bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2021‒05‒23
thirty-four papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Cancer Discov. 2021 Mar;11(3): 660-677
      Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate of approximately 9%. An improved understanding of PDAC initiation and progression is paramount for discovering strategies to better detect and combat this disease. Although transcriptomic analyses have uncovered distinct molecular subtypes of human PDAC, the factors that influence subtype development remain unclear. Here, we interrogate the impact of cell of origin and different Trp53 alleles on tumor evolution, using a panel of tractable genetically engineered mouse models. Oncogenic KRAS expression, coupled with Trp53 deletion or point mutation, drives PDAC from both acinar and ductal cells. Gene-expression analysis reveals further that ductal cell-derived and acinar cell-derived tumor signatures are enriched in basal-like and classical subtypes of human PDAC, respectively. These findings highlight cell of origin as one factor that influences PDAC molecular subtypes and provide insight into the fundamental impact that the very earliest events in carcinogenesis can have on cancer evolution. SIGNIFICANCE: Although human PDAC has been classified into different molecular subtypes, the etiology of these distinct subtypes remains unclear. Using mouse genetics, we reveal that cell of origin is an important determinant of PDAC molecular subtype. Deciphering the biology underlying pancreatic cancer subtypes may reveal meaningful distinctions that could improve clinical intervention.This article is highlighted in the In This Issue feature, p. 521.
  2. J Cell Sci. 2020 Jan 01. pii: jcs.240200. [Epub ahead of print]
      Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under Gln deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that SNAP29 regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of synaptosome-associated protein 29 (SNAP29) interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.
    Keywords:  CTPS filaments; Histidine; Intermediate filaments; SNAP29
  3. Cell Metab. 2021 May 17. pii: S1550-4131(21)00183-2. [Epub ahead of print]
      Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of aging. However, the physical characteristics of the extracellular matrix are also altered in cancerous and aging tissues. Here, we demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via solute carrier family 9 member A1-dependent ion exchange and heat shock factor 1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.
    Keywords:  UPRmt; adhesion; aging; cancer; extracellular matrix; mechanical stress; mechanotabolism; metabolism; oxidative stress; tension
  4. J Biol Chem. 2021 May 14. pii: S0021-9258(21)00573-1. [Epub ahead of print] 100780
      Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
    Keywords:  CRISPR/Cas; mammalian target of rapamycin (mTOR); nucleoside/nucleotide biosynthesis; nucleoside/nucleotide metabolism; nucleotide; phosphoribosylformylglycinamidine synthase (PFAS); tuberous sclerosis complex (TSC)
  5. Cancer Sci. 2021 May 19.
      The small GTPases RalA and RalB are members of the Ras family, and activated downstream of Ras. Ral proteins are found in GTP-bound active and GDP-bound inactive forms. The activation process is executed by guanine nucleotide exchange factors while inactivation is mediated by GTPase-activating proteins (GAPs). RalGAPs are complexes that consist of a catalytic α1 or α2 subunit together with a common β subunit. Several reports implicate the importance of Ral in pancreatic ductal adenocarcinoma (PDAC). However, there are few reports on the relationship between levels of RalGAP expression and malignancy in PDAC. We generated RalGAPβ-deficient PDAC cells by CRISPR-Cas9 genome editing to investigate how increased Ral activity affects malignant phenotypes of PDAC cells. RalGAPβ-deficient PDAC cells exhibited several-fold higher Ral activity relative to control cells. They had a high migratory and invasive capacity. The RalGAPβ-deficient cells grew more rapidly than control cells when injected subcutaneously into nude mice. When injected into the spleen, the RalGAPβ-deficient cells formed larger splenic tumors with more liver metastases, and unlike controls, they disseminated into the abdominal cavity. These results indicate that RalGAPβ deficiency in PDAC cells contributes to high activities of RalA and RalB, leading to enhanced cell migration and invasion in vitro, and tumor growth and metastasis in vivo.
    Keywords:  Pancreatic ductal adenocarcinoma; Ral; RalGAP; Ras; small GTPase
  6. J Exp Biol. 2020 Jan 01. pii: jeb.222513. [Epub ahead of print]
      Telomeres are DNA structures that protect chromosome ends. However, telomeres shorten during cell replication and at critically low lengths can reduce cell replicative potential, induce cell senescence and decrease fitness. Stress exposure, which elevates glucocorticoid hormone concentrations, can exacerbate telomere attrition. This phenomenon has been attributed to increased oxidative stress generated by glucocorticoids ('oxidative stress hypothesis'). We recently suggested that glucocorticoids could increase telomere attrition during stressful periods by reducing the resources available for telomere maintenance through changes in the metabolic machinery ('metabolic telomere attrition hypothesis'). Here we tested whether experimental increases in glucocorticoid levels affected telomere length and mitochondrial function in wild great tit (Parus major) nestlings during the energy-demanding early growth. We monitored resulting corticosterone (Cort) concentrations in plasma, and in red blood cells, telomere lengths and mitochondrial metabolism (metabolic rate, proton leak, oxidative phosphorylation, maximal mitochondrial capacity and mitochondrial inefficiency). We assessed oxidative damage caused by reactive oxygen species (ROS) metabolites as well as the total non-enzymatic antioxidant protection in plasma. Compared with control (Ctrl) nestlings, Cort-nestlings had higher baseline corticosterone, shorter telomeres and higher mitochondrial metabolic rate. Importantly, Cort-nestlings showed increased mitochondrial proton leak, leading to a decreased ATP production efficiency. Treatment groups did not differ in oxidative damage or antioxidants. Hence, glucocorticoid-induced telomere attrition is associated with changes in mitochondrial metabolism, but not with ROS production. These findings support the hypothesis that shortening of telomere length during stressful periods is mediated by glucocorticoids through metabolic rearrangements.
    Keywords:  Glucocorticoid receptor; Metabolism; Mitochondria; Nr3c1; Oxidative stress; Proton leak; Telomere
  7. iScience. 2021 May 21. 24(5): 102412
      Beginning of metastasis, cancer cells detach from the primary tumor and they can survive even under loss of anchorage; however, the detachment-elicited mechanisms have remained unknown. Here, we found that Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells is dynamically translocated from intracellular vesicles to the plasma membrane when the attached cells are detached and that this mechanism contributes to the survival of the detached (floating) cancer cells. α3NaK was detected in the plasma membrane of floating cancer cells in peritoneal fluids of patients, while it was in the cytoplasm of the cells in primary tumor tissues. On cancer cell detachment, we also found the focal-adhesion-kinase-dependent Ca2+ response that induces the α3NaK translocation via nicotinic acid adenine dinucleotide phosphate pathway. Activation of AMP-activated protein kinase was associated with the translocated α3NaK in the plasma membrane. Collectively, our study identifies a unique mechanism for survival of detached cancer cells, opening up new opportunities for development of cancer medicines.
    Keywords:  Cancer; Cell Biology
  8. J Cell Sci. 2020 Jan 01. pii: jcs.248492. [Epub ahead of print]
      Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard mitochondrial proteome and function despite increased aggregation during proteasome-inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supra-organizations of respiratory complexes (RCs) in proteasome-inhibited cells simultaneous to pelletable aggregation of RC-subunits inside mitochondria. Complex-II (CII) and CV-subunits are increasingly incorporated into oligomers. CI, CIII and CIV-subunits are engaged into supercomplex formation. We unravel unique quinary-states of supercomplexes at early-stress that exhibit plasticity and inequivalence of constituent RCs. Core stoichiometry of CI and CIII is preserved whereas CIV-composition varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC-subunits into holocomplex and supercomplexes re-establish steady-state stoichiometry. Overall, the mechanism of increased supra-organization of RCs mimics the cooperative unfolding and folding pathways for protein-folding, restricted to RCs only and not observed for any other mitochondrial protein complexes.
    Keywords:  Increased supercomplex; Multistep proteome remodelling; Proteostasis; Quinary supercomplex; Respiratory complex biogenesis; Two-dimensional complexome profiling
  9. J Cell Sci. 2020 Jan 01. pii: jcs.239335. [Epub ahead of print]
      Lysosomal exocytosis and resealing of damaged plasma membrane are essential for cellular homeostasis and tumor invasion. However, very little is known of the molecular machinery that regulates these physiological processes. Moreover, no mutations in any of the known regulators of lysosomal exocytosis in primary tumors of patients have been characterized. Here we demonstrate that RNF167-a, a lysosomal associated ubiquitin ligase, negatively regulates lysosomal exocytosis by inducing perinuclear clustering of lysosomes. Importantly, we also characterized a set of novel natural mutations in RNF167-a, which are commonly found in diverse tumor types. We found that RNF167-a-K97N mutant, unlike the wild-type, localizes in the cytoplasm and does not promote perinuclear lysosomal clustering and that cells expressing RNF167-a-K97N exhibit dispersed lysosomes, increased exocytosis, and enhanced plasma membrane repair. Interestingly, these functional features of RNF167-a-K97N were shared with a naturally occurring short version of RNF167, i.e. isoform b. In brief, the results presented here reveal a novel role of RNF167-a as well as its natural variants, RNF167-a-K97N and RNF167-b as an upstream regulator of lysosomal exocytosis and plasma membrane resealing.
    Keywords:  Isoform b; Lysosomal exocytosis; Mutant; Plasma membrane repair; RNF167; Tumor; Ubiquitin ligase
  10. J Cell Sci. 2020 Jan 01. pii: jcs.242859. [Epub ahead of print]
      Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we established an assay to quantitatively evaluate the albumin uptake by CUBAM using cells expressing full-length CUBN, which elucidated the crucial role of C-terminal part of CUBN and endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrated that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. While NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when co-expressed with AMN. NVL2 knockdown significantly impaired the internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins which is involved in endocytic processes.
    Keywords:  Albumin; Amnionless; Cubilin; Endocytosis; Nuclear valosin-containing protein like
  11. J Cell Sci. 2020 Jan 01. pii: jcs.247379. [Epub ahead of print]
      Survivin is a cancer-associated protein that is pivotal for cellular life and death: it is an essential mitotic protein and an inhibitor of apoptosis. In cancer cells, a small pool of survivin localises to the mitochondria, the function of which remains to be elucidated. Here, we report that mitochondrial survivin inhibits the selective form of autophagy, called "mitophagy", causing an accumulation of respiratory defective mitochondria. Mechanistically the data reveal that survivin prevents recruitment of the E3-ubiquitin ligase Parkin to mitochondria and their subsequent recognition by the autophagosome. The data also demonstrate that cells in which mitophagy has been blocked by survivin expression have an increased dependency on glycolysis. As these effects were found exclusively in cancer cells they suggest that the primary act of mitochondrial survivin is to steer cells towards the implementation of the Warburg transition by inhibiting mitochondrial turnover, which enables them to adapt and survive.
    Keywords:  Cancer; Mitochondria; Mitophagy; Respiration; Survivin
  12. J Cell Sci. 2020 Jan 01. pii: jcs.245589. [Epub ahead of print]
      Heat shock response (HSR) is a conserved cellular defensive response against stresses such as temperature, oxidative stress, and heavy metals. A significant group of players in HSR is the set of molecular chaperones, known as heat shock proteins (HSPs) that assist in the refolding of unfolded proteins and prevent the accumulation of damaged proteins. HSP genes are activated by the HSF1 transcription factor-a master regulator of the HSR pathway. A variety of stressors activates HSF1, but the key molecular players and the process that directly contribute to the HSF1 activation remains unclear. In this study, we show that heat shock induces perinuclear clustering of mitochondria in mammalian cells, and this clustering is essential for the activation of HSR. We also show that this perinuclear clustering of mitochondria results in the increased levels of ROS in the nucleus, leading to the activation of hypoxia-inducible factor-1α (HIF-1α). Finally, we provide evidence to suggest that HIF-1α is one of the critical regulators of HSF1 and that HIF-1α is essential for the activation of HSR during a heat shock.
    Keywords:  Chaperones; Hypoxia response; Mitochondrial transport; Oxidative stress; Stress response; Transcriptional regulation
  13. J Cell Sci. 2020 Jan 01. pii: jcs.253401. [Epub ahead of print]
      Cyclophilin A (CyPA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family that has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. CyPA mediates immunosuppressive action of the cyclic undecapeptide cyclosporine A and is also involved in multiple cellular processes such as protein folding, intracellular trafficking, signal transduction, and transcriptional regulation. CyPA is abundantly expressed in cancer cells, and due to its chaperone nature, its expression is induced upon the onset of stress. In this study, it is demonstrated that a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus when cells are stimulated with stressors. CyPA shows antiapoptotic action per se and the capability of forming ternary complexes with cytochrome c and the small acidic cochaperone p23, the latter interaction being independent of the usual association of p23 with the heat-shock protein of 90-kDa, Hsp90. These CyPA•p23 complexes enhance the antiapoptotic response of the cell, suggesting that both proteins form a functional unit whose high level of expression plays a significant role in cell survival.
    Keywords:  Apoptosis; Cyclophilin A; Immunophilin; Mitochondria; Trafficking; p23
  14. Elife. 2021 May 17. pii: e67172. [Epub ahead of print]10
      RAS genes are commonly mutated in human cancer. Despite many possible mutations, individual cancer types often have a 'tropism' towards a specific subset of RAS mutations. As driver mutations, these patterns ostensibly originate from normal cells. High oncogenic RAS activity causes oncogenic stress and different oncogenic mutations can impart different levels of activity, suggesting a relationship between oncoprotein activity and RAS mutation tropism. Here, we show that changing rare codons to common in the murine Kras gene to increase protein expression shifts tumors induced by the carcinogen urethane from arising from canonical Q61 to biochemically less active G12 Kras driver mutations, despite the carcinogen still being biased towards generating Q61 mutations. Conversely, inactivating the tumor suppressor p53 to blunt oncogenic stress partially reversed this effect, restoring Q61 mutations. One interpretation of these findings is that the RAS mutation tropism of urethane arises from selection in normal cells for specific mutations that impart a narrow window of signaling that promotes proliferation without causing oncogenic stress.
    Keywords:  RAS; cancer biology; carcinogenesis; codon bias; mouse; oncogenesis; protooncogenes; tumor initiation
  15. J Cell Sci. 2020 Jan 01. pii: jcs.248336. [Epub ahead of print]
      Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.
    Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17
  16. J Cell Sci. 2020 Jan 01. pii: jcs.250944. [Epub ahead of print]
      Both functional and dysfunctional mitochondria are known to underlie tumor progression. Here, we establish use of the proto-oncogene Drosophila Homeodomain-interacting protein kinase (Hipk) as a new tool to address this paradox. We find that, in Hipk-overexpressing tumor-like cells, mitochondria accumulate and switch from fragmented to highly fused interconnected morphologies. Moreover, elevated Hipk promotes mitochondrial membrane hyperpolarization. These mitochondrial changes are at least in part driven by the upregulation of Myc. Furthermore, we show that the altered mitochondrial energetics, but not morphology, is required for Hipk tumor-like growth as knockdown of pdsw (NDUFB10 in mammals; a Complex I subunit) abrogates the growth. Knockdown of ATPsynβ (a Complex V subunit), which produces higher levels of reactive oxygen species (ROS) than pdsw knockdown, instead synergizes with Hipk to potentiate JNK activation and the downstream induction of Matrix metalloproteinases. Accordingly, ATPsynβ knockdown suppresses Hipk tumor-like growth only when ROS scavengers are co-expressed. Altogether, our work presents an in vivo tumor model featuring the accumulation of hyperfused and hyperpolarized mitochondria, and reveals respiratory Complex subunit-dependent, opposing effects on tumorigenic outcomes.
    Keywords:  Drosophila; Energetics; Hipk; Mitochondria; Myc; ROS
  17. Mol Cancer Ther. 2021 May 17. pii: molcanther.1043.2019. [Epub ahead of print]
      Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDKs) that induce cell cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared to normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6 inhibition (CDK4/6i) results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared to monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro. We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared to treatment with vehicle or individual agents alone in Ptf1acre/+;LSL-KRASG12D/+;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.
  18. J Cell Sci. 2020 Jan 01. pii: jcs.250191. [Epub ahead of print]
      The PKC signaling is a highly conserved signaling module, which plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death via various signaling pathways, including MAPK. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High heat-stress (HHS) induced Pmk1 MAPK activation and Pck2/PKC translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity and the constitutively active Pck2 promotes its translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment into SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in the reduced Pck2 abundance and kinase activity in the soluble fraction. Collectively, MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.
    Keywords:  Heat stress; MAPK signaling; PKC; Phase separation; Spatiotemporal regulation; Stress granules
  19. J Cell Sci. 2020 Jan 01. pii: jcs.240374. [Epub ahead of print]
      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
  20. EMBO J. 2021 May 21. e105990
      Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIβ-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.
    Keywords:  Niemann-Pick Type C; mTORC; membrane contact sites; neurodegeneration; phosphoinositides
  21. Elife. 2021 May 19. pii: e67776. [Epub ahead of print]10
      To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.
    Keywords:  cell biology; developmental biology; mouse
  22. Proc Natl Acad Sci U S A. 2021 May 25. pii: e2016904118. [Epub ahead of print]118(21):
      Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.
    Keywords:  BioID; KRAS; NF1; PDAC; RSK
  23. EMBO J. 2021 May 17. e106777
      The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.
    Keywords:  apoptosis; arginine methylation; pancreatic cancer; post-translational modification; tumor suppression
  24. Curr Opin Cell Biol. 2021 May 18. pii: S0955-0674(21)00048-X. [Epub ahead of print]72 28-35
      Microenvironmental cues in tumors induce in a wide variety of cellular states that subsequently lead to cancer cells with distinct cellular identity, behavior, and fate. Recent literature suggests that the ability to change cellular states, a process defined as cell state plasticity, enable cells to rapidly adapt to their changing environment during tumor progression and metastasis. In this review, we will discuss how recent high-resolution intravital microscopy studies have been instrumental to reveal the real-time dynamics of tumor cell state plasticity during the different steps of the metastatic cascade. In addition, we will highlight the role of tumor plasticity during anticancer treatment response, and how plasticity can be used as a potential druggable target.
  25. Clin Transl Sci. 2021 May 18.
      SMAD4, a tumor suppressor gene, is lost in up to 60%-90% of pancreatic adenocarcinomas (PDAs). Loss of SMAD4 allows tumor progression by upregulating autophagy, a cell survival mechanism that counteracts apoptosis and allows intracellular recycling of macromolecules. Hydroxychloroquine (HCQ) is an autophagy inhibitor. We studied whether HCQ treatment in SMAD4 deficient PDA may prevent therapeutic resistance induced by autophagy upregulation. We retrospectively analyzed the SMAD4 status of patients with PDA enrolled in two prospective clinical trials evaluating pre-operative HCQ. The first dose escalation trial demonstrated the safety of preoperative gemcitabine with HCQ (NCT01128296). More recently, a randomized trial of gemcitabine/nab-paclitaxel +/- HCQ evaluated Evans Grade histopathologic response (NCT01978184). The effect of SMAD4 loss on response to HCQ and chemotherapy was studied for association with clinical outcome. Fisher's exact test and log-rank test were used to assess response and survival. Fifty-two patients receiving HCQ with neoadjuvant chemotherapy were studied. Twenty-five patients had SMAD4 loss (48%). 76% of HCQ-treated patients with SMAD4 loss obtained a histopathologic response greater than or equal to 2A, compared with only 37% with SMAD4 intact (p = 0.006). Although loss of SMAD4 has been associated with worse outcomes, in the current study, loss of SMAD4 was not associated with a detriment in median overall survival in HCQ-treated patients (34.43 months in SMAD4 loss vs. 27.27 months in SMAD4 intact, p = 0.18). The addition of HCQ to neoadjuvant chemotherapy in patients with PDA may improve treatment response in those with SMAD4 loss. Further study of the relationship among SMAD4, autophagy, and treatment outcomes in PDA is warranted.
  26. Elife. 2021 May 20. pii: e61011. [Epub ahead of print]10
      How cells with different genetic makeups compete in tissues is an outstanding question in developmental biology and cancer research. Studies in recent years have revealed that cell competition can either be driven by short-range biochemical signalling or by long-range mechanical stresses in the tissue. To date, cell competition has generally been characterised at the population scale, leaving the single-cell-level mechanisms of competition elusive. Here, we use high time-resolution experimental data to construct a multi-scale agent-based model for epithelial cell competition and use it to gain a conceptual understanding of the cellular factors that governs competition in cell populations within tissues. We find that a key determinant of mechanical competition is the difference in homeostatic density between winners and losers, while differences in growth rates and tissue organisation do not affect competition end result. In contrast, the outcome and kinetics of biochemical competition is strongly influenced by local tissue organisation. Indeed, when loser cells are homogenously mixed with winners at the onset of competition, they are eradicated; however, when they are spatially separated, winner and loser cells coexist for long times. These findings suggest distinct biophysical origins for mechanical and biochemical modes of cell competition.
    Keywords:  MDCK; biophysics; cell biology; cell competition; none; physics of living systems; simulation
  27. Nat Rev Cancer. 2021 May 17.
      The genetic information of human cells is stored in the context of chromatin, which is subjected to DNA methylation and various histone modifications. Such a 'language' of chromatin modification constitutes a fundamental means of gene and (epi)genome regulation, underlying a myriad of cellular and developmental processes. In recent years, mounting evidence has demonstrated that miswriting, misreading or mis-erasing of the modification language embedded in chromatin represents a common, sometimes early and pivotal, event across a wide range of human cancers, contributing to oncogenesis through the induction of epigenetic, transcriptomic and phenotypic alterations. It is increasingly clear that cancer-related metabolic perturbations and oncohistone mutations also directly impact chromatin modification, thereby promoting cancerous transformation. Phase separation-based deregulation of chromatin modulators and chromatin structure is also emerging to be an important underpinning of tumorigenesis. Understanding the various molecular pathways that underscore a misregulated chromatin language in cancer, together with discovery and development of more effective drugs to target these chromatin-related vulnerabilities, will enhance treatment of human malignancies.
  28. Sci Adv. 2021 May;pii: eabd7455. [Epub ahead of print]7(21):
      The PDL1-PD1 immune checkpoint inhibits T cell activation, and its blockade is effective in a subset of patients. Studies are investigating how checkpoints are hijacked by cancer cells and why most patients remain resistant to immunotherapy. Epithelial mesenchymal transition (EMT), which drives tumor cell invasion via the Zeb1 transcription factor, is linked to immunotherapy resistance. In addition, M2-polarized tumor-associated macrophages (TAMs), which inhibit T cell migration and activation, may also cause immunotherapy resistance. How EMT in invading cancer cells is linked to therapy resistance and events driving TAM M2 polarization are therefore important questions. We show that Zeb1 links these two resistance pathways because it is required for PDL1 expression on invading lung cancer cells, and it also induces CD47 on these invading cells, which drives M2 polarization of adjacent TAMs. Resulting reprogramming of the microenvironment around invading cells shields them from the hostile inflammatory environment surrounding tumors.
  29. Aging (Albany NY). 2021 May 13. 13
      The natural aging process is carried out by a progressive loss of homeostasis leading to a functional decline in cells and tissues. The accumulation of these changes stem from a multifactorial process on which both external (environmental and social) and internal (genetic and biological) risk factors contribute to the development of adult chronic diseases, including type 2 diabetes mellitus (T2D). Strategies that can slow cellular aging include changes in diet, lifestyle and drugs that modulate intracellular signaling. Exercise is a promising lifestyle intervention that has shown antiaging effects by extending lifespan and healthspan through decreasing the nine hallmarks of aging and age-associated inflammation. Herein, we review the effects of exercise to attenuate aging from a clinical to a cellular level, listing its effects upon various tissues and systems as well as its capacity to reverse many of the hallmarks of aging. Additionally, we suggest AMPK as a central regulator of the cellular effects of exercise due to its integrative effects in different tissues. These concepts are especially relevant in the setting of T2D, where cellular aging is accelerated and exercise can counteract these effects through the reviewed antiaging mechanisms.
    Keywords:  AMPK; aging; exercise; type 2 diabetes
  30. Nat Rev Gastroenterol Hepatol. 2021 May 17.
      Pancreatic cancer is a leading cause of cancer death worldwide and its global burden has more than doubled over the past 25 years. The highest incidence regions for pancreatic cancer include North America, Europe and Australia, and although much of this increase is due to ageing worldwide populations, there are key modifiable risk factors for pancreatic cancer such as cigarette smoking, obesity, diabetes and alcohol intake. The prevalence of these risk factors is increasing in many global regions, resulting in increasing age-adjusted incidence rates for pancreatic cancer, but the relative contribution from these risk factors varies globally due to variation in the underlying prevalence and prevention strategies. Inherited genetic factors, although not directly modifiable, are an important component of pancreatic cancer risk, and include pathogenic variants in hereditary cancer genes, genes associated with hereditary pancreatitis, as well as common variants identified in genome-wide association studies. Identification of the genetic changes that underlie pancreatic cancer not only provides insight into the aetiology of this cancer but also provides an opportunity to guide early detection strategies. The goal of this Review is to provide an up-to-date overview of the established modifiable and inherited risk factors for pancreatic cancer.
  31. Aging Cell. 2021 May 21. e13357
      Telomere attrition has been proposed as a biomarker and causal factor in aging. In addition to causing cellular senescence and apoptosis, telomere shortening has been found to affect gene expression in subtelomeric regions. Here, we analyzed the distribution of age-related differentially expressed genes from the GTEx RNA sequencing database of 54 tissue types from 979 human subjects and found significantly more upregulated than downregulated genes in subtelomeric regions as compared to the genome-wide average. Our data demonstrate spatial relationships between telomeres and gene expression in aging.
    Keywords:  aging; gene expression; telomere shortening
  32. Clin Exp Metastasis. 2021 May 17.
      There is abundant evidence that the phenotype of cells the tumor at the stromal interface is distinct from the tumor cells that are within the core. Molecular phenotyping of cells at the edge show that they express higher levels of proteins associated with elevated glycolytic metabolism, including GLUT-1, HIF-1, and CA-IX. An end product of glycolysis is the production of acid, and acidosis of tumors is strongly associated with increased metastatic potential across a wide variety of tumor types. The molecular machinery promoting this export of acid is being defined, with close collaboration between carbonic anhydrases, sodium dependent bicarbonate and monocarboxylate transporters. Neutralization of this acidity can prevent local invasion and metastasis, and this has led to the "acid-mediated invasion hypothesis" wherein export of acid from the tumor into the stroma leads to matrix remodeling, which can promote local invasion.
    Keywords:  Acid-mediated invasion; Acidosis; CAIX
  33. J Gastrointest Oncol. 2021 Apr;12(2): 464-473
      Background: Nano-liposomal irinotecan (nal-IRI) plus 5-fluorouracil/leucovorin (5-FU/LV) is the regimen of choice in the 2nd line setting for advanced pancreatic adenocarcinoma (PAC). However, real-world data is limited. Our objectives were to elicit the real-word effectiveness and safety of this combination as an advanced line of therapy in pancreatic cancer patients and analyze the impact of prior lines of therapy on survival outcomes with this regimen.Methods: We conducted a retrospective cohort study of 58 patients with locally advanced unresectable or metastatic PAC, who were treated with at least one dose of nal-IRI + 5-FU/LV following cancer progression on prior therapies between August 2015 and December 2018 at the Kansas University Medical Center (KUMC) and University of Alabama at Birmingham (UAB).
    Results: Median OS was 5.4 (range, 4.2-7) months. Disease control rate (DCR) was highest (84%) for patients given nal-IRI + 5-FU/LV as 2nd line agent after progression on a 1st line gemcitabine-based regimen. However, no significant survival difference was observed between those given nal-IRI + 5-FU/LV after 1st line or beyond the 2nd line (P=0.17). Among those given nal-IRI + 5-FU/LV as 2nd line, use of gemcitabine-inclusive chemotherapy as the 1st line agent did not impact survival (P=0.68). Prior irinotecan exposure and baseline CA 19-9 level did not affect the overall survival (OS) but patients with a higher CA 19-9 level had a significant risk of progression (HR =3.2, P=0.02). Grade 3/4 toxicities were reported in only 19% patients.
    Conclusions: Our report suggests that nal-IRI + 5-FU/LV offers a modest survival benefit with a tolerable safety profile as an advanced line of treatment in patients with advanced PAC.
    Keywords:  2nd line treatment; Liposomal irinotecan; MM-398; nano-liposomal irinotecan (nal-IRI); pancreatic cancer
  34. Nat Commun. 2021 05 17. 12(1): 2862
      Dietary restriction (DR) decreases body weight, improves health, and extends lifespan. DR can be achieved by controlling how much and/or when food is provided, as well as by adjusting nutritional composition. Because these factors are often combined during DR, it is unclear which are necessary for beneficial effects. Several drugs have been utilized that target nutrient-sensing gene pathways, many of which change expression throughout the day, suggesting that the timing of drug administration is critical. Here, we discuss how dietary and pharmacological interventions promote a healthy lifespan by influencing energy intake and circadian rhythms.