bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021–02–14
fourteen papers selected by
Ralitsa Radostinova Madsen, University College London
  1. Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.
  2. Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells.
  3. Phosphoinositide 3-Kinase Signaling in the Tumor Microenvironment: What Do We Need to Consider When Treating Chronic Lymphocytic Leukemia With PI3K Inhibitors?
  4. A direct fluorometric activity assay for lipid kinases and phosphatases.
  5. Both Hypoxia-Inducible Factor 1 and MAPK Signaling Pathway Attenuate PI3K/AKT via Suppression of Reactive Oxygen Species in Human Pluripotent Stem Cells.
  6. Metformin effectively treats Tsc1 deletion-caused kidney pathology by upregulating AMPK phosphorylation.
  7. MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.
  8. How does mTOR sense glucose starvation? AMPK is the usual suspect.
  9. The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure.
  10. How NPC1 Loss Twists the TORCque of Lysosomes.
  11. New-found brake calibrates insulin action in β-cells.
  12. Cell memory of epithelial-mesenchymal plasticity in cancer.
  13. A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer.
  14. Role of PDK1 in skeletal muscle hypertrophy induced by mechanical load.
  1. BMC Cancer. 2021 Feb 06. 21(1): 136
    Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.
    Khanh B Tran, Sharada Kolekar, Anower Jabed, Patrick Jaynes, Jen-Hsing Shih, Qian Wang, Jack U Flanagan, Gordon W Rewcastle, Bruce C Baguley, Peter R Shepherd.
       BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.
    METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.
    RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.
    CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.
    Keywords:  A66; BEZ-235; Drug resistance; IC 87114; KU-0063794; Melanoma; PI 3-kinase; PI3Kα; PI3Kδ; PIK3CA; PIK3CD; PIK3CG; VPS34; mTOR
    DOI:  https://doi.org/10.1186/s12885-021-07826-4
  2. Cell Metab. 2021 Feb 04. pii: S1550-4131(21)00014-0. [Epub ahead of print]
    Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells.
    Nont Kosaisawe, Breanne Sparta, Michael Pargett, Carolyn K Teragawa, John G Albeck.
      Cell-to-cell heterogeneity in metabolism plays an unknown role in physiology and pharmacology. To functionally characterize cellular variability in metabolism, we treated cells with inhibitors of oxidative phosphorylation (OXPHOS) and monitored their responses with live-cell reporters for ATP, ADP/ATP, or activity of the energy-sensing kinase AMPK. Across multiple OXPHOS inhibitors and cell types, we identified a subpopulation of cells resistant to activation of AMPK and reduction of ADP/ATP ratio. This resistant state persists transiently for at least several hours and can be inherited during cell divisions. OXPHOS inhibition suppresses the mTORC1 and ERK growth signaling pathways in sensitive cells, but not in resistant cells. Resistance is linked to a multi-factorial combination of increased glucose uptake, reduced protein biosynthesis, and G0/G1 cell-cycle status. Our results reveal dynamic fluctuations in cellular energetic balance and provide a basis for measuring and predicting the distribution of cellular responses to OXPHOS inhibition.
    Keywords:  AKT; FRET; PI3K; adenosine mono-phosphate-regulated protein kinase; electron transport chain; insulin signaling; mammalian target of rapamycin; metabolic cycle; oligomycin; oscillation; translation regulation
    DOI:  https://doi.org/10.1016/j.cmet.2021.01.014
  3. Front Immunol. 2020 ;11 595818
    Phosphoinositide 3-Kinase Signaling in the Tumor Microenvironment: What Do We Need to Consider When Treating Chronic Lymphocytic Leukemia With PI3K Inhibitors?
    Ebru Aydin, Sebastian Faehling, Mariam Saleh, Laura Llaó Cid, Martina Seiffert, Philipp M Roessner.
      Phosphoinositide 3-kinases (PI3Ks) and their downstream proteins constitute a signaling pathway that is involved in both normal cell growth and malignant transformation of cells. Under physiological conditions, PI3K signaling regulates various cellular functions such as apoptosis, survival, proliferation, and growth, depending on the extracellular signals. A deterioration of these extracellular signals caused by mutational damage in oncogenes or growth factor receptors may result in hyperactivation of this signaling cascade, which is recognized as a hallmark of cancer. Although higher activation of PI3K pathway is common in many types of cancer, it has been therapeutically targeted for the first time in chronic lymphocytic leukemia (CLL), demonstrating its significance in B-cell receptor (BCR) signaling and malignant B-cell expansion. The biological activity of the PI3K pathway is not only limited to cancer cells but is also crucial for many components of the tumor microenvironment, as PI3K signaling regulates cytokine responses, and ensures the development and function of immune cells. Therefore, the success or failure of the PI3K inhibition is strongly related to microenvironmental stimuli. In this review, we outline the impacts of PI3K inhibition on the tumor microenvironment with a specific focus on CLL. Acknowledging the effects of PI3K inhibitor-based therapies on the tumor microenvironment in CLL can serve as a rationale for improved drug development, explain treatment-associated adverse events, and suggest novel combinatory treatment strategies in CLL.
    Keywords:  chronic lymphocytic leukemia; idelalisib; phosphoinositide 3-kinase (PI3K); phosphoinositide 3-kinase (PI3K) inhibition; tumor microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2020.595818
  4. J Lipid Res. 2020 Jun;pii: S0022-2275(20)43603-X. [Epub ahead of print]61(6): 945-952
    A direct fluorometric activity assay for lipid kinases and phosphatases.
    Jiachen Sun, Indira Singaram, Mona Hoseini Soflaee, Wonhwa Cho.
      Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.
    Keywords:  enzyme kinetics; high-throughput inhibitor screening; lipid phosphatases; phosphoinositide 3-kinase/phosphatase and tensin homolog; ratiometric sensor; real-time activity assay
    DOI:  https://doi.org/10.1194/jlr.D120000794
  5. Front Cell Dev Biol. 2020 ;8 607444
    Both Hypoxia-Inducible Factor 1 and MAPK Signaling Pathway Attenuate PI3K/AKT via Suppression of Reactive Oxygen Species in Human Pluripotent Stem Cells.
    Petr Fojtík, Deborah Beckerová, Katerina Holomková, Martin Šenfluk, Vladimir Rotrekl.
      Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.
    Keywords:  HIF-1; MAPK; PI3K/AKT; hPSCs; hypoxia; reactive oxygen species
    DOI:  https://doi.org/10.3389/fcell.2020.607444
  6. Cell Death Discov. 2020 Jun 15. 6(1): 52
    Metformin effectively treats Tsc1 deletion-caused kidney pathology by upregulating AMPK phosphorylation.
    Yili Fang, Fang Li, Chenyang Qi, Xing Mao, Feng Wang, Zhonghua Zhao, Jian-Kang Chen, Zhigang Zhang, Huijuan Wu.
      Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in multiple organs, with most patients developing polycystic kidney disease and leading to a decline of renal function. TSC is caused by loss-of-function mutations in either Tsc1 or Tsc2 gene, but currently, there is no effective treatment for aberrant kidney growth in TSC patients. By generating a renal proximal tubule-specific Tsc1 gene-knockout (Tsc1ptKO) mouse model, we observed that Tsc1ptKO mice developed aberrantly enlarged kidneys primarily due to hypertrophy and proliferation of proximal tubule cells, along with some cystogenesis, interstitial inflammation, and fibrosis. Mechanistic studies revealed inhibition of AMP-activated protein kinase (AMPK) phosphorylation at Thr-172 and activation of Akt phosphorylation at Ser-473 and Thr-308. We therefore treated Tsc1ptKO mice with the AMPK activator, metformin, by daily intraperitoneal injection. Our results indicated that metformin increased the AMPK phosphorylation, but decreased the Akt phosphorylation. These signaling modulations resulted in inhibition of proliferation and induction of apoptosis in the renal proximal tubule cells of Tsc1ptKO mice. Importantly, metformin treatment effectively prevented aberrant kidney enlargement and cyst growth, inhibited inflammatory response, attenuated interstitial fibrosis, and protected renal function. The effects of metformin were further confirmed by in vitro experiments. In conclusion, this study indicates a potential therapeutic effect of metformin on Tsc1 deletion-induced kidney pathology, although currently metformin is primarily prescribed to treat patients with type 2 diabetes.
    DOI:  https://doi.org/10.1038/s41420-020-0285-0
  7. PLoS One. 2021 ;16(2): e0245179
    MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.
    Clothilde Philouze, Sophie Turban, Beatrice Cremers, Audrey Caliez, Gwladys Lamarche, Catherine Bernard, Nicolas Provost, Philippe Delerive.
      In type 2 diabetes (T2D), both muscle and liver are severely resistant to insulin action. Muscle insulin resistance accounts for more than 80% of the impairment in total body glucose disposal in T2D patients and is often characterized by an impaired insulin signaling. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein initially identified as a key regulator of cell membrane repair machinery has been suggested to be a critical regulator of muscle insulin signaling pathway by acting as ubiquitin E3 ligase targeting both the insulin receptor and insulin receptor substrate 1 (IRS1). Here, we show using in vitro and in vivo approaches that MG53 is not a critical regulator of insulin signaling and glucose homeostasis. First, MG53 expression is not consistently regulated in skeletal muscle from various preclinical models of insulin resistance. Second, MG53 gene knock-down in muscle cells does not lead to impaired insulin response as measured by Akt phosphorylation on Serine 473 and glucose uptake. Third, recombinant human MG53 does not alter insulin response in both differentiated C2C12 and human skeletal muscle cells. Fourth, ectopic expression of MG53 in HEK293 cells lacking endogenous MG53 expression fails to alter insulin response as measured by Akt phosphorylation. Finally, both male and female mg53 -/- mice were not resistant to high fat induced obesity and glucose intolerance compared to wild-type mice. Taken together, these results strongly suggest that MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.
    DOI:  https://doi.org/10.1371/journal.pone.0245179
  8. Cell Death Discov. 2020 Apr 22. 6(1): 27
    How does mTOR sense glucose starvation? AMPK is the usual suspect.
    Gabriel Leprivier, Barak Rotblat.
      Glucose is a major requirement for biological life. Its concentration is constantly sensed at the cellular level, allowing for adequate responses to any changes of glucose availability. Such responses are mediated by key sensors and signaling pathway components that adapt cellular metabolism to glucose levels. One of the major hubs of these responses is mechanistic target of rapamycin (mTOR) kinase, which forms the mTORC1 and mTORC2 protein complexes. Under physiological glucose concentrations, mTORC1 is activated and stimulates a number of proteins and enzymes involved in anabolic processes, while restricting the autophagic process. Conversely, when glucose levels are low, mTORC1 is inhibited, in turn leading to the repression of numerous anabolic processes, sparing ATP and antioxidants. Understanding how mTORC1 activity is regulated by glucose is not only important to better delineate the biological function of mTOR, but also to highlight potential therapeutic strategies for treating diseases characterized by deregulated glucose availability, as is the case of cancer. In this perspective, we depict the different sensors and upstream proteins responsible of controlling mTORC1 activity in response to changes in glucose concentration. This includes the major energy sensor AMP-activated protein kinase (AMPK), as well as other independent players. The impact of such modes of regulation of mTORC1 on cellular processes is also discussed.
    DOI:  https://doi.org/10.1038/s41420-020-0260-9
  9. J Biol Chem. 2021 Feb 07. pii: S0021-9258(21)00167-8. [Epub ahead of print] 100395
    The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure.
    Tzu-Chieh Chen, Taiyi Kuo, Mohamad Dandan, Rebecca A Lee, Maggie Chang, Sneha D Villivalam, Szu-Chi Liao, Damian Costello, Mahalakshmi Shankaran, Hussein Mohammed, Sona Kang, Marc K Hellerstein, Jen-Chywan Wang.
      Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remains unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone, a synthetic glucocorticoid. Treating wild type (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue and gastrocnemius muscle. This DEX effect was diminished in gastrocnemius muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in gastrocnemius muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced gastrocnemius myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle.
    Keywords:  Glucocorticoids; Pik3r1; glucocorticoid receptor; insulin resistance; protein synthesis; skeletal muscle; striated muscle
    DOI:  https://doi.org/10.1016/j.jbc.2021.100395
  10. Dev Cell. 2021 Feb 08. pii: S1534-5807(21)00038-1. [Epub ahead of print]56(3): 251-252
    How NPC1 Loss Twists the TORCque of Lysosomes.
    W Mike Henne.
      Niemann-Pick type C is a neurological disorder caused by mutations in the lysosome cholesterol exporter NPC1. In this issue of Developmental Cell, Davis et al. dissect how NPC1 loss elevates mTORC1 signaling, and demonstrate that suppression of mTORC1 signaling can rescue disease pathology in NPC1-deficient cell models.
    DOI:  https://doi.org/10.1016/j.devcel.2021.01.007
  11. Nature. 2021 02;590(7845): 221-223
    New-found brake calibrates insulin action in β-cells.
    Rohit N Kulkarni.
      
    Keywords:  Diabetes; Metabolism
    DOI:  https://doi.org/10.1038/d41586-021-00141-9
  12. Curr Opin Cell Biol. 2021 Feb 09. pii: S0955-0674(21)00001-6. [Epub ahead of print]69 103-110
    Cell memory of epithelial-mesenchymal plasticity in cancer.
    Jordi Berenguer, Toni Celià-Terrassa.
      Fundamental biological processes of cell identity and cell fate determination are controlled by complex regulatory networks. These processes require molecular mechanisms that confer cellular phenotypic memory and state persistence. In this minireview, we will summarize mechanisms of cell memory based on regulatory hysteretic feedback loops and explore epigenetic mechanisms widely represented in nature, with special focus on epithelial-to-mesenchymal plasticity. We will also discuss the functional consequences of cell memory and epithelial-to-mesenchymal plasticity dynamics during development and cancer metastasis.
    Keywords:  Cell plasticity; Cell state; Cellular memory; Dynamics; EMT; Feedback loops; Hysteresis; Multistability
    DOI:  https://doi.org/10.1016/j.ceb.2021.01.001
  13. Nat Biotechnol. 2021 Feb 11.
    A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer.
    Daniel S C Butler, Caterina Cafaro, Johannes Putze, Murphy Lam Yim Wan, Thi Hien Tran, Ines Ambite, Shahram Ahmadi, Sven Kjellström, Charlotte Welinder, Sing Ming Chao, Ulrich Dobrindt, Catharina Svanborg.
      Is the oncogene MYC upregulated or hyperactive? In the majority of human cancers, finding agents that target c-MYC has proved difficult. Here we report specific bacterial effector molecules that inhibit cellular MYC (c-MYC) in human cells. We show that uropathogenic Escherichia coli (UPEC) degrade the c-MYC protein and attenuate MYC expression in both human cells and animal tissues. c-MYC protein was rapidly degraded by both cell-free bacterial lysates and the purified bacterial protease Lon. In mice, intravesical or peroral delivery of Lon protease delayed tumor progression and increased survival in MYC-dependent bladder and colon cancer models, respectively. These results suggest that bacteria have evolved strategies to control c-MYC tissue levels in the host and that the Lon protease shows promise for therapeutic targeting of c-MYC in cancer.
    DOI:  https://doi.org/10.1038/s41587-020-00805-3
  14. Sci Rep. 2021 Feb 10. 11(1): 3447
    Role of PDK1 in skeletal muscle hypertrophy induced by mechanical load.
    Naoki Kuramoto, Kazuhiro Nomura, Daisuke Kohno, Tadahiro Kitamura, Gerard Karsenty, Tetsuya Hosooka, Wataru Ogawa.
      Phosphatidylinositol 3-kinase (PI3K) plays an important role in protein metabolism and cell growth. We here show that mice (M-PDK1KO mice) with skeletal muscle-specific deficiency of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key component of PI3K signaling pathway, manifest a reduced skeletal muscle mass under the static condition as well as impairment of mechanical load-induced muscle hypertrophy. Whereas mechanical load-induced changes in gene expression were not affected, the phosphorylation of ribosomal protein S6 kinase (S6K) and S6 induced by mechanical load was attenuated in skeletal muscle of M-PDK1KO mice, suggesting that PDK1 regulates muscle hypertrophy not through changes in gene expression but through stimulation of kinase cascades such as the S6K-S6 axis, which plays a key role in protein synthesis. Administration of the β2-adrenergic receptor (AR) agonist clenbuterol activated the S6K-S6 axis in skeletal muscle and induced muscle hypertrophy in mice. These effects of clenbuterol were attenuated in M-PDK1KO mice, and mechanical load-induced activation of the S6K-S6 axis and muscle hypertrophy were inhibited in mice with skeletal muscle-specific deficiency of β2-AR. Our results suggest that PDK1 regulates skeletal muscle mass under the static condition and that it contributes to mechanical load-induced muscle hypertrophy, at least in part by mediating signaling from β2-AR.
    DOI:  https://doi.org/10.1038/s41598-021-83098-z
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