bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2022‒07‒03
33 papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Gastroenterology. 2022 Jun 28. pii: S0016-5085(22)00682-5. [Epub ahead of print]
      BACKGROUND & AIMS: Rapid deconditioning, also called cachexia, and metabolic reprogramming are two hallmarks of pancreatic cancer. ACSS2 is an acetyl-coA synthetase that contributes to lipid synthesis and epigenetic reprogramming. However, the role of ACSS2 on the non-selective macropinocytosis and cancer cachexia in pancreatic cancer remains elusive. In this study, we demonstrate that ACSS2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer.METHODS: Clinical significance of ACSS2 was analyzed using human pancreatic cancer patient samples. ACSS2 knockout cells were established utilizing CRISPR-Cas9 system. Single-cell RNA sequencing data from genetically engineered mouse models was analyzed. Macropinocytotic index was evaluated by dextran uptake assay. ChIP assay was performed to validate transcriptional activation. ACSS2 mediated tumor progression and muscle wasting were examined in orthotopic xenograft models.
    RESULTS: Metabolic stress induced ACSS2 expression, which is associated with worse prognosis in pancreatic cancer. ACSS2 knockout significantly suppressed cell proliferation in 2D and 3D models. Macropinocytosis associated genes are upregulated in tumor tissues and are correlated to worse prognosis. ACSS2 knockout inhibited macropinocytosis. We identified ZIP4 as a downstream target of ACSS2, and knockdown of ZIP4 reversed ACSS2 induced macropinocytosis. ACSS2 upregulated ZIP4 through ETV4 mediated transcriptional activation. ZIP4 induces macropinocytosis through CREB activated SDC1 and DNM2. Meanwhile, ZIP4 drives muscle wasting and cachexia via GSK3β mediated secretion of TRAIL. ACSS2 knockout attenuated muscle wasting and extended survival in orthotopic mouse models.
    CONCLUSIONS: ACSS2-mediated metabolic reprogramming activates ZIP4 pathway, and promotes macropinocytosis via SDC1/DNM2 and drives muscle wasting through GSK3β/TRAIL axis, which potentially provides additional nutrients for macropinocytosis in pancreatic cancer.
    Keywords:  Cachexia; Macropinocytosis; Metabolic Stress; Muscle Wasting
  2. Mol Cell. 2022 Jun 18. pii: S1097-2765(22)00541-X. [Epub ahead of print]
      Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.
    Keywords:  PKM2; cancer metabolism; glucose oxidation; metastasis; methionine oxidation; pancreatic cancer; redox signaling
  3. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]
      Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
    Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy
  4. Nature. 2022 Jun 29.
      Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.
  5. Cancer Discov. 2022 Jun 30. pii: candisc.0043.2022-1-10 21:36:20.420. [Epub ahead of print]
      Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC we now demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in PDAC patients. Together, our data reveal that maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC.
  6. Nature. 2022 Jun 29.
      Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
  7. Nat Commun. 2022 Jun 25. 13(1): 3652
      Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous "control" lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.
  8. Science. 2022 Jul;377(6601): 47-56
      The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.
  9. Cancer Discov. 2022 Jun 30. pii: cd.22.0044. [Epub ahead of print]
      The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacological inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor, Trametinib (MEKi), we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition.
  10. Nat Metab. 2022 Jun;4(6): 693-710
      Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.
  11. Mol Cell. 2022 Jun 24. pii: S1097-2765(22)00544-5. [Epub ahead of print]
      Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.
    Keywords:  SLC4A7/NBCn1; bicarbonate metabolism; mTOR signaling; purine metabolism; pyrimidine metabolism
  12. J Clin Invest. 2022 Jul 01. pii: e158447. [Epub ahead of print]132(13):
      Mitochondrial dysfunction and cell senescence are hallmarks of aging and are closely interconnected. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity per mitochondrion together with a decreased mitochondrial membrane potential, typically accompanied by increased production of oxygen free radicals, is a cause and a consequence of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. Here, we summarize pathways that cause mitochondrial dysfunction in senescence and aging and discuss the major consequences of mitochondrial dysfunction and how these consequences contribute to senescence and aging. We also highlight the potential of senescence-associated mitochondrial dysfunction as an antiaging and antisenescence intervention target, proposing the combination of multiple interventions converging onto mitochondrial dysfunction as novel, potent senolytics.
  13. Nature. 2022 Jun 29.
      Ageing is intimately connected to the induction of cell senescence1,2, but why this is so remains poorly understood. A key challenge is the identification of pathways that normally suppress senescence, are lost during ageing and are functionally relevant to oppose ageing3. Here we connected the structural and functional decline of ageing tissues to attenuated function of the master effectors of cellular mechanosignalling YAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation of YAP/TAZ in these cells leads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation of YAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent that inhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signalling relies on the unexpected role of YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which is involved in building the peri-nuclear actin cap. The findings demonstrate that declining YAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STING signalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limiting senescence-associated inflammation and improving healthy ageing.
  14. Nat Cancer. 2022 Jun 30.
      Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.
  15. J Cell Sci. 2022 Jul 01. pii: jcs.259090. [Epub ahead of print]
      Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the therapeutic strategies targeting this vulnerability are still inefficient and have unacceptable side effects in clinical trials. Therefore, developing biomarkers to predict therapeutic efficacy would be essential to improve the selective targeting of cancer cells. Here, we found that the cell lines sensitive to glucose deprivation have high expression of cystine/glutamate antiporter xCT. We found that cystine uptake and glutamate export through xCT contributed to rapid NADPH depletion under glucose deprivation. This collapse of the redox system oxidized and inactivated AMPK, a major regulator of metabolic adaptation, resulting in a metabolic catastrophe and cell death. While this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. Taken together, these findings suggest a novel cross-talk between AMPK and xCT for the metabolism and signal transduction and reveal a metabolic vulnerability in xCT-high expressing cancer cells to glucose deprivation.
    Keywords:  AMPK; Cystine; Glucose starvation; NADPH; SLC7A11; xCT
  16. J Clin Invest. 2022 Jul 01. pii: e158452. [Epub ahead of print]132(13):
      Recent improvements in cancer treatment have increased the lifespan of pediatric and adult cancer survivors. However, cancer treatments accelerate aging in survivors, which manifests clinically as the premature onset of chronic diseases, such as endocrinopathies, osteoporosis, cardiac dysfunction, subsequent cancers, and geriatric syndromes of frailty, among others. Therefore, cancer treatment-induced early aging accounts for significant morbidity, mortality, and health expenditures among cancer survivors. One major mechanism driving this accelerated aging is cellular senescence; cancer treatments induce cellular senescence in tumor cells and in normal, nontumor tissue, thereby helping mediate the onset of several chronic diseases. Studies on clinical monitoring and therapeutic targeting of cellular senescence have made considerable progress in recent years. Large-scale clinical trials are currently evaluating senotherapeutic drugs, which inhibit or eliminate senescent cells to ameliorate cancer treatment-related aging. In this article, we survey the recent literature on phenotypes and mechanisms of aging in cancer survivors and provide an up-to-date review of the major preclinical and translational evidence on cellular senescence as a mechanism of accelerated aging in cancer survivors, as well as insight into the potential of senotherapeutic drugs. However, only with time will the clinical effect of senotherapies on cancer survivors be visible.
  17. Aging Cell. 2022 Jul 02. e13664
      Although chronological age correlates with various age-related diseases and conditions, it does not adequately reflect an individual's functional capacity, well-being, or mortality risk. In contrast, biological age provides information about overall health and indicates how rapidly or slowly a person is aging. Estimates of biological age are thought to be provided by aging clocks, which are computational models (e.g., elastic net) that use a set of inputs (e.g., DNA methylation sites) to make a prediction. In the past decade, aging clock studies have shown that several age-related diseases, social variables, and mental health conditions associate with an increase in predicted biological age relative to chronological age. This phenomenon of age acceleration is linked to a higher risk of premature mortality. More recent research has demonstrated that predicted biological age is sensitive to specific interventions. Human trials have reported that caloric restriction, a plant-based diet, lifestyle changes involving exercise, a drug regime including metformin, and vitamin D3 supplementation are all capable of slowing down or reversing an aging clock. Non-interventional studies have connected high-quality sleep, physical activity, a healthy diet, and other factors to age deceleration. Specific molecules have been associated with the reduction or reversal of predicted biological age, such as the antihypertensive drug doxazosin or the metabolite alpha-ketoglutarate. Although rigorous clinical trials are needed to validate these initial findings, existing data suggest that aging clocks are malleable in humans. Additional research is warranted to better understand these computational models and the clinical significance of lowering or reversing their outputs.
    Keywords:  aging clock; biological age; epigenetic age; healthspan; lifespan; longevity; machine learning; mortality
  18. Pancreatology. 2022 May 31. pii: S1424-3903(22)00170-3. [Epub ahead of print]
      BACKGROUND: First-degree relatives (FDRs) of patients with pancreatic ductal adenocarcinoma (PDAC) have elevated PDAC risk, partially due to germline genetic variants. We evaluated the potential effectiveness of genetic testing to target MRI-based screening among FDRs.METHODS: We used a microsimulation model of PDAC, calibrated to Surveillance, Epidemiology, and End Results (SEER) data, to estimate the potential life expectancy (LE) gain of screening for each of the following groups of FDRs: individuals who test positive for each of eight variants associated with elevated PDAC risk (e.g., BRCA2, CDKN2A); individuals who test negative; and individuals who do not test. Screening was assumed to take place if LE gains were achievable. We simulated multiple screening approaches, defined by starting age and frequency. Sensitivity analysis evaluated changes in results given varying model assumptions.
    RESULTS: For women, 92% of mutation carriers had projected LE gains from screening for PDAC, if screening strategies (start age, frequency) were optimized. Among carriers, LE gains ranged from 0.1 days (ATM+ women screened once at age 70) to 510 days (STK11+ women screened annually from age 40). For men, LE gains were projected for all mutation carriers, ranging from 0.2 days (BRCA1+ men screened once at age 70) to 620 days (STK11+ men screened annually from age 40). For men and women who did not undergo genetic testing, or for whom testing showed no variant, screening yielded small LE benefit (0-2.1 days).
    CONCLUSIONS: Genetic testing of FDRs can inform targeted PDAC screening by identifying which FDRs may benefit.
    Keywords:  Cancer screening; Genetic testing; Pancreatic cancer; Simulation modeling
  19. Methods Mol Biol. 2022 ;2497 1-10
      Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.
    Keywords:  Electron transport chain; Mitochondria; Mitochondrial membrane potential; Mitochondrial metabolism; NADH; Oxygen consumption; Respiratory complex; TMRM; Warburg Metabolism
  20. Nat Metab. 2022 Jun;4(6): 651-662
      Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.
  21. Physiol Rev. 2022 Jun 30.
      The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.
    Keywords:  Lymphatic system; cancer lymphangiogenesis; lymph node; lymphatic endothelial cells; lymphatic metastasis
  22. Methods Mol Biol. 2022 ;2497 11-61
      The mitochondrial membrane potential (ΔψM) is the major component of the bioenergetic driving force responsible for most cellular ATP produced, and it controls a host of biological processes. In intact cells, assay readouts with commonly used fluorescence ΔψM probes are distorted by factors other than ΔψM. Here, we describe a protocol to calculate both ΔψM and plasma membrane potential (ΔψP) in absolute millivolts in intact single cells, or in populations of adherent, cultured cells. Our approach generates unbiased data that allows comparison of ΔψM between cell types with different geometry and ΔψP, and to follow ΔψM in time when ΔψP fluctuates. The experimental paradigm results in fluorescence microscopy time courses using a pair of cationic and anionic probes with internal calibration points that are subsequently computationally converted to millivolts on an absolute scale. The assay is compatible with wide field, confocal or two-photon microscopy. The method given here is optimized for a multiplexed, partial 96-well microplate format to record ΔψP and ΔψM responses for three consecutive treatment additions.
    Keywords:  Bis-oxonol; Cell culture; Cellular heterogeneity; Fluorescence microscopy; Live cell microscopy; MitoTracker; Mitochondrial biogenesis; Mitochondrial membrane potential; Plasma membrane potential; Single cell; TMRM; Tetramethylrhodamine methyl ester
  23. Nat Commun. 2022 Jun 29. 13(1): 3749
      Insulin sensitivity progressively declines with age. Currently, the mechanism underlying age-associated insulin resistance remains unknown. Here, we identify membrane-bound matrix metalloproteinase 14 (MT1-MMP/MMP14) as a central regulator of insulin sensitivity during ageing. Ageing promotes MMP14 activation in insulin-sensitive tissues, which cleaves Insulin Receptor to suppress insulin signaling. MT1-MMP inhibition restores Insulin Receptor expression, improving insulin sensitivity in aged mice. The cleavage of Insulin Receptor by MT1-MMP also contributes to obesity-induced insulin resistance and inhibition of MT1-MMP activities normalizes metabolic dysfunctions in diabetic mouse models. Conversely, overexpression of MT1-MMP in the liver reduces the level of Insulin Receptor, impairing hepatic insulin sensitivity in young mice. The soluble Insulin Receptor and circulating MT1-MMP are positively correlated in plasma from aged human subjects and non-human primates. Our findings provide mechanistic insights into regulation of insulin sensitivity during physiological ageing and highlight MT1-MMP as a promising target for therapeutic avenue against diabetes.
  24. Nat Cardiovasc Res. 2022 Mar;1(3): 211-222
      Heart failure with preserved ejection fraction (HFpEF) is increasing in prevalence worldwide, already accounting for at least half of all heart failure (HF). As most patients with HFpEF are obese with metabolic syndrome, metabolic stress has been implicated in syndrome pathogenesis. Recently, compelling evidence for bidirectional crosstalk between metabolic stress and chronic inflammation has emerged, and alterations in systemic and cardiac immune responses are held to participate in HFpEF pathophysiology. Indeed, based on both preclinical and clinical evidence, comorbidity-driven systemic inflammation, coupled with metabolic stress, have been implicated together in HFpEF pathogenesis. As metabolic alterations impact immune function(s) in HFpEF, major changes in immune cell metabolism are also recognized in HFpEF and in HFpEF-predisposing conditions. Both arms of immunity - innate and adaptive - are implicated in the cardiomyocyte response in HFpEF. Indeed, we submit that crosstalk among adipose tissue, the immune system, and the heart represents a critical component of HFpEF pathobiology. Here, we review recent evidence in support of immunometabolic mechanisms as drivers of HFpEF pathogenesis, discuss pivotal biological mechanisms underlying the syndrome, and highlight questions requiring additional inquiry.
    Keywords:  HFpEF; immune system; metabolism
  25. Proc Natl Acad Sci U S A. 2022 Jul 12. 119(28): e2115867119
      Liver regeneration is a well-orchestrated process that is typically studied in animal models. Although previous animal studies have offered many insights into liver regeneration, human biology is less well understood. To this end, we developed a three-dimensional (3D) platform called structurally vascularized hepatic ensembles for analyzing regeneration (SHEAR) to model multiple aspects of human liver regeneration. SHEAR enables control over hemodynamic alterations to mimic those that occur during liver injury and regeneration and supports the administration of biochemical inputs such as cytokines and paracrine interactions with endothelial cells. We found that exposing the endothelium-lined channel to fluid flow led to increased secretion of regeneration-associated factors. Stimulation with relevant cytokines not only amplified the secretory response, but also induced cell-cycle entry of primary human hepatocytes (PHHs) embedded within the device. Further, we identified endothelial-derived mediators that are sufficient to initiate proliferation of PHHs in this context. Collectively, the data presented here underscore the importance of multicellular models that can recapitulate high-level tissue functions and demonstrate that the SHEAR device can be used to discover and validate conditions that promote human liver regeneration.
    Keywords:  hepatocyte; regeneration; vascular
  26. Gut. 2022 Jun 27. pii: gutjnl-2021-326522. [Epub ahead of print]
      OBJECTIVE: Prior studies identified clinical factors associated with increased risk of pancreatic ductal adenocarcinoma (PDAC). However, little is known regarding their time-varying nature, which could inform earlier diagnosis. This study assessed temporality of body mass index (BMI), blood-based markers, comorbidities and medication use with PDAC risk .DESIGN: We performed a population-based nested case-control study of 28 137 PDAC cases and 261 219 matched-controls in England. We described the associations of biomarkers with risk of PDAC using fractional polynomials and 5-year time trends using joinpoint regression. Associations with comorbidities and medication use were evaluated using conditional logistic regression.
    RESULTS: Risk of PDAC increased with raised HbA1c, liver markers, white blood cell and platelets, while following a U-shaped relationship for BMI and haemoglobin. Five-year trends showed biphasic BMI decrease and HbA1c increase prior to PDAC; early-gradual changes 2-3 years prior, followed by late-rapid changes 1-2 years prior. Liver markers and blood counts (white blood cell, platelets) showed monophasic rapid-increase approximately 1 year prior. Recent diagnosis of pancreatic cyst, pancreatitis, type 2 diabetes and initiation of certain glucose-lowering and acid-regulating therapies were associated with highest risk of PDAC.
    CONCLUSION: Risk of PDAC increased with raised HbA1c, liver markers, white blood cell and platelets, while followed a U-shaped relationship for BMI and haemoglobin. BMI and HbA1c derange biphasically approximately 3 years prior while liver markers and blood counts (white blood cell, platelets) derange monophasically approximately 1 year prior to PDAC. Profiling these in combination with their temporality could inform earlier PDAC diagnosis.
    Keywords:  cancer epidemiology; pancreatic cancer
  27. J Physiol Biochem. 2022 Jun 29.
      Pancreatic ductal adenocarcinoma (PDAC) is the deadliest of the common cancers. A major hallmark of PDAC is an abundant and dense fibrotic stroma, the result of a disproportionate deposition of extracellular matrix (ECM) proteins. Cancer-associated fibroblasts (CAFs) are the main mediators of PDAC desmoplasia. CAFs represent a heterogenous group of activated fibroblasts with different origins and activation mechanisms. microRNAs (miRNAs) are small non-coding RNAs with critical activity during tumour development and resistance to chemotherapy. Increasing evidence has revealed that miRNAs play a relevant role in the differentiation of normal fibroblasts into CAFs in PDAC. In this review, we discuss recent findings on the role of miRNAs in the activation of CAFs during the progression of PDAC and its response to therapy, as well as the potential role that PDAC-derived exosomal miRNAs may play in the activation of hepatic stellate cells (HSCs) and formation of liver metastasis. Since targeting of CAF activation may be a viable strategy for PDAC therapy, and miRNAs have emerged as potential therapeutic targets, understanding the biology underpinning miRNA-mediated tumour cell-CAF interactions is an important component in guiding rational approaches to treating this deadly disease.
    Keywords:  Cancer-associated fibroblasts (CAFs); Pancreatic cancer; Response to therapy; Tumour pathogenesis; microRNAs
  28. Nat Cell Biol. 2022 Jun 30.
      CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.
  29. Methods Mol Biol. 2022 ;2497 185-206
      Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).
    Keywords:  Bioenergetics; Cellular respiration; Mitochondrial respiration; Permeabilized cells; Seahorse XF Extracellular Flux Analyzer
  30. Biochim Biophys Acta Biomembr. 2022 Jun 23. pii: S0005-2736(22)00133-X. [Epub ahead of print] 183995
      By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lβ (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.
    Keywords:  Asymmetric lipid bilayer; Coexistence of liquid phases; Induced ordered domains; Lipid packing/order
  31. Nat Struct Mol Biol. 2022 Jun 30.
      Oxidative stress is a primary cause of cellular senescence and contributes to the etiology of numerous human diseases. Oxidative damage to telomeric DNA has been proposed to cause premature senescence by accelerating telomere shortening. Here, we tested this model directly using a precision chemoptogenetic tool to produce the common lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblasts and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results, we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions that drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.
  32. J Gastrointest Surg. 2022 Jun 29.
      BACKGROUND: Chemotherapy (CTX) is associated with improved survival for patients undergoing resection for pancreatic ductal adenocarcinoma (PDAC). The current study evaluated the influence of tumor location on receipt of CTX.METHODS: The NCDB (2006-2017) was queried to identify patients with clinical stage I-III PDAC. Predictors of receipt of CTX, sequencing of CTX, and overall survival (OS) were analyzed.
    RESULTS: Among 14,557 patients who underwent resection for PDAC 3,453 (24%) did not receive CTX. On multivariable analysis, patients with tail tumors were 15% less likely to receive CTX (OR 0.85, 95% CI 0.747-0.968) and 58% less likely to receive neoadjuvant CTX (OR 0.42, 95% CI 0.351-0.509) relative to patients with head/neck tumors. For patients with body tumors, there was no difference in rates of administration or sequence of CTX. For patients with resected tail tumors, median OS was 29.9 vs 18.9 months (p < 0.001) between those who did and did not receive CTX. For patients with tail tumors, independent predictors of not receiving CTX, regardless of sequence, were increasing age (OR 0.95, 95% CI 0.935-0.965), increasing post-op length of stay (OR 0.95, 95% CI 0.932-0.968), and 30-day post-op readmission (OR 0.46, 95% CI 0.315-0.670).
    CONCLUSIONS: In patients with clinical stage I-III PDAC, tumor location within the tail was independently associated with not receiving CTX. Given the marked improvement in OS when CTX is administered, strategies aimed at increasing the number of these patients who receive CTX are necessary.
    Keywords:  Multimodal Therapy; Pancreatic cancer