bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020–05–17
fifty-one papers selected by
Kıvanç Görgülü, Technical University of Munich



  1. Nat Rev Gastroenterol Hepatol. 2020 May 11.
      Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related mortality in the Western world with limited therapeutic options and dismal long-term survival. The neoplastic epithelium exists within a dense stroma, which is recognized as a critical mediator of disease progression through direct effects on cancer cells and indirect effects on the tumour immune microenvironment. The three dominant entities in the PDAC stroma are extracellular matrix (ECM), vasculature and cancer-associated fibroblasts (CAFs). The ECM can function as a barrier to effective drug delivery to PDAC cancer cells, and a multitude of strategies to target the ECM have been attempted in the past decade. The tumour vasculature is a complex system and, although multiple anti-angiogenesis agents have already failed late-stage clinical trials in PDAC, other vasculature-targeting approaches aimed at vessel normalization and tumour immunosensitization have shown promise in preclinical models. Lastly, PDAC CAFs participate in active cross-talk with cancer cells within the tumour microenvironment. The existence of intratumoural CAF heterogeneity represents a paradigm shift in PDAC CAF biology, with myofibroblastic and inflammatory CAF subtypes that likely make distinct contributions to PDAC progression. In this Review, we discuss our current understanding of the three principal constituents of PDAC stroma, their effect on the prevalent immune landscape and promising therapeutic targets within this compartment.
    DOI:  https://doi.org/10.1038/s41575-020-0300-1
  2. Cancer Prev Res (Phila). 2020 May 14. pii: canprevres.0053.2020. [Epub ahead of print]
      Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated microRNAs (miRNAs) starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanINs) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted pro-tumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas (TCGA) identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.
    DOI:  https://doi.org/10.1158/1940-6207.CAPR-20-0053
  3. Nat Chem Biol. 2020 May 11.
      Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.
    DOI:  https://doi.org/10.1038/s41589-020-0533-x
  4. Cell Metab. 2020 May 05. pii: S1550-4131(20)30197-2. [Epub ahead of print]
      Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes.
    Keywords:  T cells; autophagy; inflammaging; metformin; mitochondria
    DOI:  https://doi.org/10.1016/j.cmet.2020.04.015
  5. Cell Stem Cell. 2020 May 10. pii: S1934-5909(20)30151-X. [Epub ahead of print]
      Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
    Keywords:  CRC; Cancer Stem Cell; biosynthetic capacity; plasticity; stem cell hierarchy
    DOI:  https://doi.org/10.1016/j.stem.2020.04.012
  6. Nat Rev Clin Oncol. 2020 May 12.
      Metastatic pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumours despite the use of multi-agent conventional chemotherapy regimens. Such poor outcomes have fuelled ongoing efforts to exploit the tumour microenvironment (TME) for therapy, but strategies aimed at deconstructing the surrounding desmoplastic stroma and targeting the immunosuppressive pathways have largely failed. In fact, evidence has now shown that the stroma is multi-faceted, which illustrates the complexity of exploring features of the TME as isolated targets. In this Review, we describe ways in which the PDAC microenvironment has been targeted and note the current understanding of the clinical outcomes that have unexpectedly contradicted preclinical observations. We also consider the more sophisticated therapeutic strategies under active investigation - multi-modal treatment approaches and exploitation of biologically integrated targets - which aim to remodel the TME against PDAC.
    DOI:  https://doi.org/10.1038/s41571-020-0363-5
  7. Cell Metab. 2020 May 07. pii: S1550-4131(20)30236-9. [Epub ahead of print]
      Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy. Additionally, disulfiram treatment reversed established diet-induced obesity and metabolic dysfunctions in middle-aged mice. Reductions in feeding efficiency and increases in energy expenditure were associated with body weight regulation in response to long-term disulfiram treatment. Loss of fat tissue and an increase in liver fenestrations were also observed in rats on disulfiram. Given the potent anti-obesogenic effects in rodents, repurposing disulfiram in the clinic could represent a new strategy to treat obesity and its metabolic comorbidities.
    Keywords:  beta-cell hyperplasia; fibrosis; hepatic steatosis; inflammation; insulin resistance; obesity; weight gain
    DOI:  https://doi.org/10.1016/j.cmet.2020.04.019
  8. Nat Metab. 2019 Dec;1(12): 1209-1218
      The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes Parp9, remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam +/- mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.
    DOI:  https://doi.org/10.1038/s42255-019-0150-8
  9. Elife. 2020 May 11. pii: e54095. [Epub ahead of print]9
      Weight loss and anorexia are common symptoms in cancer patients that occur prior to initiation of cancer therapy. Inflammation in the brain is a driver of these symptoms, yet cellular sources of neuroinflammation during malignancy are unknown. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), we observed early and robust myeloid cell infiltration into the brain. Infiltrating immune cells were predominately neutrophils, which accumulated at a unique central nervous system entry portal called the velum interpositum, where they expressed CCR2. Pharmacologic CCR2 blockade and genetic deletion of Ccr2 both resulted in significantly decreased brain-infiltrating myeloid cells as well as attenuated cachexia during PDAC. Lastly, intracerebroventricular blockade of the purinergic receptor P2RX7 during PDAC abolished immune cell recruitment to the brain and attenuated anorexia. Our data demonstrate a novel function for the CCR2/CCL2 axis in recruiting neutrophils to the brain, which drives anorexia and muscle catabolism.
    Keywords:  immunology; inflammation; mouse; neuroscience
    DOI:  https://doi.org/10.7554/eLife.54095
  10. Nat Metab. 2019 Sep 30. 1 966-974
      During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.
    DOI:  https://doi.org/10.1038/s42255-019-0115-y
  11. Proc Natl Acad Sci U S A. 2020 May 15. pii: 201916584. [Epub ahead of print]
      The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.
    Keywords:  FKBP8; membrane contact site; membrane protein topology; mitochondria-associated membrane (MAM); proximity labeling
    DOI:  https://doi.org/10.1073/pnas.1916584117
  12. Nat Commun. 2020 May 11. 11(1): 2332
      Fasting-mimicking diets delay tumor progression and sensitize a wide range of tumors to chemotherapy, but their therapeutic potential in combination with non-cytotoxic compounds is poorly understood. Here we show that vitamin C anticancer activity is limited by the up-regulation of the stress-inducible protein heme-oxygenase-1. The fasting-mimicking diet selectivity reverses vitamin C-induced up-regulation of heme-oxygenase-1 and ferritin in KRAS-mutant cancer cells, consequently increasing reactive iron, oxygen species, and cell death; an effect further potentiated by chemotherapy. In support of a potential role of ferritin in colorectal cancer progression, an analysis of The Cancer Genome Atlas Database indicates that KRAS mutated colorectal cancer patients with low intratumor ferritin mRNA levels display longer 3- and 5-year overall survival. Collectively, our data indicate that the combination of a fasting-mimicking diet and vitamin C represents a promising low toxicity intervention to be tested in randomized clinical trials against colorectal cancer and possibly other KRAS mutated tumors.
    DOI:  https://doi.org/10.1038/s41467-020-16243-3
  13. J Cell Sci. 2020 May 14. pii: jcs.239004. [Epub ahead of print]
      Functional recovery after peripheral nerve damage is dependent on the reprogramming of differentiated Schwann cells (dSC) into repair Schwann cell (rSC), which promotes axonal regeneration and tissue homeostasis. Transition into a repair phenotype requires expression of c-Jun and Sox2, which transcriptionally mediates inhibition of the dSC program of myelination and activate a non-cell autonomous repair program, characterized by the secretion of neuronal survival and regenerative molecules, formation of a cellular scaffold to guide regenerating axons, and activation of an innate immune response. Also, rSC release exosomes that are internalized by peripheral neurons, promoting axonal regeneration. Here, we demonstrate that SC reprogramming is accompanied by a shift in the capacity of their secreted exosomes to promote neurite growth, which is dependent on the rSC expression of c-Jun and Sox2. Furthermore, increased expression of miRNA-21 is responsible for the pro-regenerative capacity of rSC exosomes, which is associated to PTEN downregulation and PI3-kinase activation in neurons. We propose that modification of exosomal cargo constitutes another important feature of the repair program of SC, contributing to axonal regeneration and functional recovery after nerve injury.
    Keywords:  Axonal outgrowth; Axonal regeneration; Exosomes; MiRNA-21; Schwann cell
    DOI:  https://doi.org/10.1242/jcs.239004
  14. Nat Med. 2020 May;26(5): 693-698
      Although elevated plasma interleukin-8 (pIL-8) has been associated with poor outcome to immune checkpoint blockade 1, this has not been comprehensively evaluated in large randomized studies. Here we analyzed circulating pIL-8 and IL8 gene expression in peripheral blood mononuclear cells and tumors of patients treated with atezolizumab (anti-PD-L1 monoclonal antibody) from multiple randomized trials representing 1,445 patients with metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma. High levels of IL-8 in plasma, peripheral blood mononuclear cells and tumors were associated with decreased efficacy of atezolizumab in patients with mUC and metastatic renal cell carcinoma, even in tumors that were classically CD8+ T cell inflamed. Low baseline pIL-8 in patients with mUC was associated with increased response to atezolizumab and chemotherapy. Patients with mUC who experienced on-treatment decreases in pIL-8 exhibited improved overall survival when treated with atezolizumab but not with chemotherapy. Single-cell RNA sequencing of the immune compartment showed that IL8 is primarily expressed in circulating and intratumoral myeloid cells and that high IL8 expression is associated with downregulation of the antigen-presentation machinery. Therapies that can reverse the impacts of IL-8-mediated myeloid inflammation will be essential for improving outcomes of patients treated with immune checkpoint inhibitors.
    DOI:  https://doi.org/10.1038/s41591-020-0860-1
  15. Cancer Res. 2020 May 11. pii: canres.3534.2019. [Epub ahead of print]
      Although fibrotic stroma forms an integral component of pancreatic diseases, whether fibroblasts programmed by different types of pancreatic diseases are phenotypically distinct remains unknown. Here we show that fibroblasts isolated from patients with pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), periampullary tumors (PAT), and adjacent normal (NA) tissue (N=34) have distinct mRNA and miRNA profiles. Compared to NA-fibroblasts, PDAC-associated fibroblasts were generally less sensitive to an anti-fibrotic stimulus (NPPB) and more responsive to positive regulators of activation such as TGFβ1 and WNT. Of the disease-associated fibroblasts examined, PDAC- and CP-derived fibroblasts shared greatest similarity, yet PDAC-associated fibroblasts expressed higher levels of Tenascin C (TNC), a finding attributable to miR-137, a novel regulator of TNC. TNC protein and transcript levels were higher in PDAC tissue versus CP tissue and were associated with greater levels of stromal activation, and conditioned media from TNC-depleted PDAC-associated fibroblasts modestly increased both PDAC cell proliferation and PDAC cell migration, indicating that stromal TNC may have inhibitory effects on PDAC cells. Finally, circulating TNC levels were higher in patients with PDAC compared to CP. Our characterization of pancreatic fibroblast programming as disease-specific has consequences for therapeutic targeting and for the manner in which fibroblasts are used in research.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3534
  16. J Invest Dermatol. 2020 May 07. pii: S0022-202X(20)31406-8. [Epub ahead of print]
      Melanomas frequently harbor activating NRAS mutations leading to activation of MEK-ERK1/2 signaling; however, clinical efficacy of inhibitors to this pathway are limited by resistance. Tumors re-wire metabolic pathways in response to stress signals such as targeted inhibitors and drug resistance, but most therapeutic resistant pre-clinical models are generated in conditions that lack physiological metabolism. We generated human NRAS mutant melanoma xenografts that were resistant to the MEK inhibitor (MEKi), PD0325901, in vivo. MEKi-resistant (MEKiR) cells showed cross-resistance to the structurally distinct MEKi, trametinib, and elevated ERK1/2 phosphorylation and downstream signaling. Additionally, we observed upregulation of the serine synthesis pathway and phosphoglycerate dehydrogenase (PHGDH), a key enzyme in this pathway. Suppressing PHGDH in MEKiR cells together with MEKi treatment decreased oxidative stress tolerance and cell proliferation. Together, our data suggest targeting PHGDH as a potential strategy in overcoming MEKi resistance.
    DOI:  https://doi.org/10.1016/j.jid.2020.02.047
  17. Nat Commun. 2020 May 11. 11(1): 2333
      Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTPM/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP+/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTPM/LOH cells show an increased in vivo tumor-initiating ability compared with AKTPNull and AKTP+/M cells. RNAseq analyses reveal that inflammatory and growth factor/MAPK pathways are specifically activated in AKTPM/LOH cells, while the stem cell signature is upregulated in both AKTPM/LOH and AKTPNull cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination.
    DOI:  https://doi.org/10.1038/s41467-020-16245-1
  18. Nat Commun. 2020 May 12. 11(1): 2362
      Due to their bacterial ancestry, many components of mitochondria share structural similarities with bacteria. Release of molecular danger signals from injured cell mitochondria (mitochondria-derived damage-associated molecular patterns, mito-DAMPs) triggers a potent inflammatory response, but their role in fibrosis is unknown. Using liver fibrosis resistant/susceptible mouse strain system, we demonstrate that mito-DAMPs released from injured hepatocyte mitochondria (with mtDNA as major active component) directly activate hepatic stellate cells, the fibrogenic cell in the liver, and drive liver scarring. The release of mito-DAMPs is controlled by efferocytosis of dying hepatocytes by phagocytic resident liver macrophages and infiltrating Gr-1(+) myeloid cells. Circulating mito-DAMPs are markedly increased in human patients with non-alcoholic steatohepatitis (NASH) and significant liver fibrosis. Our study identifies specific pathway driving liver fibrosis, with important diagnostic and therapeutic implications. Targeting mito-DAMP release from hepatocytes and/or modulating the phagocytic function of macrophages represents a promising antifibrotic strategy.
    DOI:  https://doi.org/10.1038/s41467-020-16092-0
  19. Cell Death Dis. 2020 May 15. 11(5): 375
      Age-dependent decrease of mitochondrial energy production and cellular redox imbalance play significant roles in age-related hearing loss (ARHL). Lactate dehydrogenase B (LDHB) is a key glycolytic enzyme that catalyzes the interconversion of pyruvate and lactate. LDH activity and isoenzyme patterns are known to be changed with aging, but the role of LDHB in ARHL has not been studied yet. Here, we found that LDHB knockout mice showed hearing loss at high frequencies, which is the typical feature of ARHL. LDHB knockdown caused downregulation of mitochondrial functions in auditory cell line, University of Bristol/organ of Corti 1 (UB/OC1) with decreased NAD+ and increased hypoxia inducing factor-1α. LDHB knockdown also enhanced the death of UB/OC1 cells with ototoxic gentamicin treatment. On the contrary, the induction of LDHB expression caused enhanced mitochondrial functions, including changes in mitochondrial respiratory subunits, mitochondrial membrane potentials, ATP, and the NAD+/NADH ratio. Thus, we concluded that suppression of LDHB activity may be closely related with the early onset or progression of ARHL.
    DOI:  https://doi.org/10.1038/s41419-020-2577-y
  20. Sci Immunol. 2020 May 15. pii: eaaz6894. [Epub ahead of print]5(47):
      During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.
    DOI:  https://doi.org/10.1126/sciimmunol.aaz6894
  21. Elife. 2020 May 12. pii: e55008. [Epub ahead of print]9
      The 'building-block' model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.
    Keywords:  cell biology; human; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.55008
  22. J Cell Biol. 2020 Jun 01. pii: e201906153. [Epub ahead of print]219(6):
      Desmosomes are cell-cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are "locked in" to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.
    DOI:  https://doi.org/10.1083/jcb.201906153
  23. EMBO Mol Med. 2020 May 11. e11571
      Small molecular PD-1 inhibitors are lacking in current immuno-oncology clinic. PD-1/PD-L1 antibody inhibitors currently approved for clinical usage block interaction between PD-L1 and PD-1 to enhance cytotoxicity of CD8+ cytotoxic T lymphocyte (CTL). Whether other steps along the PD-1 signaling pathway can be targeted remains to be determined. Here, we report that methylene blue (MB), an FDA-approved chemical for treating methemoglobinemia, potently inhibits PD-1 signaling. MB enhances the cytotoxicity, activation, cell proliferation, and cytokine-secreting activity of CTL inhibited by PD-1. Mechanistically, MB blocks interaction between Y248-phosphorylated immunoreceptor tyrosine-based switch motif (ITSM) of human PD-1 and SHP2. MB enables activated CTL to shrink PD-L1 expressing tumor allografts and autochthonous lung cancers in a transgenic mouse model. MB also effectively counteracts the PD-1 signaling on human T cells isolated from peripheral blood of healthy donors. Thus, we identify an FDA-approved chemical capable of potently inhibiting the function of PD-1. Equally important, our work sheds light on a novel strategy to develop inhibitors targeting PD-1 signaling axis.
    Keywords:  PD-1; immunotherapy; methylene blue; small molecular inhibitor; transgenic mouse model
    DOI:  https://doi.org/10.15252/emmm.201911571
  24. Trends Biochem Sci. 2020 Jun;pii: S0968-0004(20)30059-1. [Epub ahead of print]45(6): 545-546
      
    DOI:  https://doi.org/10.1016/j.tibs.2020.02.011
  25. Mol Syst Biol. 2020 May;16(5): e9156
      Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver-identity (LIVER-ID) transcription factor (TF) network, initiated by rapid LIVER-ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER-ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co-recruitment of LIVER-ID TFs and decommissioning of BRD4 super-enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER-ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients.
    Keywords:  NFIL3; PAR-bZIP; liver injury; sepsis; super-enhancer
    DOI:  https://doi.org/10.15252/msb.20199156
  26. F1000Res. 2019 ;8 1775
      Single-cell sequencing reveals cellular heterogeneity but not cell localization. However, by combining single-cell transcriptomic data with a reference atlas of a small set of genes, it would be possible to predict the position of individual cells and reconstruct the spatial expression profile of thousands of genes reported in the single-cell study. To develop new algorithms for this purpose, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) consortium organized a crowd-sourced competition known as DREAM Single Cell Transcriptomics Challenge (SCTC). In the spirit of this framework, we describe here the proposed procedures for adequate reference genes selection, and an iterative procedure to predict spatial expression profile of other genes.
    Keywords:  DREAM Challenge; Drosophila Embryo; Gene expression Patterns; Single-Cell RNA sequencing
    DOI:  https://doi.org/10.12688/f1000research.20715.1
  27. J Biol Chem. 2020 May 11. pii: jbc.AW120.011105. [Epub ahead of print]
      Excess fatty acid accumulation in non-adipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid-induced cell death and remodels metabolism in vivo opens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology.
    Keywords:  RNA; diabetes; diabetes complications; fatty acid; genetic screens; lipotoxicity; non-coding RNAs; snoRNAs; triacylglycerol
    DOI:  https://doi.org/10.1074/jbc.AW120.011105
  28. Methods Mol Biol. 2020 ;2144 187-200
      The cellular recycling process of autophagy is essential for survival, development, and homeostasis. Autophagy also plays an important role in aging and has been linked to longevity in many species, including the nematode C. elegans. Study of the physiological roles of autophagy during C. elegans aging requires methods for the spatiotemporal analysis of autophagy. Here we describe a method for assessing autophagic flux in multiple tissues of C. elegans by quantifying the pool of autophagic vesicles using fluorescently labelled Atg8/LGG-1 reporters upon autophagy inhibition using bafilomycin A1 (BafA). This methodology has revealed that autophagic activity varies in different cell types of C. elegans during aging.
    Keywords:  Aging; Autophagic flux; Bafilomycin A; C. elegans
    DOI:  https://doi.org/10.1007/978-1-0716-0592-9_17
  29. Cell Rep. 2020 May 12. pii: S2211-1247(20)30585-4. [Epub ahead of print]31(6): 107632
      Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. Here, we show that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. We discover a feedback mechanism involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Our findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer.
    Keywords:  DDR; DNA damage response; Drosophila intestinal stem cell; ISC; MEF; O-GlcNAc transferase; O-GlcNAcase; O-GlcNAcylation; OGA; OGT; mESC; mouse embryonic fibroblast; mouse embryonic stem cell
    DOI:  https://doi.org/10.1016/j.celrep.2020.107632
  30. EMBO Mol Med. 2020 May 13. e11217
      Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors.
    Keywords:   BCRA ; OXPHOS ; PARP inhibitors; cancer metabolism; metformin
    DOI:  https://doi.org/10.15252/emmm.201911217
  31. Nat Commun. 2020 May 15. 11(1): 2416
      Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxia-repressed miR-142-3p, which targets HIF1A, LOX and ITGA5, causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients.
    DOI:  https://doi.org/10.1038/s41467-020-16199-4
  32. Trends Biochem Sci. 2020 May 06. pii: S0968-0004(20)30092-X. [Epub ahead of print]
      Many chromatin-modifying enzymes require metabolic cofactors to support their catalytic activities, providing a direct path for fluctuations in metabolite availability to regulate the epigenome. Over the past decade, our knowledge of this link has grown significantly. What began with studies showing that cofactor availability drives global abundances of chromatin modifications has transitioned to discoveries highlighting metabolic enzymes as loci-specific regulators of gene expression. Here, we cover our current understanding of mechanisms that facilitate the dynamic and complex relationship between metabolism and the epigenome, focusing on the roles of essential metabolic and chromatin associated enzymes. We discuss physiological conditions where availability of these epimetabolites is dynamically altered, highlighting known links to the epigenome and proposing other plausible connections.
    Keywords:  acetylation; acylation; chromatin; circadian cycles; dietary challenges; methylation
    DOI:  https://doi.org/10.1016/j.tibs.2020.04.002
  33. Cell. 2020 May 05. pii: S0092-8674(20)30481-5. [Epub ahead of print]
      Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.
    Keywords:  activation domain; condensate; intrinscially disordered region; phase separation; repeat expansion; synpolydactyly; transcription factor; transcriptional condensate
    DOI:  https://doi.org/10.1016/j.cell.2020.04.018
  34. Aging (Albany NY). 2020 05 10. 12
      Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.
    Keywords:  BMAL1; P53; colorectal cancer1; mTOR; senescence
    DOI:  https://doi.org/10.18632/aging.103124
  35. Science. 2020 May 15. 368(6492): 731-736
      The establishment of reproductive barriers between populations can fuel the evolution of new species. A genetic framework for this process posits that "incompatible" interactions between genes can evolve that result in reduced survival or reproduction in hybrids. However, progress has been slow in identifying individual genes that underlie hybrid incompatibilities. We used a combination of approaches to map the genes that drive the development of an incompatibility that causes melanoma in swordtail fish hybrids. One of the genes involved in this incompatibility also causes melanoma in hybrids between distantly related species. Moreover, this melanoma reduces survival in the wild, likely because of progressive degradation of the fin. This work identifies genes underlying a vertebrate hybrid incompatibility and provides a glimpse into the action of these genes in natural hybrid populations.
    DOI:  https://doi.org/10.1126/science.aba5216
  36. Elife. 2020 May 11. pii: e56090. [Epub ahead of print]9
      Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway.
    Keywords:  D. melanogaster; S. cerevisiae; biochemistry; cell biology; chemical biology
    DOI:  https://doi.org/10.7554/eLife.56090
  37. Mol Metab. 2020 May 07. pii: S2212-8778(20)30082-X. [Epub ahead of print] 101008
       OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) conveys information from ingested nutrients to peripheral tissues, signaling energy availability. The GIP Receptor (GIPR) is also expressed in the bone marrow, notably in cells of the myeloid lineage. However the importance of gain and loss of GIPR signaling for diverse hematopoietic responses remains unclear.
    METHODS: We assessed the expression of the Gipr in BM lineages and examined functional roles for the GIPR in control of hematopoiesis. Bone marrow responses were studied in (i) mice fed regular or energy-rich diets, (ii) mice treated with hematopoietic stressors including acute 5-fluorouracil (5-FU), Lipopolysaccharide (LPS), and Pam3CysSerLys4 (Pam3CSK4)), with or without pharmacological administration of a GIPR agonist and (iii) mice with global (Gipr-/-) or selective deletion of the GIPR (GiprTie2-/-) with and without bone marrow transplantation (BMT).
    RESULTS: Gipr is expressed within T cells, myeloid cells and myeloid precursors, however these cell populations were not different in peripheral blood, spleen or BM of Gipr-/- and GiprTie2-/- mice. Nevertheless, gain and loss of function studies revealed that GIPR signaling controls expression of BM Toll-like receptor (TLR) and Notch-related genes regulating hematopoiesis. Loss of the BM GIPR attenuates the extent of adipose tissue inflammation, and dysregulates the hematopoietic response to BMT. GIPR agonism modified BM gene expression profiles following 5-FU and PAM3CSK4 whereas loss of the Gipr altered the hematopoietic responses to energy excess, two TLR ligands and 5-FU. However, the magnitude of the cellular changes in hematopoiesis in response to gain or loss of GIPR signaling was relatively modest.
    CONCLUSION: These studies identify a functional gut hormone-BM axis positioned for transduction of signals linking nutrient availability to the control of TLR and Notch genes regulating hematopoiesis. Nevertheless, stimulation or loss of GIPR signaling has minimal impact on basal hematopoiesis or the physiological response to hematopoietic stress.
    Keywords:  Glucose-dependant insulinotropic polypeptide receptor; bone marrow; hematopoiesis; inflammation; myeloid cells
    DOI:  https://doi.org/10.1016/j.molmet.2020.101008
  38. J Cell Sci. 2020 May 14. 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
    DOI:  https://doi.org/10.1242/jcs.239335
  39. J Cell Mol Med. 2020 May 11.
      Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate-specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow-up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.
    Keywords:  PSMA; brain metastasis; glioblastoma; proliferating microvasculature
    DOI:  https://doi.org/10.1111/jcmm.15350
  40. Sci Signal. 2020 May 12. pii: eaaz1965. [Epub ahead of print]13(631):
      Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.
    DOI:  https://doi.org/10.1126/scisignal.aaz1965
  41. J Invest Dermatol. 2020 May 11. pii: S0022-202X(20)31454-8. [Epub ahead of print]
      Fibroblasts produce collagens and other proteins that form the bulk of the extracellular matrix (ECM) in connective tissues. Emerging data point to functional heterogeneity of fibroblasts. However, the lack of sub-type specific markers hinders our understanding of the different roles of fibroblasts in ECM biology, wound healing, diseases and aging. We have investigated the utility of the cell surface protein CD26 (dipeptidyl peptidase-4) to identify functionally distinct fibroblast subpopulations in human skin. Using flow cytometry and immunohistology, we find that CD26, in combination with the cell surface glycoprotein CD90, identifies a distinct subpopulation of cells which express relatively high levels of type I collagen (COL1A1), a hallmark of fibroblasts. Importantly, the population of CD26+ fibroblasts is selectively increased following wounding of human skin. These cells account for the majority of COL1A1 expression during the ECM remodeling phase of healing. The proportion of CD26+ fibroblasts in the skin of young and aged individuals is similar, indicating that loss of collagen production during aging does not involve selective reduction of CD26+ fibroblasts. In culture, the majority of freshly isolated CD26- fibroblasts gain expression of CD26+. Taken together, these data provide a foundation for targeting CD26+ fibroblasts to modulate wound healing in human skin.
    DOI:  https://doi.org/10.1016/j.jid.2020.04.010
  42. Cancer Discov. 2020 May 13. pii: CD-20-0657. [Epub ahead of print]
      The coronavirus SARS-CoV-2 has created a global pandemic that has killed more than a quarter million people since December 2019, halted commerce, and disrupted our ability to research cancer in the laboratory and clinic and care for our patients. A return toward a functioning society can be facilitated by the active participation of cancer researchers to diagnose and treat SARS-CoV-2 infected patients, and the direct and indirect benefits of our involvement cannot be overstated.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0657
  43. Circulation. 2020 May 12.
      Background: Metabolic disorders such as obesity and diabetes can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than VEGF-VEGFR-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. Methods: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-KO) and their wild-type littermates (WT) to a fast-food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We employed crosslinking immunoprecipitation (iCLIP)- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy and obese/Type 2 diabetic donors. Results: We identified an anti-obesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an anti-angiogenic and pro-inflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from obese and/or type 2 diabetic donors, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. Conclusions: Our study suggests a novel mechanism by which ECs, through AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.
    Keywords:  Argonaute; Thrombspondin; browning; diabetes; metabolic syndrome
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.119.041231
  44. Nat Genet. 2020 May 11.
      The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.
    DOI:  https://doi.org/10.1038/s41588-020-0618-1
  45. Autophagy. 2020 May 13. 1-17
      Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.
    ABBREVIATIONS: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.
    Keywords:  Brown adipocyte; inflammasome; pink1; transcriptional activation; white adipocyte
    DOI:  https://doi.org/10.1080/15548627.2020.1753002
  46. J Biol Chem. 2020 May 14. pii: jbc.RA120.013223. [Epub ahead of print]
      The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.
    Keywords:  Akt PKB; CRISPR/Cas; MK2206; amino acid homeostasis; kinase signaling; lysosomal acidification; nutient sensing; pH regulation; proton transport; vacuolar ATPase
    DOI:  https://doi.org/10.1074/jbc.RA120.013223
  47. Mol Cell. 2020 May 05. pii: S1097-2765(20)30266-5. [Epub ahead of print]
      Precise control of the RNA polymerase II (RNA Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Despite previous work to understand individual transcription steps, we reveal a mechanism that integrates RNA Pol II cycle transitions. Surprisingly, KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters, guaranteeing continuous progression of RNA Pol II entry to and exit from the pause state. Upon chromatin docking, KAP1 first associates with RNA Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands, enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by tumor cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.
    Keywords:  CDK9; KAP1; RNA polymerase II; SMAD; TGF-β; TRIM28; cancer; chromatin reader; epigenetics; pausing
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.024
  48. Trends Biochem Sci. 2020 May 11. pii: S0968-0004(20)30091-8. [Epub ahead of print]
      While targeting of proteins synthesized in the cytosol to any organelle is complex, mitochondria present the most challenging of destinations. First, import of nuclear-encoded proteins needs to be balanced with production of mitochondrial-encoded ones. Moreover, as mitochondria are divided into distinct subdomains, their proteins harbor a number of different targeting signals and biophysical properties. While translocation into the mitochondrial membranes has been well studied, the cytosolic steps of protein import remain poorly understood. Here, we review current knowledge on mRNA and protein targeting to mitochondria, as well as recent advances in our understanding of the cellular programs that respond to accumulation of mitochondrial precursor proteins in the cytosol, thus linking defects in targeting-capacity to signaling.
    Keywords:  RNA-binding proteins; chaperones; mitochondrial precursor; mitochondrial protein import; mitochondrial targeting sequence; nascent-chain associated complex
    DOI:  https://doi.org/10.1016/j.tibs.2020.04.001
  49. Elife. 2020 May 11. pii: e56898. [Epub ahead of print]9
      How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor, Hunchback acts in NB7-1 neuronal stem cells to control how many NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng et al., 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Here, we perform additional temporal transcription factor manipulations-prolonging expression of Hunchback in NB3-1, as well as precociously expressing Pdm and Castor in NB7-1. We use confocal microscopy, calcium imaging, and electrophysiology to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. Our data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.
    Keywords:  D. melanogaster; developmental biology; neuroscience
    DOI:  https://doi.org/10.7554/eLife.56898
  50. Oncogene. 2020 May 11.
      Kaposi's sarcoma (KS) caused by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly angiogenic and invasive vascular tumor and the most common AIDS-associated cancer. KSHV-encoded viral interleukin-6 (vIL-6) is implicated in the development of KSHV-induced malignancies; however, the mechanisms underlying vIL-6-induced angiogenesis and tumorigenesis remain undefined. Here, we show that vIL-6 promotes angiogenesis, cell proliferation, and invasion by downregulating caveolin 1 (CAV1) that plays a pivotal and versatile role in multiple cancer-associated processes. Mechanistically, vIL-6 signaling led to the phosphorylation and acetylation of STAT3 that targeted DNA methyltransferase 1 (DNMT1) in a sequential manner. Specifically, the vIL-6-induced phosphorylated form of STAT3 transcriptionally activated DNMT1 expression. Furthermore, vIL-6-induced acetylated form of STAT3 interacted with DNMT1 to form a transcription factor complex that bound to and methylated the CAV1 promoter, leading to CAV1 expression silencing. In fact, downregulation of CAV1 expression resulted in the activation of AKT signaling, promoting cell invasion, and growth transformation induced by KSHV. Finally, genetic deletion of vIL-6 from the KSHV genome abolished KSHV-induced cellular transformation and impaired angiogenesis. Our results reveal that vIL-6 epigenetically silences CAV1 expression to promote angiogenesis and tumorigenesis by regulating the formation of STAT3-DNMT1 complex. These novel findings define a mechanism by which KSHV inhibits the CAV1 pathway and establish the scientific basis for targeting this pathway to treat KSHV-associated cancers.
    DOI:  https://doi.org/10.1038/s41388-020-1317-1