deficient mice.
Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability.
PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy.
Integrin and autocrine IGF2 pathways control fasting insulin secretion in β-cells.
Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response.
Extrachromosomal DNA (ecDNA) in cancer pathogenesis.
PI3K/Akt pathway and Nanog maintain cancer stem cells in sarcomas.
The Source of Glycolytic Intermediates in Mammalian Tissues.
Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Science. 2021 01 22. 371(6527): 405-410

Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity.

Ke Xu, Na Yin, Min Peng, Efstathios G Stamatiades, Amy Shyu, Peng Li, Xian Zhang, Mytrang H Do, Zhaoquan Wang, Kristelle J Capistrano, Chun Chou, Andrew G Levine, Alexander Y Rudensky, Ming O Li.

Infection triggers expansion and effector differentiation of T cells specific for microbial antigens in association with metabolic reprograming. We found that the glycolytic enzyme lactate dehydrogenase A (LDHA) is induced in CD8+ T effector cells through phosphoinositide 3-kinase (PI3K) signaling. In turn, ablation of LDHA inhibits PI3K-dependent phosphorylation of Akt and its transcription factor target Foxo1, causing defective antimicrobial immunity. LDHA deficiency cripples cellular redox control and diminishes adenosine triphosphate (ATP) production in effector T cells, resulting in attenuated PI3K signaling. Thus, nutrient metabolism and growth factor signaling are highly integrated processes, with glycolytic ATP serving as a rheostat to gauge PI3K-Akt-Foxo1 signaling in the control of T cell immunity. Such a bioenergetic mechanism for the regulation of signaling may explain the Warburg effect.

DOI: https://doi.org/10.1126/science.abb2683
Methods Mol Biol. 2021 ;2251 73-89

Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes.

Rebecca Cabral-Dias, Yasmin Awadeh, Roberto J Botelho, Costin N Antonescu.

  The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane compartments as a result of dynamic phosphorylation and dephosphorylation by lipid kinases and phosphatases, and/or by vesicle-mediated transport. Several PIPs are found within the plasma membrane, such as phosphatidylinositol-4-phosphate [PI(4)P], phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and these control many aspects of cell physiology, including receptor signaling and membrane traffic. As a result, measurement of the cell surface abundance of these PIPs is a valuable resource to allow understanding of the regulation and function of these cell surface lipids. Here, we describe methods based on quantification of the localization of genetically encoded fluorescent PIP probes to the cell surface by either spinning disc confocal microscopy or total internal reflection fluorescence microscopy that allow detection of changes in cell surface levels of PI(4,5)P2, PI(3,4)P2, and PIP3. These methods can also be applied to the measurement of other PIPs or lipid species at the cell surface, and thus represent a useful resource for the study of the cell biology of PIPs.

Keywords:  Biosensors; Fluorescence; Lipids; Membrane; Microscopy; Signaling; Transfection

DOI:  https://doi.org/10.1007/978-1-0716-1142-5_5
J Biol Chem. 2020 Dec 09. pii: S0021-9258(20)00150-7. [Epub ahead of print]296 100157

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Roberta L Beauchamp, Serkan Erdin, Luke Witt, Justin T Jordan, Scott R Plotkin, James F Gusella, Vijaya Ramesh.

  Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

Keywords:  Akt PKB; NF2; NRG1-ERBB3; brain tumor; dual mTORC1/mTORC2 inhibition; insulin-like growth factor (IGF) receptor 1; mammalian target of rapamycin (mTOR); meningioma; signaling; tumor suppressor gene

DOI:  https://doi.org/10.1074/jbc.RA120.014960
Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2022120118. [Epub ahead of print]118(4):

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.

Kendall J Condon, Jose M Orozco, Charles H Adelmann, Jessica B Spinelli, Pim W van der Helm, Justin M Roberts, Tenzin Kunchok, David M Sabatini.

  In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

Keywords:  CRISPR-Cas9 screen; mTORC1; mitochondria

DOI:  https://doi.org/10.1073/pnas.2022120118
J Lipid Res. 2020 Dec;pii: S0022-2275(20)60019-0. [Epub ahead of print]61(12): 1565-1576

Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice.

Abudukadier Abulizi, Daniel F Vatner, Zhang Ye, Yongliang Wang, Joao-Paulo Camporez, Dongyan Zhang, Mario Kahn, Kun Lyu, Alaa Sirwi, Gary W Cline, M Mahmood Hussain, Patricia Aspichueta, Varman T Samuel, Gerald I Shulman.

  Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp-/- mice.

Keywords:  diabetes; drug therapy; lipids; liver; liver microsomal triglyceride transfer protein; liver-targeted mitochondrial uncoupler; metabolic disease; nonalcoholic fatty liver disease

DOI:  https://doi.org/10.1194/jlr.RA119000586
Nat Commun. 2021 01 21. 12(1): 508

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24.

Xiongwen Cao, Alexandra Khitun, Yang Luo, Zhenkun Na, Thitima Phoodokmai, Khomkrit Sappakhaw, Elizabeth Olatunji, Chayasith Uttamapinant, Sarah A Slavoff.

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

DOI: https://doi.org/10.1038/s41467-020-20841-6
Sci Rep. 2021 Jan 18. 11(1): 1720

PTEN inhibitor bpV(HOpic) confers protection against ionizing radiation.

Ankit Chauhan, Dhananjay Kumar Sah, Neeraj Kumari, Namita Kalra, Ravi Soni, Anant Narayan Bhatt.

Exposure to Ionizing radiation (IR) poses a severe threat to human health. Therefore, there is an urgent need to develop potent and safe radioprotective agents for radio-nuclear emergencies. Phosphatidylinositol-3-kinase (PI3K) mediates its cytoprotective signaling against IR by phosphorylating membrane phospholipids to phosphatidylinositol 3,4,5 triphosphate, PIP3, that serve as a docking site for AKT. Phosphatase and Tensin Homolog on chromosome 10 (PTEN) antagonizes PI3K activity by dephosphorylating PIP3, thus suppressing PI3K/AKT signaling that could prevent IR induced cytotoxicity. The current study was undertaken to investigate the radioprotective potential of PTEN inhibitor (PTENi), bpV(HOpic). The cell cytotoxicity, proliferation index, and clonogenic survival assays were performed for assessing the radioprotective potential of bpV(HOpic). A safe dose of bpV(HOpic) was shown to be radioprotective in three radiosensitive tissue origin cells. Further, bpV(HOpic) significantly reduced the IR-induced apoptosis and associated pro-death signaling. A faster and better DNA repair kinetics was also observed in bpV(HOpic) pretreated cells exposed to IR. Additionally, bpV(HOpic) decreased the IR-induced oxidative stress and significantly enhanced the antioxidant defense mechanism in cells. The radioprotective effect of bpV(HOpic) was found to be AKT dependant and primarily regulated by the enhanced glycolysis and associated signaling. Furthermore, this in-vitro observation was verified in-vivo, where administration of bpV(HOpic) in C57BL/6 mice resulted in AKT activation and conferred survival advantage against IR-induced mortality. These results imply that bpV(HOpic) ameliorates IR-induced oxidative stress and cell death by inducing AKT signaling mediated antioxidant defense system and DNA repair pathways, thus strengthening its potential to be used as a radiation countermeasure.

DOI: https://doi.org/10.1038/s41598-020-80754-8
Diabetol Int. 2021 Jan;12(1): 62-67

Insulin resistance and exaggerated insulin sensitivity triggered by single-gene mutations in the insulin signaling pathway.

Ryo Kushi, Yushi Hirota, Wataru Ogawa.

Whereas the genetic basis of insulin sensitivity is determined by variation in multiple genes, mutations of single genes can give rise to profound changes in such sensitivity. Mutations of the insulin receptor gene (INSR)-which trigger type A insulin resistance, Rabson-Mendenhall, or Donohue syndromes-and those of the gene for the p85α regulatory subunit of phosphoinositide 3-kinase (PIK3R1), which give rise to SHORT syndrome, are the most common and second most common causes, respectively, of single-gene insulin resistance. Loss-of-function mutations of the genes for the protein kinase Akt2 (AKT2) or for TBC1 domain family member 4 (TBC1D4) have been identified in families with severe insulin resistance. Gain-of-function mutations of the gene for protein tyrosine phosphatase nonreceptor type 11 (PTPN11), which negatively regulates insulin receptor signaling, give rise to Noonan syndrome, and some individuals with this syndrome manifest insulin resistance. Gain-of-function mutations of the gene for the p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA) have been identified in individuals with segmental overgrowth or megalencephaly, some of whom also manifest spontaneous hypoglycemia. A gain-of-function mutation of AKT2 was also found in individuals with recurrent hypoglycemia. Loss-of-function mutations of the gene for phosphatase and tensin homolog (PTEN), another negative regulator of insulin signaling, give rise to Cowden syndrome in association with exaggerated metabolic actions of insulin. Clinical manifestations of individuals with such mutations of genes related to insulin signaling thus provide insight into the essential function of such genes in the human body.

DOI: https://doi.org/10.1007/s13340-020-00455-5
Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31636-3. [Epub ahead of print]34(3): 108647

Systematic alteration of in vitro metabolic environments reveals empirical growth relationships in cancer cell phenotypes.

Karl Kochanowski, Timur Sander, Hannes Link, Jeremy Chang, Steven J Altschuler, Lani F Wu.

  Cancer cells, like microbes, live in complex metabolic environments. Recent evidence suggests that microbial behavior across metabolic environments is well described by simple empirical growth relationships, or growth laws. Do such empirical growth relationships also exist in cancer cells? To test this question, we develop a high-throughput approach to extract quantitative measurements of cancer cell behaviors in systematically altered metabolic environments. Using this approach, we examine relationships between growth and three frequently studied cancer phenotypes: drug-treatment survival, cell migration, and lactate overflow. Drug-treatment survival follows simple linear growth relationships, which differ quantitatively between chemotherapeutics and EGFR inhibition. Cell migration follows a weak grow-and-go growth relationship, with substantial deviation in some environments. Finally, lactate overflow is mostly decoupled from growth rate and is instead determined by the cells' ability to maintain high sugar uptake rates. Altogether, this work provides a quantitative approach for formulating empirical growth laws of cancer.

Keywords:  cancer; growth law; metabolic environment; metabolism

DOI:  https://doi.org/10.1016/j.celrep.2020.108647
Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2003193118. [Epub ahead of print]118(4):

Mdm2 phosphorylation by Akt regulates the p53 response to oxidative stress to promote cell proliferation and tumorigenesis.

Loretah Chibaya, Baktiar Karim, Hong Zhang, Stephen N Jones.

  We have shown previously that phosphorylation of Mdm2 by ATM and c-Abl regulates Mdm2-p53 signaling and alters the effects of DNA damage in mice, including bone marrow failure and tumorigenesis induced by ionizing radiation. Here, we examine the physiological effects of Mdm2 phosphorylation by Akt, another DNA damage effector kinase. Surprisingly, Akt phosphorylation of Mdm2 does not alter the p53-mediated effects of ionizing radiation in cells or mice but regulates the p53 response to oxidative stress. Akt phosphorylation of Mdm2 serine residue 183 increases nuclear Mdm2 stability, decreases p53 levels, and prevents senescence in primary cells exposed to reactive oxidative species (ROS). Using multiple mouse models of ROS-induced cancer, we show that Mdm2 phosphorylation by Akt reduces senescence to promote KrasG12D-driven lung cancers and carcinogen-induced papilloma and hepatocellular carcinomas. Collectively, we document a unique physiologic role for Akt-Mdm2-p53 signaling in regulating cell growth and tumorigenesis in response to oxidative stress.

Keywords:  Akt; Mdm2; Tp53; senescence; tumorigenesis

DOI:  https://doi.org/10.1073/pnas.2003193118
Aging Cell. 2021 Jan 23. e13302

Unlike dietary restriction, rapamycin fails to extend lifespan and reduce transcription stress in progeroid DNA repair-deficient mice.

María B Birkisdóttir, Dick Jaarsma, Renata M C Brandt, Sander Barnhoorn, Nicole van Vliet, Sandra Imholz, Conny T van Oostrom, Bhawani Nagarajah, Eliana Portilla Fernández, Anton J M Roks, Ype Elgersma, Harry van Steeg, José A Ferreira, Jeroen L A Pennings, Jan H J Hoeijmakers, Wilbert P Vermeij, Martijn E T Dollé.

  Dietary restriction (DR) and rapamycin extend healthspan and life span across multiple species. We have recently shown that DR in progeroid DNA repair-deficient mice dramatically extended healthspan and trippled life span. Here, we show that rapamycin, while significantly lowering mTOR signaling, failed to improve life span nor healthspan of DNA repair-deficient Ercc1∆/- mice, contrary to DR tested in parallel. Rapamycin interventions focusing on dosage, gender, and timing all were unable to alter life span. Even genetically modifying mTOR signaling failed to increase life span of DNA repair-deficient mice. The absence of effects by rapamycin on P53 in brain and transcription stress in liver is in sharp contrast with results obtained by DR, and appoints reducing DNA damage and transcription stress as an important mode of action of DR, lacking by rapamycin. Together, this indicates that mTOR inhibition does not mediate the beneficial effects of DR in progeroid mice, revealing that DR and rapamycin strongly differ in their modes of action.

Keywords:  DNA damage repair; aging; dietary restriction; rapamycin; transcription stress

DOI:  https://doi.org/10.1111/acel.13302
Biochem Biophys Res Commun. 2021 Jan 19. pii: S0006-291X(21)00068-1. [Epub ahead of print]541 84-89

Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability.

Seishu Gen, Yu Matsumoto, Tsukasa Suzuki, Jun Inoue, Yuji Yamamoto.

  Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein that is partially regulated by insulin, energy, oxygen, and growth factors. Mutations in the TSC2 gene and loss of TSC2 promote cell growth by the mammalian target of rapamycin complex 1 (mTORC1) activation. Furthermore, S-adenosylmethionine (SAM) sensor upstream of mTORC1 indirectly inhibits mTORC1 activity via the methionine metabolite SAM. Here, we investigated the effects of methionine on insulin/TSC2/mTORC1 activity. Our results showed that methionine affected TSC2 stability and abolished TSC2 localization to the lysosome. Moreover, activation of insulin signaling contributed to TSC2 degradation in a methionine deprivation-dependent manner. Thus, methionine and insulin crosstalk occurred via TSC2.

Keywords:  AKT; Amino acid; Mammalian target of rapamycin complex 1; Methionine; S-adenosylmethionine; Tuberous sclerosis complex 2

DOI:  https://doi.org/10.1016/j.bbrc.2021.01.033
PLoS One. 2021 ;16(1): e0245715

PI3k inhibitors (BKM120 and BYL719) as radiosensitizers for head and neck squamous cell carcinoma during radiotherapy.

Fu-Cheng Chuang, Chih-Chun Wang, Jian-Han Chen, Tzer-Zen Hwang, Shyh-An Yeh, Yu-Chieh Su.

Approximately 500,000 new cases of head and neck squamous cell carcinoma (HNSCC) are reported annually. Radiation therapy is an important treatment for oral squamous cell carcinoma (OSCC). The survival rate of patients with HNSCC remained low (50%) in decades because of radiation therapy failure caused by the radioresistance of HNSCC cells. This study aimed to identify PI3K inhibitors that can enhance radiosensitivity. Results showed that pan-Phosphoinositide 3-kinases (PI3K) inhibitor BKM120 and class I α-specific PI3K inhibitor BYL719 dose-dependently reduced the growth of OSCC cells but not that of radioresistant OML1-R cells. The combination treatment of BKM120 or BYL719 with radiation showed an enhanced inhibitory effect on OSCC cells and radioresistant OML1-R cells. Furthermore, the enhanced inhibitory effect of the combination treatment was confirmed in patient-derived OSCC cells. The triple combination treatment of mTOR inhibitor AZD2014 and BKM120 or AZD2014 and BYL719 with radiation showed a significantly enhanced inhibitory effect on radioresistant OML1-R cells. These results suggest that the PI3K inhibitors are potential therapeutic agents with radiosensitivity for patients with OSCC.

DOI: https://doi.org/10.1371/journal.pone.0245715
J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50472-X. [Epub ahead of print]295(49): 16510-16528

Integrin and autocrine IGF2 pathways control fasting insulin secretion in β-cells.

Caroline Arous, Maria Luisa Mizgier, Katharina Rickenbach, Michel Pinget, Karim Bouzakri, Bernhard Wehrle-Haller.

  Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2-AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)-dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Keywords:  AKT isoform; Akt PKB; IGF1 receptor signaling; IGF2; beta cell (B-cell); insulin; insulin receptor signaling; insulin secretion; insulin-like growth factor (IGF); insulin/insulin-like growth factor 1 (IGF1)-receptor signaling; integrin

DOI:  https://doi.org/10.1074/jbc.RA120.012957
Nat Commun. 2021 01 20. 12(1): 476

Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response.

Wenjun Wang, Jianshuang Li, Junyang Tan, Miaomiao Wang, Jing Yang, Zhi-Min Zhang, Chuanzhou Li, Alexei G Basnakian, Hong-Wen Tang, Norbert Perrimon, Qinghua Zhou.

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.

DOI: https://doi.org/10.1038/s41467-020-20780-2
Curr Opin Genet Dev. 2021 Jan 18. pii: S0959-437X(21)00002-2. [Epub ahead of print]66 78-82

Extrachromosomal DNA (ecDNA) in cancer pathogenesis.

Sihan Wu, Vineet Bafna, Paul S Mischel.

In cancer, oncogenes and surrounding regulatory regions can untether themselves from chromosomes, forming extrachromosomal DNA particles (ecDNAs). Because of their non-chromosomal inheritance, ecDNA drives high oncogene copy number and intratumoral genetic heterogeneity, endowing tumors with the ability to rapidly change their genomes, accelerating tumor evolution, and contributing to therapeutic resistance. Further, the circular topology of ecDNA leads to enhanced chromatin accessibility, altered gene regulation, and massive oncogene transcription, driving tumor growth and progression, and placing ecDNA at the interface of cancer genomics and epigenetics. Recent studies show that ecDNA is a common event in many of the most aggressive forms of cancer, potentially challenging our current precision oncology approaches. In this review, we discuss what is known about ecDNA and its biological and clinical impact, highlighting new research and suggesting the promise, and some of the challenges ahead for the field.

DOI: https://doi.org/10.1016/j.gde.2021.01.001
Oncogenesis. 2021 Jan 19. 10(1): 12

PI3K/Akt pathway and Nanog maintain cancer stem cells in sarcomas.

Changhwan Yoon, Jun Lu, Brendan C Yi, Kevin K Chang, M Celeste Simon, Sandra Ryeom, Sam S Yoon.

The self-renewal transcription factor Nanog and the phosphoinositide 3-kinase (PI3K)-Akt pathway are known to be essential for maintenance of mesenchymal stem cells. We evaluated their contribution to the maintenance of CD133(+) cancer stem-like cells (CSCs) and spheroid-forming cells in patient-derived cell lines from three human sarcoma subtypes: HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and DDLS8817 dedifferentiated liposarcoma. Levels of Nanog and activated Akt were significantly higher in sarcoma cells grown as spheroids or sorted for CD133 expression to enrich for CSCs. shRNA knockdown of Nanog decreased spheroid formation 10- to 14-fold, and reversed resistance to both doxorubicin and radiation in vitro and in H1080 flank xenografts. In the HT1080 xenograft model, doxorubicin and Nanog knockdown reduced tumor growth by 34% and 45%, respectively, and the combination reduced tumor growth by 74%. Using a human phospho-kinase antibody array, Akt1/2 signaling, known to regulate Nanog, was found to be highly activated in sarcoma spheroid cells compared with monolayer cells. Pharmacologic inhibition of Akt using LY294002 and Akt1/2 knockdown using shRNA in sarcoma CSCs decreased Nanog expression and spheroid formation and reversed chemotherapy resistance. Akt1/2 inhibition combined with doxorubicin treatment of HT1080 flank xenografts reduced tumor growth by 73%. Finally, in a human sarcoma tumor microarray, expression of CD133, Nanog, and phospho-Akt were 1.8- to 6.8-fold higher in tumor tissue compared with normal tissue. Together, these results indicate that the Akt1/2-Nanog pathway is critical for maintenance of sarcoma CSCs and spheroid-forming cells, supporting further exploration of this pathway as a therapeutic target in sarcoma.

DOI: https://doi.org/10.1038/s41389-020-00300-z
Cell Metab. 2021 Jan 18. pii: S1550-4131(20)30728-2. [Epub ahead of print]

The Source of Glycolytic Intermediates in Mammalian Tissues.

Tara TeSlaa, Caroline R Bartman, Connor S R Jankowski, Zhaoyue Zhang, Xincheng Xu, Xi Xing, Lin Wang, Wenyun Lu, Sheng Hui, Joshua D Rabinowitz.

  Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use 13C-tracing in mice to quantify glycolytic intermediate sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.

Keywords:  compartmentalized metabolism; glucose homeostasis; glycogen; glycolysis; glycolytic intermediates; glycolytic specialist; isotope tracing; metabolic heterogeneity; red muscle

DOI:  https://doi.org/10.1016/j.cmet.2020.12.020
Methods Mol Biol. 2021 ;2251 1-17

Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Noah Steinfeld, Sai Srinivas Panapakkam Giridharan, Emily J Kauffman, Lois S Weisman.

  Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

Keywords:  PI3,4,5P3; PI3,4P2; PI3,5P2; PI3P; PI4,5P2; PI4P; PI5P; Phosphatidylinositol; Phosphoinositide lipids; PtdIns; Radioactive metabolic labeling

DOI:  https://doi.org/10.1007/978-1-0716-1142-5_1