bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒06‒28
twenty-nine papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Nature. 2020 Jun 24.
    Aitken SJ, Anderson CJ, Connor F, Pich O, Sundaram V, Feig C, Rayner TF, Lukk M, Aitken S, Luft J, Kentepozidou E, Arnedo-Pac C, Beentjes SV, Davies SE, Drews RM, Ewing A, Kaiser VB, Khamseh A, López-Arribillaga E, Redmond AM, Santoyo-Lopez J, Sentís I, Talmane L, Yates AD, , Semple CA, López-Bigas N, Flicek P, Odom DT, Taylor MS.
      Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.
  2. Cancer Cell. 2020 Jun 09. pii: S1535-6108(20)30268-3. [Epub ahead of print]
    Kudo Y, Sugimoto M, Arias E, Kasashima H, Cordes T, Linares JF, Duran A, Nakanishi Y, Nakanishi N, L'Hermitte A, Campos A, Senni N, Rooslid T, Roberts LR, Cuervo AM, Metallo CM, Karin M, Diaz-Meco MT, Moscat J.
      Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.
    Keywords:  NRF2; PKCζ; PKCι; PKCλ; atypical PKC; autophagy; hepatocellular carcinoma; metabolic reprogramming; oxidative phosphorylation; reactive oxygen species
  3. Cancer Discov. 2020 Jun 22. pii: CD-19-1228. [Epub ahead of print]
    Ngo B, Kim E, Osorio-Vasquez V, Doll S, Bustraan S, Liang RJ, Luengo A, Davidson SM, Ali A, Ferraro GB, Fischer GM, Eskandari R, Kang DS, Ni J, Plasger A, Rajasekhar VK, Kastenhuber ER, Bacha S, Sriram RK, Stein BD, Bakhoum SF, Snuderl M, Cotzia P, Healey JH, Mainolfi N, Suri V, Friedman A, Manfredi M, Sabatini DM, Jones DR, Yu M, Zhao JJ, Jain RK, Keshari KR, Davies MA, Vander Heiden MG, Hernando E, Mann M, Cantley LC, Pacold ME.
      A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.
  4. Nat Med. 2020 Jun 22.
    Izar B, Tirosh I, Stover EH, Wakiro I, Cuoco MS, Alter I, Rodman C, Leeson R, Su MJ, Shah P, Iwanicki M, Walker SR, Kanodia A, Melms JC, Mei S, Lin JR, Porter CBM, Slyper M, Waldman J, Jerby-Arnon L, Ashenberg O, Brinker TJ, Mills C, Rogava M, Vigneau S, Sorger PK, Garraway LA, Konstantinopoulos PA, Liu JF, Matulonis U, Johnson BE, Rozenblatt-Rosen O, Rotem A, Regev A.
      Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.
  5. Dev Cell. 2020 Jun 12. pii: S1534-5807(20)30420-2. [Epub ahead of print]
    Fattet L, Jung HY, Matsumoto MW, Aubol BE, Kumar A, Adams JA, Chen AC, Sah RL, Engler AJ, Pasquale EB, Yang J.
      Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.
    Keywords:  ECM stiffness and breast cancer; EMT; EPHA2; LYN; TWIST1; epithelial-mesenchymal transition; matrix stiffness; mechanotransduction; metastasis
  6. Sci Adv. 2020 Jun;6(24): eaba3458
    Wu J, Weisshaar N, Hotz-Wagenblatt A, Madi A, Ma S, Mieg A, Hering M, Mohr K, Schlimbach T, Borgers H, Cui G.
      CD8+ T cells become functionally impaired or "exhausted" in chronic infections, accompanied by unwanted body weight reduction and muscle mass loss. Whether muscle regulates T cell exhaustion remains incompletely understood. We report that mouse skeletal muscle increased interleukin (IL)-15 production during LCMV clone 13 chronic infection. Muscle-specific ablation of Il15 enhanced the CD8+ T cell exhaustion phenotype. Muscle-derived IL-15 was required to maintain a population of CD8+CD103+ muscle-infiltrating lymphocytes (MILs). MILs resided in a less inflamed microenvironment, expressed more T cell factor 1 (Tcf1), and had higher proliferative potential than splenic T cells. MILs differentiated into functional effector T cells after reentering lymphoid tissues. Increasing muscle mass via muscle-specific inhibition of TGFβ signaling enhanced IL-15 production and antiviral CD8+ T cell responses. We conclude that skeletal muscle antagonizes T cell exhaustion by protecting T cell proliferative potential from inflammation and replenishing the effector T cell progeny pool in lymphoid organs.
  7. J Clin Invest. 2020 Jun 22. pii: 132513. [Epub ahead of print]
    Cochrane CR, Vaghjiani V, Szczepny A, Jayasekara WSN, Gonzalez-Rajal A, Kikuchi K, McCaughan GW, Burgess A, Gough DJ, Watkins DN, Cain JE.
      Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.
    Keywords:  Autophagy; Cell Biology; Lung cancer; Oncology
  8. Sci Signal. 2020 Jun 23. pii: eaba1015. [Epub ahead of print]13(637):
    Thakore P, Pritchard HAT, Griffin CS, Yamasaki E, Drumm BT, Lane C, Sanders KM, Feng Earley Y, Earley S.
      TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.
  9. Cancer. 2020 Jun 23.
    McIntyre CA, Lawrence SA, Richards AL, Chou JF, Wong W, Capanu M, Berger MF, Donoghue MTA, Yu KH, Varghese AM, Kelsen DP, Park W, Balachandran VP, Kingham TP, D'Angelica MI, Drebin JA, Jarnagin WR, Iacobuzio-Donahue CA, Allen PJ, O'Reilly EM.
      BACKGROUND: KRAS, TP53, CDKN2A, and SMAD4 are established driver genes in pancreatic ductal adenocarcinoma (PDAC). This study was aimed at determining whether the mutational status of driver genes and those involved in DNA repair pathways are associated with clinical outcomes for individuals who undergo resection.METHODS: Eligible individuals were those who underwent resection of PDAC and consented to targeted sequencing of their primary tumor via Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). Genomic alterations were determined on the basis of MSK-IMPACT results from formalin-fixed, paraffin-embedded samples. Associations between genomic alterations and clinical outcomes were assessed.
    RESULTS: Targeted sequencing was performed on 283 primary tumors resected between 2004 and 2017. The median follow-up was 23 months among survivors. Alterations in KRAS and TP53 were associated with worse overall survival (OS) in comparison to wild type (median for KRAS, 38.8 months [95% CI, 33.0-45.5 months] vs 91.0 months [95% CI, 34.8 months to not available (NA)]; P = .043; median for TP53, 37.4 months [95% CI, 32.1-42.8 months] vs 65.0 months [95% CI, 33.0 months to NA]; P = .035). KRAS G12D mutations were associated with worse OS (median, 31.6 months [95% CI, 25.3-45.5 months] vs 39.2 months [95% CI, 37.4-75.2 months]; P = .012). TP53 truncating mutations (median, 39.6 months [95% CI, 32.4-75.2 months] vs 33.9 months [95% CI, 24.0-39.0 months]; P = .020) and those associated with loss of heterozygosity (median, 26.6 months [95% CI, 21.6-44.2 months] vs 39.2 months [95% CI, 34.5-49.1 months]; P = .048) had decreased OS. TP53 alterations were independently associated with OS in a multivariate analysis (hazard ratio, 1.54; 95% CI, 1.01-2.33; P = .042). Individuals with germline alterations in homologous recombination deficiency (HRD) genes had improved OS in comparison with those without them (median, not reached vs 37.0 months; 95% CI, 33.0-49.8 months; P = .035).
    CONCLUSIONS: In patients with resected PDAC, genomic alterations in KRAS and TP53 are associated with worse outcomes, whereas alterations in HRD genes are associated with a favorable prognosis. Further studies are needed to better define these alterations as biomarkers in resected PDAC.
    Keywords:  driver gene alterations; homologous recombination; pancreatic ductal adenocarcinoma; resection; survival outcomes
  10. Cell Metab. 2020 Jun 22. pii: S1550-4131(20)30307-7. [Epub ahead of print]
    Gnad T, Navarro G, Lahesmaa M, Reverte-Salisa L, Copperi F, Cordomi A, Naumann J, Hochhäuser A, Haufs-Brusberg S, Wenzel D, Suhr F, Jespersen NZ, Scheele C, Tsvilovskyy V, Brinkmann C, Rittweger J, Dani C, Kranz M, Deuther-Conrad W, Eltzschig HK, Niemi T, Taittonen M, Brust P, Nuutila P, Pardo L, Fleischmann BK, Blüher M, Franco R, Bloch W, Virtanen KA, Pfeifer A.
      The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
    Keywords:  GPCR; adenosine; adenosine receptor A2B; aging; brown adipose tissue; browning; energy metabolism; muscle; obesity; sarcopenia
  11. Aging Cell. 2020 Jun 22.
    Tanaka H, Igata T, Etoh K, Koga T, Takebayashi SI, Nakao M.
      Senescent cells may possess the intrinsic programs of metabolic and epigenomic remodeling, but the molecular mechanism remains to be clarified. Using an RNAi-based screen of chromatin regulators, we found that knockdown of the NSD2/WHSC1/MMSET methyltransferase induced cellular senescence that augmented mitochondrial mass and oxidative phosphorylation in primary human fibroblasts. Transcriptome analysis showed that loss of NSD2 downregulated the expression of cell cycle-related genes in a retinoblastoma protein (RB)-mediated manner. Chromatin immunoprecipitation analyses further revealed that NSD2 was enriched at the gene bodies of actively transcribed genes, including cell cycle-related genes, and that loss of NSD2 decreased the levels of histone H3 lysine 36 trimethylation (H3K36me3) at these gene loci. Consistent with these findings, oncogene-induced or replicative senescent cells showed reduced NSD2 expression together with lower H3K36me3 levels at NSD2-enriched genes. In addition, we found that NSD2 gene was upregulated by serum stimulation and required for the induction of cell cycle-related genes. Indeed, in both mouse and human tissues and human cancer cell lines, the expression levels of NSD2 were positively correlated with those of cell cycle-related genes. These data reveal that NSD2 plays a pivotal role in epigenomic maintenance and cell cycle control to prevent cellular senescence.
    Keywords:  H3K36 methylation; NSD2/WHSC1/MMSET; cell cycle control; retinoblastoma; senescence-associated epigenomic remodeling; senescence-associated metabolic remodeling
  12. Mol Biol Cell. 2020 Jun 24. mbcE19100589
    Zhang H, Zhao R, Tones J, Liu M, Dilley R, Chenoweth DM, Greenberg RA, Lampson MA.
      Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically-induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere binding protein sumoylation nucleates APB condensation via SUMO-SIM (SUMO interaction motif) interactions, and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering independent of sumoylation. Our findings introduce a chemical dimerization approach to manipulate phase separation and demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
  13. Cell Metab. 2020 Jun 17. pii: S1550-4131(20)30305-3. [Epub ahead of print]
    Fafián-Labora JA, Rodríguez-Navarro JA, O'Loghlen A.
      Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging.
    Keywords:  4-HNE; EV; GSH; GST; ROS; SASP; aging; extracellular vesicles; glutathione metabolism; glutathione-S-transferase; lipid peroxidation; rejuvenation; senescence; senescence-associated secretory phenotype
  14. EMBO J. 2020 Jun 22. e103790
    Dumas AA, Pomella N, Rosser G, Guglielmi L, Vinel C, Millner TO, Rees J, Aley N, Sheer D, Wei J, Marisetty A, Heimberger AB, Bowman RL, Brandner S, Joyce JA, Marino S.
      Tumour-associated microglia/macrophages (TAM) are the most numerous non-neoplastic populations in the tumour microenvironment in glioblastoma multiforme (GBM), the most common malignant brain tumour in adulthood. The mTOR pathway, an important regulator of cell survival/proliferation, is upregulated in GBM, but little is known about the potential role of this pathway in TAM. Here, we show that GBM-initiating cells induce mTOR signalling in the microglia but not bone marrow-derived macrophages in both in vitro and in vivo GBM mouse models. mTOR-dependent regulation of STAT3 and NF-κB activity promotes an immunosuppressive microglial phenotype. This hinders effector T-cell infiltration, proliferation and immune reactivity, thereby contributing to tumour immune evasion and promoting tumour growth in mouse models. The translational value of our results is demonstrated in whole transcriptome datasets of human GBM and in a novel in vitro model, whereby expanded-potential stem cells (EPSC)-derived microglia-like cells are conditioned by syngeneic patient-derived GBM-initiating cells. These results raise the possibility that microglia could be the primary target of mTOR inhibition, rather than the intrinsic tumour cells in GBM.
    Keywords:   TAM ; mTOR ; T cells; glioblastoma; microglia
  15. Proc Natl Acad Sci U S A. 2020 Jun 25. pii: 202004223. [Epub ahead of print]
    Greenway R, Barts N, Henpita C, Brown AP, Arias Rodriguez L, Rodríguez Peña CM, Arndt S, Lau GY, Murphy MP, Wu L, Lin D, Tobler M, Kelley JL, Shaw JH.
      Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.
    Keywords:  adaptive evolution; comparative physiology; ecological genomics; hydrogen sulfide; phylogenetic comparative analysis
  16. Proc Natl Acad Sci U S A. 2020 Jun 24. pii: 201922197. [Epub ahead of print]
    Mu L, Kang JH, Olcum S, Payer KR, Calistri NL, Kimmerling RJ, Manalis SR, Miettinen TP.
      Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.
    Keywords:  cell cycle; cell growth; cell size; mass measurement; transport limitation
  17. Mol Cancer Res. 2020 Jun 22. pii: molcanres.0291.2020. [Epub ahead of print]
    Lazar I, Fabre B, Feng Y, Khateb A, Turko P, Martinez-Gomez MJ, Frederick DT, Levesque MP, Feld L, Zhang G, Zhang T, James B, Shklover J, Avitan Hersh E, Livneh I, Scortegagna M, Brown K, Larsson O, Topisirovic I, Wolfenson H, Herlyn M, Flaherty K, Dummer R, Ronai ZA.
      Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX, a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX down-regulation in melanoma cell lines perturbed nuclear organization, decreased cell viability and promoted senescence-associated phenotypes. Moreover, SPANX knockdown in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX-knockdown in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naive melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. Implications: SPANX, a testis protein, interacts with LAMNA and controls nuclear architecture and melanoma growth.
  18. Nat Metab. 2019 Nov;1(11): 1127-1140
    Wang C, Haas MA, Yang F, Yeo S, Okamoto T, Chen S, Wen J, Sarma P, Plas DR, Guan JL.
      Although mTORC1 negatively regulates autophagy in cultured cells, how autophagy impacts mTORC1 signaling, in particular in vivo, is less clear. Here we show that autophagy supports mTORC1 hyperactivation in NSCs lacking Tsc1, thereby promoting defects in NSC maintenance, differentiation, tumourigenesis, and the formation of the neurodevelopmental lesion of Tuberous Sclerosis Complex (TSC). Analysing mice that lack Tsc1 and the essential autophagy gene Fip200 in NSCs we find that TSC-deficient cells require autophagy to maintain mTORC1 hyperactivation under energy stress conditions, likely to provide lipids via lipophagy to serve as an alternative energy source for OXPHOS. In vivo, inhibition of lipophagy or its downstream catabolic pathway reverses defective phenotypes caused by Tsc1-null NSCs and reduces tumorigenesis in mouse models. These results reveal a cooperative function of selective autophagy in coupling energy availability with TSC pathogenesis and suggest a potential new therapeutic strategy to treat TSC patients.
    Keywords:  autophagy; energy stress; lipid catabolism; mTORC1; neural stem cells; tumorigenesis
  19. J Clin Invest. 2020 Jun 23. pii: 137660. [Epub ahead of print]
    Dodhiawala PB, Khurana N, Zhang D, Cheng Y, Li L, Wei Q, Seehra K, Jiang H, Grierson PM, Wang-Gillam A, Lim KH.
      NF-kB transcription factors, driven by the IRAK-IKK cascade, confer treatment resistance in pancreatic ductal adenocarcinoma (PDAC), a cancer characterized by near universal KRAS mutation. Through reverse-phase protein array and RNAseq we discovered IRAK4 also contributes substantially to MAPK activation in KRAS-mutant PDAC. IRAK4 ablation completely blocked RAS-induced transformation of human and murine cells. Mechanistically, expression of mutant KRAS stimulated an inflammatory, autocrine IL-1b signaling loop that activated IRAK4 and MAPK pathway. Downstream of IRAK4, we uncovered TPL2/MAP3K8 as the essential kinase that propels both MAPK and NF-kB cascades. Inhibition of TPL2 blocked both MAPK and NF-kB signaling, and suppressed KRAS-mutant cell growth. To counter chemotherapy-induced genotoxic stress, PDAC cells upregulated TLR9, which activated pro-survival IRAK4-TPL2 signaling. Accordingly, TPL2 inhibitor synergized with chemotherapy to curb PDAC growth in vivo. Finally, from TCGA we characterized two MAP3K8 point mutations that hyperactivate MAPK and NF-kB cascades by impeding TPL2 protein degradation. Cancer cell lines naturally harboring these MAP3K8 mutations are strikingly sensitive to TPL2 inhibition, underscoring the need to identify these potentially targetable mutations in patients. Overall, our study establishes TPL2 as a promising therapeutic target in RAS- and MAP3K8-mutant cancers and strongly prompts development of TPL2 inhibitors for pre-clinical and clinical studies.
    Keywords:  Inflammation; NF-kappaB; Oncogenes; Oncology; Protein kinases
  20. Proc Natl Acad Sci U S A. 2020 Jun 22. pii: 201922535. [Epub ahead of print]
    Mushtaq M, Kovalevska L, Darekar S, Abramsson A, Zetterberg H, Kashuba V, Klein G, Arsenian-Henriksson M, Kashuba E.
      Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization of Rb1 -/- primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at the S18-2 promoter region and that the S18-2 expression is positively correlated with KLF4 levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.
    Keywords:  cell immortalization; embryogenesis; stemness and differentiation; tumorigenesis
  21. Trends Mol Med. 2020 Jul;pii: S1471-4914(20)30077-0. [Epub ahead of print]26(7): 630-638
    Pignolo RJ, Passos JF, Khosla S, Tchkonia T, Kirkland JL.
      Cellular senescence is a primary aging process and tumor suppressive mechanism characterized by irreversible growth arrest, apoptosis resistance, production of a senescence-associated secretory phenotype (SASP), mitochondrial dysfunction, and alterations in DNA and chromatin. In preclinical aging models, accumulation of senescent cells is associated with multiple chronic diseases and disorders, geriatric syndromes, multimorbidity, and accelerated aging phenotypes. In animals, genetic and pharmacologic reduction of senescent cell burden results in the prevention, delay, and/or alleviation of a variety of aging-related diseases and sequelae. Early clinical trials have thus far focused on safety and target engagement of senolytic agents that clear senescent cells. We hypothesize that these pharmacologic interventions may have transformative effects on geriatric medicine.
  22. Sci Transl Med. 2020 Jun 24. pii: eaay9013. [Epub ahead of print]12(549):
    Li W, Lu L, Lu J, Wang X, Yang C, Jin J, Wu L, Hong X, Li F, Cao D, Yang Y, Wu M, Su B, Cheng J, Yang X, Di W, Deng L.
      Although cGAS-STING-mediated DNA sensing in tumor cells or phagocytes is central for launching antitumor immunity, the role of intrinsic cGAS-STING activation in T cells remains unknown. Here, we observed that peripheral blood CD8+ T cells from patients with cancer showed remarkably compromised expression of the cGAS-STING cascade. We demonstrated that the cGAS-STING cascade in adoptively transferred CD8+ T cells was essential for antitumor immune responses in the context of T cell therapy in mice. Mechanistically, cell-autonomous cGAS and STING promoted the maintenance of stem cell-like CD8+ T cells, in part, by regulating the transcription factor TCF1 expression. Moreover, autocrine cGAS-STING-mediated type I interferon signaling augmented stem cell-like CD8+ T cell differentiation program mainly by restraining Akt activity. In addition, genomic DNA was selectively enriched in the cytosol of mouse CD8+ T cells upon in vitro and in vivo stimulation. STING agonism enhanced the formation of stem-like central memory CD8+ T cells from patients with cancer and potentiated antitumor responses of CAR-T cell therapy in a xenograft model. These findings advance our understanding of inherent cGAS-STING activation in T cells and provide insight into the development of improved T cell therapy by harnessing the cGAS-STING pathway for cancer immunotherapy.
  23. Proc Natl Acad Sci U S A. 2020 Jun 22. pii: 202007400. [Epub ahead of print]
    Grones P, Majda M, Doyle SM, Van Damme D, Robert S.
      Puzzle-shaped pavement cells provide a powerful model system to investigate the cellular and subcellular processes underlying complex cell-shape determination in plants. To better understand pavement cell-shape acquisition and the role of auxin in this process, we focused on the spirals of young stomatal lineage ground cells of Arabidopsis leaf epidermis. The predictability of lobe formation in these cells allowed us to demonstrate that the auxin response gradient forms within the cells of the spiral and fluctuates based on the particular stage of lobe development. We revealed that specific localization of auxin transporters at the different membranes of these young cells changes during the course of lobe formation, suggesting that these fluctuating auxin response gradients are orchestrated via auxin transport to control lobe formation and determine pavement cell shape.
    Keywords:  Arabidopsis; auxin response gradient; auxin transporter; lobe formation; pavement cells
  24. Biochemistry (Mosc). 2020 Apr;85(4): 393-408
    Nesterov SV, Yaguzhinsky LS, Podoprigora GI, Nartsissov YR.
      In this review, we discuss the principles of regulation and synchronization of metabolic processes in mammalian cells using a two-component model of cell metabolism consisting of a controlling signaling system that regulates major enzymatic cascades and executive metabolic system that directly performs biosynthetic reactions. This approach has allowed us to distinguish two transitional metabolic states (from catabolism to anabolism and vice versa) accompanied by major rearrangements in the signaling system. The signaling system of natural amino acids was selected, because amino acids are involved in both signaling and executive metabolic subsystems of general cell metabolism. We have developed a graphical representation of metabolic events that allowed us to demonstrate the succession of processes occurring in both metabolic subsystems during complete metabolic cycle in a non-dividing cell. An important revealed feature of the amino acid signaling system is that the signaling properties of amino acid are determined not only by their molecular structure, but also by the location within the cell. Four major signaling groups of amino acids have been identified that localize to lysosomes, mitochondria, cytosol, and extracellular space adjacent to the plasma membrane. Although these amino acids groups are similar in the composition, they have different receptors. We also proposed a scheme for the metabolism regulation by amino acids signaling that can serve as a basis for developing more complete spatio-temporal picture of metabolic regulation involving a wide variety of intracellular signaling cascades.
  25. Clin Cancer Res. 2020 Jun 26. pii: clincanres.1025.2020. [Epub ahead of print]
    Wu AA, Bever KM, Ho WJ, Fertig EJ, Niu N, Zheng L, Parkinson RM, Durham JN, Onners BL, Ferguson A, Wilt C, Ko AH, Wang-Gillam A, Laheru DA, Anders RA, Thompson ED, Sugar EA, Jaffee EM, Le DT.
      PURPOSE: This phase 2 study tested granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells (GVAX) and ipilimumab in metastatic pancreatic ductal adenocarcinoma (PDA) in the maintenance setting.METHODS: Patients with PDA who were treated with front-line chemotherapy consisting of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) in the metastatic setting and had ongoing response or stable disease after 8-12 doses were eligible. Patients were randomized 1:1 to treatment with GVAX and ipilimumab given every 3 weeks for 4 doses then every 8 weeks (Arm A) or to FOLFIRINOX continuation (Arm B). The primary objective was to compare overall survival (OS) between the two arms.
    RESULTS: Eighty-two patients were included in the final analysis (Arm A: 40; Arm B: 42). The study was stopped for futility after interim analysis. Median overall survival (OS) was 9.38 months (95% CI: 5.0, 12.2) for Arm A and 14.7 months (95% CI: 11.6, 20.0) for Arm B (HR 1.75, p=0.019). Using immune related-response criteria, 2 partial responses (5.7%) were observed in Arm A and 4 (13.8%) in Arm B. GVAX + ipilimumab promoted T cell differentiation into effector memory phenotypes both in the periphery and in the tumor microenvironment and increased M1 macrophages in the tumor.
    CONCLUSIONS: GVAX and ipilimumab maintenance therapy did not improve OS over continuation of chemotherapy and resulted in a numerically inferior survival in metastatic PDA. However, clinical responses and biological effects on immune cells were observed. Further study of novel combinations in the maintenance treatment of metastatic PDA is feasible.
  26. Cancer Res. 2020 Jun 24. pii: canres.3281.2019. [Epub ahead of print]
    Wang F, Qi XM, Wertz R, Mortensen M, Hagen C, Evans J, Sheinin Y, James M, Liu P, Tsai S, Thomas J, Mackinnon A, Dwinell M, Myers CR, Bartrons R, Fu L, Chen G.
      KRAS is mutated in most pancreatic ductal adenocarcinomas (PDAC) and yet remains undruggable. Here we report that p38gamma MAPK, which promotes PDAC tumorigenesis by linking KRAS signaling and aerobic glycolysis (also called the Warburg effect), is a novel therapeutic target. p38gamma interacted with a glycolytic activator PFKFB3 that was dependent on mutated KRAS. KRAS transformation and overexpression of p38gamma increased expression of PFKFB3 and glucose transporter GLUT2; conversely, silencing mutant KRAS and p38gamma decreased PFKFB3 and GLUT2 expression. p38gamma phosphorylated PFKFB3 at S467, stabilized PFKFB3, and promoted their interaction with GLUT2. Pancreatic knockout (KO) of p38gamma decreased p-PFKFB3/PFKFB3/GLUT2 protein levels, reduced aerobic glycolysis, and inhibited PDAC tumorigenesis in KPC mice. PFKFB3 and GLUT2 depended on p38gamma to stimulate glycolysis and PDAC growth and p38gamma required PFKFB3/S467 to promote these activities. A p38gamma inhibitor cooperated with a PFKFB3 inhibitor to blunt aerobic glycolysis and PDAC growth, which was dependent on p38gamma. Moreover, overexpression of p38gamma, p-PFKFB3, PFKFB3, and GLUT2 in PDAC predicted poor clinical prognosis. These results indicate that p38gamma links KRAS oncogene signaling and aerobic glycolysis to promote pancreatic tumorigenesis through PFKFB3 and GLUT2, and that p38gamma and PFKFB3 may be targeted for therapeutic intervention in PDAC.
  27. Nat Protoc. 2020 Jun 26.
    Font-Tello A, Kesten N, Xie Y, Taing L, Varešlija D, Young LS, Hamid AA, Van Allen EM, Sweeney CJ, Gjini E, Lako A, Hodi FS, Bellmunt J, Brown M, Cejas P, Long HW.
      Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.
  28. J Cell Sci. 2020 Jun 23. pii: jcs.240374. [Epub ahead of print]
    Lee RG, Gao J, Siira SJ, Shearwood AM, Ermer JA, Hofferek V, Mathews JC, Zheng M, Reid GE, Rackham O, Filipovska A.
      The mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the cardiolipin biosynthesis gene Crls1 to investigate the effects of cardiolipin loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by reduced uncoordinated mitochondrial translation rates and impaired respiratory supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of cardiolipin resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that cardiolipin is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.
    Keywords:  Mitochondrial membranes; Mitochondrial ribosomes; Protein synthesis
  29. FASEB J. 2020 Jun 27.
    Uematsu M, Kita Y, Shimizu T, Shindou H.
      Visualizing intracellular fatty acids (including free and esterified form) is very useful for understanding how and where such molecules are incorporated, stored, and metabolized within cells. However, techniques of imaging multiple intracellular fatty acids have been limited by their small size, making it difficult to label and track without changing their biological and biophysical characteristics. Here, we present a new method for simultaneously visualizing up to five atomically labeled intracellular fatty acid species. For this, we utilized the distinctive Raman spectra depending on the labeling patterns and created a new, extensible opensource software to perform by-pixel analysis of extracting original spectra from mixed ones. Our multiplex imaging method revealed that fatty acids with more double bonds tend to concentrate more efficiently at lipid droplets. This novel approach contributes to reveal not only the spatial dynamics of fatty acids, but also of any other metabolites inside cells.
    Keywords:  lipid droplet; spectral unmixing; triacylglycerol