bims-cagime 2023-05-21 papers

bims-cagime

Biomed News

on Cancer, aging and metabolism

Issue of 2023‒05‒21
37 papers selected by
Kıvanç Görgülü
Technical University of Munich

Uridine-derived ribose fuels glucose-restricted pancreatic cancer.
Blocking muscle wasting via deletion of the muscle-specific E3 ligase MuRF1 impedes pancreatic tumor growth.
Cross-Dataset Single-Cell Analysis Identifies Temporal Alterations in Cell Populations of Primary Pancreatic Tumor and Liver Metastasis.
Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence.
Impact of acute stress on murine metabolomics and metabolic flux.
Emerging roles of nucleotide metabolism in cancer.
Tumor-derived nanoseeds condition the soil for metastatic organotropism.
Periostin+ stromal cells guide lymphovascular invasion by cancer cells.
Turning Down Oxygen to Turn Up Inflammation in CAFs.
Prognosticators for Patients with Pancreatic Ductal Adenocarcinoma Who Received Neoadjuvant FOLFIRINOX or Gemcitabine/Nab-Paclitaxel Therapy and Pancreatectomy.
IL-6 is dispensable for causing cachexia in the colon carcinoma 26 model.
Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.
Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions.
Cancer cachexia as a blueprint for treating obesity.
Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.
Inflammation-Based Prognostic Scores in Pancreatic Cancer Patients-A Single-Center Analysis of 1294 Patients within the Last Decade.
Cachexia causes time-dependent activation of the inflammasome in the liver.
Towards sequence-based principles for protein phase separation predictions.
SQSTM1/P62 promotes lysophagy via formation of liquid-like condensates maintained by HSP27.
Measuring Immune Cell Movement Toward the Soluble Microenvironment of Human Tissues Using a Boyden Chamber-Based Migration Assay.
Experimental evolution for cell biology.
MRI-Based Tumor Necrosis Depiction in Pancreatic Ductal Adenocarcinoma: Can It Predict Tumor Aggressiveness?
SMARCB1 regulates the hypoxic stress response in sickle cell trait.
SnapShot: Eukaryotic ribosome biogenesis I.
Multiscale stiffness of human emphysematous precision cut lung slices.
Membrane Curvature: The Inseparable Companion of Autophagy.
Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.
Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair.
Efficacy and Imaging Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.
Serum-Free Production of Human Stem Cell-Derived Liver Spheres for Cancer Metastasis Research.
Single Breath-Hold MR Elastography for Fast Biomechanical Probing of Pancreatic Stiffness.
Generating Mitochondrial-Nuclear Exchange (MNX) Mice to Identify Mitochondrial Determinants of Cancer Metastasis.
Nerve-driven metastasis.
Partial tumor irradiation plus pembrolizumab in treating large advanced solid tumor metastases.
Lysine catabolism reprograms tumour immunity through histone crotonylation.
Fluorescence-Based Detection of Proteins and Their Interactions in Live Cells.

Nature. 2023 May 17.

Uridine-derived ribose fuels glucose-restricted pancreatic cancer.

Zeribe C Nwosu, Matthew H Ward, Peter Sajjakulnukit, Pawan Poudel, Chanthirika Ragulan, Steven Kasperek, Megan Radyk, Damien Sutton, Rosa E Menjivar, Anthony Andren, Juan J Apiz-Saab, Zachary Tolstyka, Kristee Brown, Ho-Joon Lee, Lindsey N Dzierozynski, Xi He, Hari Ps, Julia Ugras, Gift Nyamundanda, Li Zhang, Christopher J Halbrook, Eileen S Carpenter, Jiaqi Shi, Leah P Shriver, Gary J Patti, Alexander Muir, Marina Pasca di Magliano, Anguraj Sadanandam, Costas A Lyssiotis.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS-MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.

DOI: https://doi.org/10.1038/s41586-023-06073-w
Commun Biol. 2023 05 13. 6(1): 519

Blocking muscle wasting via deletion of the muscle-specific E3 ligase MuRF1 impedes pancreatic tumor growth.

Daria Neyroud, Orlando Laitano, Aneesha Dasgupta, Christopher Lopez, Rebecca E Schmitt, Jessica Z Schneider, David W Hammers, H Lee Sweeney, Glenn A Walter, Jason Doles, Sarah M Judge, Andrew R Judge.

Cancer-induced muscle wasting reduces quality of life, complicates or precludes cancer treatments, and predicts early mortality. Herein, we investigate the requirement of the muscle-specific E3 ubiquitin ligase, MuRF1, for muscle wasting induced by pancreatic cancer. Murine pancreatic cancer (KPC) cells, or saline, were injected into the pancreas of WT and MuRF1-/- mice, and tissues analyzed throughout tumor progression. KPC tumors induces progressive wasting of skeletal muscle and systemic metabolic reprogramming in WT mice, but not MuRF1-/- mice. KPC tumors from MuRF1-/- mice also grow slower, and show an accumulation of metabolites normally depleted by rapidly growing tumors. Mechanistically, MuRF1 is necessary for the KPC-induced increases in cytoskeletal and muscle contractile protein ubiquitination, and the depression of proteins that support protein synthesis. Together, these data demonstrate that MuRF1 is required for KPC-induced skeletal muscle wasting, whose deletion reprograms the systemic and tumor metabolome and delays tumor growth.

DOI: https://doi.org/10.1038/s42003-023-04902-2
Cancers (Basel). 2023 Apr 21. pii: 2396. [Epub ahead of print]15(8):

Cross-Dataset Single-Cell Analysis Identifies Temporal Alterations in Cell Populations of Primary Pancreatic Tumor and Liver Metastasis.

Daowei Yang, Rohan Moniruzzaman, Hua Wang, Huamin Wang, Yang Chen.

  Pancreatic ductal adenocarcinoma (PDAC) has a unique tumor microenvironment composed of various cell populations such as cancer cells, cancer-associated fibroblasts (CAFs), immune cells, and endothelial cells. Recently, single-cell RNA-sequencing analysis (scRNA-seq) has systemically revealed the genomic profiles of these cell populations in PDAC. However, the direct comparison of cell population composition and genomic profile between primary tumors (at both early- and late-stage) and metastatic tumors of PDAC is still lacking. In this study, we combined and analyzed recent scRNA-seq datasets of transgenic KPC mouse models with autochthonous PDAC and matched liver metastasis, revealing the unique tumor ecosystem and cell composition of liver metastasis in contrast to primary PDAC. Metastatic PDAC tumors harbor distinct cancer cell subpopulations from primary tumors. Several unique markers, including HMGA1, were identified for metastasis-enriched cancer cell subpopulations. Furthermore, metastatic tumors reveal significantly enriched granulocytic myeloid-derived suppressor cells (G-MDSCs), mature neutrophils, and granulocyte-myeloid progenitors (GMPs). A common GMP population across primary tumors, liver metastases, and healthy bone marrow was identified as the putative cell origin of tumor-associated neutrophils/granulocytes.

Keywords:  genetically engineered mouse models; liver metastasis; pancreatic cancer; single-cell RNA-sequencing analysis; tumor immunology; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers15082396
Dev Cell. 2023 May 09. pii: S1534-5807(23)00191-0. [Epub ahead of print]

Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.

Reo Kurusu, Yuki Fujimoto, Hideaki Morishita, Daisuke Noshiro, Shuhei Takada, Koji Yamano, Hideaki Tanaka, Ritsuko Arai, Shun Kageyama, Tomoko Funakoshi, Satoko Komatsu-Hirota, Hikari Taka, Saiko Kazuno, Yoshiki Miura, Masato Koike, Toshifumi Wakai, Satoshi Waguri, Nobuo N Noda, Masaaki Komatsu.

  In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

Keywords:  Mallory-Denk body; NBR1; fluorescence-activated particle sorting; hepatocellular carcinoma; liquid-liquid phase separation; non-alcoholic steatohepatitis; p62/SQSTM1; selective autophagy; vault; vault-phagy

DOI:  https://doi.org/10.1016/j.devcel.2023.04.015
J Vis Exp. 2023 Apr 28.

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence.

Felipe J Renna, Malena Herrera Lopez, Maria Manifava, Nicholas T Ktistakis, Maria I Vaccaro.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

DOI: https://doi.org/10.3791/65005
Proc Natl Acad Sci U S A. 2023 05 23. 120(21): e2301215120

Impact of acute stress on murine metabolomics and metabolic flux.

Won Dong Lee, Lingfan Liang, Jenna AbuSalim, Connor S R Jankowski, Laith Z Samarah, Michael D Neinast, Joshua D Rabinowitz.

  Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.

Keywords:  catecholamine; in vivo; isotope tracing; metabolomics; stress

DOI:  https://doi.org/10.1073/pnas.2301215120
Trends Cancer. 2023 May 10. pii: S2405-8033(23)00064-X. [Epub ahead of print]

Emerging roles of nucleotide metabolism in cancer.

Diana D Shi, Milan R Savani, Kalil G Abdullah, Samuel K McBrayer.

  Nucleotides are substrates for multiple anabolic pathways, most notably DNA and RNA synthesis. Since nucleotide synthesis inhibitors began to be used for cancer therapy in the 1950s, our understanding of how nucleotides function in tumor cells has evolved, prompting a resurgence of interest in targeting nucleotide metabolism for cancer therapy. In this review, we discuss recent advances that challenge the idea that nucleotides are mere building blocks for the genome and transcriptome and highlight ways that these metabolites support oncogenic signaling, stress resistance, and energy homeostasis in tumor cells. These findings point to a rich network of processes sustained by aberrant nucleotide metabolism in cancer and reveal new therapeutic opportunities.

Keywords:  metabolism; nucleotides; purines; pyrimidines

DOI:  https://doi.org/10.1016/j.trecan.2023.04.008
Semin Cancer Biol. 2023 May 11. pii: S1044-579X(23)00077-9. [Epub ahead of print]

Tumor-derived nanoseeds condition the soil for metastatic organotropism.

Mengying Hu, Candia M Kenific, Nancy Boudreau, David Lyden.

  Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments. The cargo incorporated into EVs is collectively shed from cancer cells and cancer-associated stromal cells. Increased understanding of how tumor EVs promote PMN establishment and detection of EVs in bodily fluids highlight how tumor EVs could serve as potential diagnostic and prognostic biomarkers, as well as provide a therapeutic target for metastasis prevention. This review focuses on tumor-derived EVs and how they direct organotropism and subsequently modulate stromal and immune microenvironments at distal sites to facilitate PMN formation. We also outline the progress made thus far towards clinical applications of tumor EVs.

Keywords:  Biomarker; Organotropism; Pre-metastatic niche reprogramming; Therapy; Tumor-derived EVs

DOI:  https://doi.org/10.1016/j.semcancer.2023.05.003
Cancer Res. 2023 May 19. pii: CAN-22-2412. [Epub ahead of print]

Periostin+ stromal cells guide lymphovascular invasion by cancer cells.

Jamie L Null, Dae Joong Kim, James V McCann, Patcharin Pramoonjago, Jay W Fox, Jianhao Zeng, Pankaj Kumar, Lincy Edatt, Chad V Pecot, Andrew C Dudley.

Cancer cell dissemination to sentinel lymph nodes associates with poor patient outcomes, particularly in breast cancer. The process by which cancer cells egress from the primary tumor upon interfacing with the lymphatic vasculature is complex and driven by dynamic interactions between cancer cells and stromal cells, including cancer associated fibroblasts (CAFs). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, since periostin is secreted, periostin-expressing CAFs are difficult to characterize in situ, limiting our understanding of their specific contribution to cancer progression. Here, we used in vivo genetic labeling and ablation to lineage trace periostin+ cells and characterize their functions during tumor growth and metastasis. Periostin-expressing CAFs were spatially found at periductal and perivascular margins, were enriched at lymphatic vessel peripheries, and were differentially activated by highly-metastatic cancer cells versus poorly-metastatic counterparts. Surprisingly, genetically depleting periostin+ CAFs slightly accelerated primary tumor growth but impaired intratumoral collagen organization and inhibited lymphatic, but not lung, metastases. Periostin ablation in CAFs impaired their ability to deposit aligned collagen matrices and inhibited cancer cell invasion through collagen and across lymphatic endothelial cell monolayers. Thus, highly-metastatic cancer cells mobilize periostin-expressing CAFs in the primary tumor site that promote collagen remodeling and collective cell invasion within lymphatic vessels and ultimately to sentinel lymph nodes.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2412
Cancer Res. 2023 May 15. 83(10): 1560-1562

Turning Down Oxygen to Turn Up Inflammation in CAFs.

Natividad R Fuentes, Cullen M Taniguchi.

Pancreatic ductal adenocarcinoma (PDAC) contains a desmoplastic stroma that limits blood perfusion and thus the delivery of nutrients, oxygen, and even therapeutics, creating a hypoxic microenvironment that resists nearly all forms of treatment, including immunomodulating therapy. Cancer-associated fibroblasts (CAF) are the main cellular components and producers of stroma in PDAC. Interestingly, CAFs exist as functionally diverse subpopulations derived from distinct lineages, some of which can be either inflammatory (iCAF) or myofibroblastic (myCAF). While previous work has suggested a link between hypoxia and the iCAF phenotype, direct experimental evidence is lacking. In this issue of Cancer Research, Schwörer and colleagues investigate the role of hypoxia and hypoxia-inducible factor-1α (HIF1α) in maintaining fibroblast heterogeneity and promoting tumor progression in PDAC. The authors use a combination of in vitro and orthotopic techniques to identify a strong role for hypoxia in combination with tumor-derived cytokines in maintaining an iCAF phenotype highlighted by IL6 expression. The authors use an innovative in vitro system to simulate oxygen gradients and use these methods to support their assertions regarding hypoxia as a proinflammatory state. These findings suggest that HIF1α promotes the generation of iCAFs, providing novel insight into CAF heterogeneity. See related article by Schwörer et al., p. 1596.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-0523
Cancers (Basel). 2023 May 04. pii: 2608. [Epub ahead of print]15(9):

Prognosticators for Patients with Pancreatic Ductal Adenocarcinoma Who Received Neoadjuvant FOLFIRINOX or Gemcitabine/Nab-Paclitaxel Therapy and Pancreatectomy.

Yi Tat Tong, Zongshan Lai, Matthew H G Katz, Laura R Prakash, Hua Wang, Deyali Chatterjee, Michael Kim, Ching-Wei D Tzeng, Jeffrey E Lee, Naruhiko Ikoma, Asif Rashid, Robert A Wolff, Dan Zhao, Eugene J Koay, Anirban Maitra, Huamin Wang.

  Neoadjuvant FOLFIRINOX and gemcitabine/nab-paclitaxel (GemNP) therapies are increasingly used to treat patients with pancreatic ductal adenocarcinoma (PDAC). However, limited data are available on their clinicopathologic prognosticators. We examined the clinicopathologic factors and survival of 213 PDAC patients who received FOLFIRINOX with 71 patients who received GemNP. The FOLFIRINOX group was younger (p < 0.01) and had a higher rate of radiation (p = 0.049), higher rate of borderline resectable and locally advanced disease (p < 0.001), higher rate of Group 1 response (p = 0.045) and lower ypN stage (p = 0.03) than the GemNP group. Within FOLFIRINOX group, radiation was associated with decreased lymph node metastasis (p = 0.01) and lower ypN stage (p = 0.01). The tumor response group, ypT, ypN, LVI and PNI, correlated significantly with both DFS and OS (p < 0.05). Patients with the ypT0/T1a/T1b tumor had better DFS (p = 0.04) and OS (p = 0.03) than those with ypT1c tumor. In multivariate analysis, the tumor response group and ypN were independently prognostic factors for DFS and OS (p < 0.05). Our study demonstrated that the FOLFIRINOX group was younger and had a better pathologic response than the GemNP group and that the tumor response group, ypN, ypT, LVI and PNI, are significant prognostic factors for survival in these patients. Our results also suggest that the tumor size of 1.0 cm is a better cut off for ypT2. Our study highlights the importance of systemic pathologic examination and the reporting of post-treatment pancreatectomies.

Keywords:  FOLFIRINOX; gemcitabine/nab-paclitaxel; lymph node metastasis; neoadjuvant therapy; pancreatic cancer; survival; tumor response grade; tumor stage

DOI:  https://doi.org/10.3390/cancers15092608
bioRxiv. 2023 May 02. pii: 2023.05.02.539076. [Epub ahead of print]

IL-6 is dispensable for causing cachexia in the colon carcinoma 26 model.

Young-Yon Kwon, Sheng Hui.

Various cytokines have been implicated in cancer cachexia. One such cytokine is IL-6, which has been deemed a key cachectic factor in mice inoculated with the colon carcinoma 26 (C26) cells, one of the most widely used models of cancer cachexia. Here to test the causal role of IL-6 in cancer cachexia, we used CRISPR/Cas9 editing to knock out IL-6 in C26 cells. We found that growth of IL-6 KO C26 tumors was dramatically delayed. Most strikingly, while IL-6 KO tumors eventually reached the similar size as wild-type tumors, cachexia still took place, despite no elevation in circulating IL-6. We further showed an increase of immune cell populations in IL-6 KO tumors and the defective IL-6 KO tumor growth was rescued in immunodeficient mice. Thus, our results invalidated IL-6 as a necessary factor for causing cachexia in the C26 model and revealed instead its important role in regulating tumor growth via immune suppression.

DOI: https://doi.org/10.1101/2023.05.02.539076
Oncogene. 2023 May 18.

Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.

Osamu Araki, Motoyuki Tsuda, Mayuki Omatsu, Mio Namikawa, Makoto Sono, Yuichi Fukunaga, Tomonori Masuda, Takaaki Yoshikawa, Munemasa Nagao, Satoshi Ogawa, Kenji Masuo, Norihiro Goto, Yu Muta, Yukiko Hiramatsu, Takahisa Maruno, Yuki Nakanishi, Sho Koyasu, Toshihiko Masui, Etsuro Hatano, Dieter Saur, Akihisa Fukuda, Hiroshi Seno.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. We previously reported that chromatin remodeler Brg1 is essential for acinar cell-derived PDAC formation in mice. However, the functional role of Brg1 in established PDAC and its metastasis remains unknown. Here, we investigated the importance of Brg1 for established PDAC by using a mouse model with a dual recombinase system. We discovered that Brg1 was a critical player for the cell survival and growth of spontaneously developed PDAC in mice. In addition, Brg1 was essential for metastasis of PDAC cells by inhibiting apoptosis in splenic injection and peritoneal dissemination models. Moreover, cancer stem-like property was compromised in PDAC cells by Brg1 ablation. Mechanistically, the hypoxia pathway was downregulated in Brg1-deleted mouse PDAC and BRG1-low human PDAC. Brg1 was essential for HIF-1α to bind to its target genes to augment the hypoxia pathway, which was important for PDAC cells to maintain their stem-like properties and to metastasize to the liver. Human PDAC cells with high BRG1 expression were more susceptible to BRG1 suppression. In conclusion, Brg1 plays a critical role for cell survival, stem-like property and metastasis of PDAC through the regulation of hypoxia pathway, and thus could be a novel therapeutic target for PDAC.

DOI: https://doi.org/10.1038/s41388-023-02716-4
Nat Metab. 2023 May 17.

Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions.

Owen S Skinner, Joan Blanco-Fernández, Russell P Goodman, Akinori Kawakami, Hongying Shen, Lajos V Kemény, Lena Joesch-Cohen, Matthew G Rees, Jennifer A Roth, David E Fisher, Vamsi K Mootha, Alexis A Jourdain.

Glucose is vital for life, serving as both a source of energy and carbon building block for growth. When glucose is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized genome-wide genetic screens and a PRISM growth assay across 482 cancer cell lines. We report that catabolism of uridine from the medium enables the growth of cells in the complete absence of glucose. While previous studies have shown that uridine can be salvaged to support pyrimidine synthesis in the setting of mitochondrial oxidative phosphorylation deficiency1, our work demonstrates that the ribose moiety of uridine or RNA can be salvaged to fulfil energy requirements via a pathway based on: (1) the phosphorylytic cleavage of uridine by uridine phosphorylase UPP1/UPP2 into uracil and ribose-1-phosphate (R1P), (2) the conversion of uridine-derived R1P into fructose-6-P and glyceraldehyde-3-P by the non-oxidative branch of the pentose phosphate pathway and (3) their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Capacity for glycolysis from uridine-derived ribose appears widespread, and we confirm its activity in cancer lineages, primary macrophages and mice in vivo. An interesting property of this pathway is that R1P enters downstream of the initial, highly regulated steps of glucose transport and upper glycolysis. We anticipate that 'uridine bypass' of upper glycolysis could be important in the context of disease and even exploited for therapeutic purposes.

DOI: https://doi.org/10.1038/s42255-023-00774-2
Trends Endocrinol Metab. 2023 May 10. pii: S1043-2760(23)00087-5. [Epub ahead of print]

Cancer cachexia as a blueprint for treating obesity.

Nikolai P Jaschke, Tilman D Rachner.

  Effective pharmacological treatments to achieve significant and sustained weight loss in obese individuals remain limited. Here, we apply a 'reverse engineering' approach to cancer cachexia, an extreme form of dysregulated energy balance resulting in net catabolism. We discuss three phenotypic features of the disease, summarize the underlying molecular checkpoints, and explore their translation to obesity research. We then provide examples for established pharmaceuticals, which follow a reverse engineering logic, and propose additional targets that may be of relevance for future studies. Finally, we argue that approaching diseases from this perspective may prove useful as a generic strategy to fuel the development of innovative therapies.

Keywords:  cancer cachexia; obesity; reverse engineering; systems biology

DOI:  https://doi.org/10.1016/j.tem.2023.04.001
Autophagy. 2023 Feb 22. 1-14

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Gal Chaim Nuta, Yuval Gilad, Nadav Goldberg, Sara Meril, Marcela Bahlsen, Silvia Carvalho, Noga Kozer, Haim Barr, Yael Fridmann Sirkis, Kamil Hercík, Petra Břehová, Radim Nencka, Shani Bialik, Miriam Eisenstein, Adi Kimchi.

  Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Keywords:  Autophagy inhibition; LC3B; cancer; drug screen; pancreatic cancer; protein-fragment complementation assay; small molecules

DOI:  https://doi.org/10.1080/15548627.2023.2178159
Cancers (Basel). 2023 Apr 19. pii: 2367. [Epub ahead of print]15(8):

Inflammation-Based Prognostic Scores in Pancreatic Cancer Patients-A Single-Center Analysis of 1294 Patients within the Last Decade.

Christopher C M Neumann, François Schneider, Georg Hilfenhaus, Loredana Vecchione, Matthäus Felsenstein, Jana Ihlow, Dominik Geisel, Steffen Sander, Johann Pratschke, Sebastian Stintzing, Ulrich Keilholz, Uwe Pelzer.

  Inflammatory properties are known to promote tumor progression leading to an impaired median overall survival (mOS). Various small studies have focused on a wide range of inflammation-based prognostic indicators. By using sufficient data from 1294 out of 2323 patients diagnosed with pancreatic cancer between 2009 and 2021 at our cancer center, inflammatory markers such as the neutrophil to lymphocyte ratio (NRL), the platelet to lymphocyte ratio (PLR), the lymphocyte to monocyte ratio (LMR) and the CRP to albumin ratio (CAR) were evaluated. We identified a new combined score, termed the inflammatory benchmark index (IBI). We performed univariate and multivariate overall survival analyses and identified optimal prognostic cut-off values for each parameter. In univariate analyses, advanced age (p < 0.001), gender (p < 0.001), tumor stage (p < 0.001), CA19-9 (p = 0.001), NLR (p = 0.001), LMR (p = 0.004), PLR (p = 0.004), CAR (p = 0.001) and IBI (p = 0.001) were identified as prognostic markers. In multivariate analyses advanced age (p < 0.001), gender (p = 0.001), tumor stage (p < 0.001), CA19-9 (p < 0.001), NLR (p = 0.001), LMR (p = 0.038), CAR (p < 0.001) and IBI (p < 0.001) were independent prognostic markers. These findings emphasize the impact of inflammation in pancreatic cancer, provide easily accessible prognostic values for the clinician, and may be useful as stratification parameters for trials aimed at patient inflammation or immune response.

Keywords:  CRP to albumin ratio (CAR); inflammatory benchmark index (IBI); inflammatory biomarkers; lymphocyte to monocyte ratio (LMR); neutrophil to lymphocyte ratio (NLR); pancreatic cancer; platelet to lymphocyte ratio (PLR)

DOI:  https://doi.org/10.3390/cancers15082367
J Cachexia Sarcopenia Muscle. 2023 May 12.

Cachexia causes time-dependent activation of the inflammasome in the liver.

Rodrigo Xavier das Neves, Alex S Yamashita, Daniela M R Riccardi, Julia Köhn-Gaone, Rodolfo G Camargo, Nelson I Neto, Daniela Caetano, Silvio P Gomes, Felipe H Santos, Joanna D C C Lima, Miguel L Batista, José Cesar Rosa-Neto, Paulo Sérgio Martins De Alcântara, Linda F Maximiano, José P Otoch, Giorgio Trinchieri, Janina E E Tirnitz-Parker, Marília Seelaender.

  BACKGROUND: Cachexia is a wasting syndrome associated with systemic inflammation and metabolic disruption. Detection of the early signs of the disease may contribute to the effective attenuation of associated symptoms. Despite playing a central role in the control of metabolism and inflammation, the liver has received little attention in cachexia. We previously described relevant disruption of metabolic pathways in the organ in an animal model of cachexia, and herein, we adopt the same model to investigate temporal onset of inflammation in the liver. The aim was thus to study inflammation in rodent liver in the well-characterized cachexia model of Walker 256 carcinosarcoma and, in addition, to describe inflammatory alterations in the liver of one cachectic colon cancer patient, as compared to one control and one weight-stable cancer patient.METHODS: Colon cancer patients (one weight stable [WSC] and one cachectic [CC]) and one patient undergoing surgery for cholelithiasis (control, n = 1) were enrolled in the study, after obtainment of fully informed consent. Eight-week-old male rats were subcutaneously inoculated with a Walker 256 carcinosarcoma cell suspension (2 × 107 cells in 1.0 mL; tumour-bearing [T]; or phosphate-buffered saline-controls [C]). The liver was excised on Days 0 (n = 5), 7 (n = 5) and 14 (n = 5) after tumour cell injection.
RESULTS: In rodent cachexia, we found progressively higher numbers of CD68+ myeloid cells in the liver along cancer-cachexia development. Similar findings are described for CC, whose liver showed infiltration of the same cell type, compared with both WSC and control patient organs. In advanced rodent cachexia, hepatic phosphorylated c-Jun N-terminal kinase protein content and the inflammasome pathway protein expression were increased in relation to baseline (P < 0.05). These changes were accompanied by augmented expression of the active interleukin-1β (IL-1β) form (P < 0.05 for both circulating and hepatic content).
CONCLUSIONS: The results show that cancer cachexia is associated with an increase in the number of myeloid cells in rodent and human liver and with modulation of hepatic inflammasome pathway. The latter contributes to the aggravation of systemic inflammation, through increased release of IL-1β.

Keywords:  cancer cachexia; inflammasome; inflammation; liver; myeloid cells

DOI:  https://doi.org/10.1002/jcsm.13236
Curr Opin Chem Biol. 2023 May 17. pii: S1367-5931(23)00055-8. [Epub ahead of print]75 102317

Towards sequence-based principles for protein phase separation predictions.

Michele Vendruscolo, Monika Fuxreiter.

  The phenomenon of protein phase separation, which underlies the formation of biomolecular condensates, has been associated with numerous cellular functions. Recent studies indicate that the amino acid sequences of most proteins may harbour not only the code for folding into the native state but also for condensing into the liquid-like droplet state and the solid-like amyloid state. Here we review the current understanding of the principles for sequence-based methods for predicting the propensity of proteins for phase separation. A guiding concept is that entropic contributions are generally more important to stabilise the droplet state than they are for the native and amyloid states. Although estimating these entropic contributions has proven difficult, we describe some progress that has been recently made in this direction. To conclude, we discuss the challenges ahead to extend sequence-based prediction methods of protein phase separation to include quantitative in vivo characterisations of this process.

Keywords:  Protein aggregation; Protein interactions; Protein phase separation; Sequence-based predictions

DOI:  https://doi.org/10.1016/j.cbpa.2023.102317
Autophagy. 2023 May 16. 1-2

SQSTM1/P62 promotes lysophagy via formation of liquid-like condensates maintained by HSP27.

Elizabeth R Gallagher, Erika L F Holzbaur.

  ABBREVIATIONS: SQSTM1/p62: Sequestosome-1; HSP27: Heat shock protein 27; LLPS: liquid-liquid phase separation; iPSC: induced pluripotent stem cell; PB1: Phox and Bem1p; FRAP: fluorescence recovery after photo-bleaching; ATG: autophagy-related; ALS: amyotrophic lateral sclerosis.

Keywords:  HSP27; Lysosomes; Sqstm1/P62; lysophagy; neurodegeneration; phase separation

DOI:  https://doi.org/10.1080/15548627.2023.2210943
Methods Mol Biol. 2023 ;2645 231-240

Measuring Immune Cell Movement Toward the Soluble Microenvironment of Human Tissues Using a Boyden Chamber-Based Migration Assay.

Eimear Mylod, Joanne Lysaght, Melissa J Conroy.

  Migration assays are used to measure cell movement toward a variety of chemoattractants in a controlled environment. Here we describe a method for a Boyden chamber-based migration assay using conditioned media generated from the tumor, liver, and visceral adipose tissue of cancer patients.

Keywords:  Boyden chamber; Cell migration; Chemokines; Chemotaxis; Immune cells

DOI:  https://doi.org/10.1007/978-1-0716-3056-3_14
Trends Cell Biol. 2023 May 13. pii: S0962-8924(23)00080-6. [Epub ahead of print]

Experimental evolution for cell biology.

Jana Helsen, Gavin Sherlock, Gautam Dey.

  Evolutionary cell biology explores the origins, principles, and core functions of cellular features and regulatory networks through the lens of evolution. This emerging field relies heavily on comparative experiments and genomic analyses that focus exclusively on extant diversity and historical events, providing limited opportunities for experimental validation. In this opinion article, we explore the potential for experimental laboratory evolution to augment the evolutionary cell biology toolbox, drawing inspiration from recent studies that combine laboratory evolution with cell biological assays. Primarily focusing on approaches for single cells, we provide a generalizable template for adapting experimental evolution protocols to provide fresh insight into long-standing questions in cell biology.

Keywords:  adaptation; evolutionary cell biology; evolutionary dynamics; evolutionary innovation; experimental design

DOI:  https://doi.org/10.1016/j.tcb.2023.04.006
Cancers (Basel). 2023 Apr 15. pii: 2313. [Epub ahead of print]15(8):

MRI-Based Tumor Necrosis Depiction in Pancreatic Ductal Adenocarcinoma: Can It Predict Tumor Aggressiveness?

Mark A Anderson, David E Knipp, Yoshifumi Noda, Sophia C Kamran, Vinit Baliyan, Hamed Kordbacheh, Theodore S Hong, Avinash Kambadakone.

  The purpose of this study was to investigate whether tumor necrosis depicted on contrast-enhanced abdominal MRI can predict tumor aggressiveness in pancreatic ductal adenocarcinoma (PDAC). In this retrospective analysis, we included 71 patients with pathology-proven PDAC who underwent contrast-enhanced MRI from 2006 to 2020. Assessment for the presence/absence of imaging detected necrosis was performed on T2-weighted and contrast-enhanced T1-weighted images. Primary tumor characteristics, regional lymphadenopathy, metastases, stage, and overall survival were analyzed. Fisher's exact and Mann-Whitney U tests were used for statistical analysis. Of the 72 primary tumors, necrosis was identified on MRI in 58.3% (42/72). Necrotic PDACs were larger (44.6 vs. 34.5 mm, p = 0.0016), had higher rates of regional lymphadenopathy (69.0% vs. 26.7%, p = 0.0007), and more frequent metastases (78.6% vs. 40.0%, p = 0.0010) than those without MRI-evident necrosis. A non-statistically significant reduction in median overall survival was observed in patients with versus without MRI-evident necrosis (15.8 vs. 38.0 months, p = 0.23). PDAC tumor necrosis depicted on MRI was associated with larger tumors and higher frequency of regional lymphadenopathy and metastases.

Keywords:  magnetic resonance imaging; necrosis; pancreatic ductal adenocarcinoma

DOI:  https://doi.org/10.3390/cancers15082313
Proc Natl Acad Sci U S A. 2023 05 23. 120(21): e2209639120

SMARCB1 regulates the hypoxic stress response in sickle cell trait.

Melinda Soeung, Luigi Perelli, Ziheng Chen, Eleonora Dondossola, I-Lin Ho, Federica Carbone, Li Zhang, Hania Khan, Courtney N Le, Cihui Zhu, Michael D Peoples, Ningping Feng, Shan Jiang, Niki Millward Zacharias, Rosalba Minelli, Daniel D Shapiro, Angela K Deem, Sisi Gao, Emily H Cheng, Donatella Lucchetti, Cheryl L Walker, Alessandro Carugo, Virginia Giuliani, Timothy P Heffernan, Andrea Viale, Nizar M Tannir, Giulio F Draetta, Pavlos Msaouel, Giannicola Genovese.

  Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Keywords:  SMARCB1; hypoxia; renal medullary carcinoma; sickle cell trait

DOI:  https://doi.org/10.1073/pnas.2209639120
Cell. 2023 May 11. pii: S0092-8674(23)00464-6. [Epub ahead of print]186(10): 2282-2282.e1

SnapShot: Eukaryotic ribosome biogenesis I.

Ed Hurt, Jingdong Cheng, Jochen Baβler, Janet Iwasa, Roland Beckmann.

Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.

DOI: https://doi.org/10.1016/j.cell.2023.04.030
Sci Adv. 2023 May 19. 9(20): eadf2535

Multiscale stiffness of human emphysematous precision cut lung slices.

Jae Hun Kim, Niccole Schaible, Joseph K Hall, Erzsébet Bartolák-Suki, Yuqing Deng, Jacob Herrmann, Adam Sonnenberg, Holger P Behrsing, Kenneth R Lutchen, Ramaswamy Krishnan, Béla Suki.

Emphysema is a debilitating disease that remodels the lung leading to reduced tissue stiffness. Thus, understanding emphysema progression requires assessing lung stiffness at both the tissue and alveolar scales. Here, we introduce an approach to determine multiscale tissue stiffness and apply it to precision-cut lung slices (PCLS). First, we established a framework for measuring stiffness of thin, disk-like samples. We then designed a device to verify this concept and validated its measuring capabilities using known samples. Next, we compared healthy and emphysematous human PCLS and found that the latter was 50% softer. Through computational network modeling, we discovered that this reduced macroscopic tissue stiffness was due to both microscopic septal wall remodeling and structural deterioration. Lastly, through protein expression profiling, we identified a wide spectrum of enzymes that can drive septal wall remodeling, which, together with mechanical forces, lead to rupture and structural deterioration of the emphysematous lung parenchyma.

DOI: https://doi.org/10.1126/sciadv.adf2535
Cells. 2023 04 11. pii: 1132. [Epub ahead of print]12(8):

Membrane Curvature: The Inseparable Companion of Autophagy.

Lei Liu, Yu Tang, Zijuan Zhou, Yuan Huang, Rui Zhang, Hao Lyu, Shuai Xiao, Dong Guo, Declan William Ali, Marek Michalak, Xing-Zhen Chen, Cefan Zhou, Jingfeng Tang.

  Autophagy is a highly conserved recycling process of eukaryotic cells that degrades protein aggregates or damaged organelles with the participation of autophagy-related proteins. Membrane bending is a key step in autophagosome membrane formation and nucleation. A variety of autophagy-related proteins (ATGs) are needed to sense and generate membrane curvature, which then complete the membrane remodeling process. The Atg1 complex, Atg2-Atg18 complex, Vps34 complex, Atg12-Atg5 conjugation system, Atg8-phosphatidylethanolamine conjugation system, and transmembrane protein Atg9 promote the production of autophagosomal membranes directly or indirectly through their specific structures to alter membrane curvature. There are three common mechanisms to explain the change in membrane curvature. For example, the BAR domain of Bif-1 senses and tethers Atg9 vesicles to change the membrane curvature of the isolation membrane (IM), and the Atg9 vesicles are reported as a source of the IM in the autophagy process. The amphiphilic helix of Bif-1 inserts directly into the phospholipid bilayer, causing membrane asymmetry, and thus changing the membrane curvature of the IM. Atg2 forms a pathway for lipid transport from the endoplasmic reticulum to the IM, and this pathway also contributes to the formation of the IM. In this review, we introduce the phenomena and causes of membrane curvature changes in the process of macroautophagy, and the mechanisms of ATGs in membrane curvature and autophagosome membrane formation.

Keywords:  Atg proteins; ER-phagy; autophagy; membrane curvature; nucleophagy; xenophagy

DOI:  https://doi.org/10.3390/cells12081132
J Cachexia Sarcopenia Muscle. 2023 May 16.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

Steffen H Raun, Mona S Ali, Xiuqing Han, Carlos Henríquez-Olguín, T C Phung Pham, Roberto Meneses-Valdés, Jonas R Knudsen, Anna C H Willemsen, Steen Larsen, Thomas E Jensen, Ramon Langen, Lykke Sylow.

  BACKGROUND: Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia.METHODS: In vastus lateralis muscle biopsies from n = 26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n = 27, WT + LLC: n = 34, mAMPK-KiDe: n = 23, mAMPK-KiDe + LLC: n = 38). Moreover, male LLC-tumour-bearing mice were treated with (n = 10)/without (n = 9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting.
RESULTS: Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, β2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, β2 and γ1), fat-free mass (α1, β2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance.
CONCLUSIONS: Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

Keywords:  AMP-activated protein kinase (AMPK); cancer cachexia; glucose metabolism; insulin resistance; skeletal muscle

DOI:  https://doi.org/10.1002/jcsm.13238
Elife. 2023 May 19. pii: e86556. [Epub ahead of print]12

Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair.

Justin Krish Williams, Jordan Matthew Ngo, Isabelle Madeline Lehman, Randy Schekman.

  Exosomes are an extracellular vesicle (EV) subtype that is secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane. Exosomes may participate in intercellular communication and have utility as disease biomarkers; however, little is known regarding the physiological stimuli that induce their secretion. Ca2+ influx promotes exosome secretion, raising the possibility that exosomes are secreted during the Ca2+-dependent plasma membrane repair of tissues damaged by mechanical stress in vivo. To determine whether exosomes are secreted upon plasma membrane damage, we developed sensitive assays to measure exosome secretion in intact and permeabilized cells. Our results suggest that exosome secretion is coupled to Ca2+-dependent plasma membrane repair. We find that annexin A6 (ANXA6), a well-known plasma membrane repair protein, is recruited to MVBs in the presence of Ca2+ and required for Ca2+-dependent exosome secretion, both in intact and in permeabilized cells. ANXA6 depletion stalls MVBs at the cell periphery, and ANXA6 truncations localize to different membranes, suggesting that ANXA6 may serve to tether MVBs to the plasma membrane. We find that cells secrete exosomes and other EVs upon plasma membrane damage and propose that repair-induced secretion may contribute to the pool of EVs present within biological fluids.

Keywords:  cell biology; human

DOI:  https://doi.org/10.7554/eLife.86556
Mol Cancer Ther. 2023 May 15. pii: MCT-22-0810. [Epub ahead of print]

Efficacy and Imaging Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.

Catherine Lee, Ziyue Karen Jiang, Simon Planken, Lisa K Manzuk, Roberto Ortiz, Michael Hall, Kavon Noorbehesht, Sripad Ram, Timothy Affolter, Gabriel E Troche, Nathan T Ihle, Theodore Johnson, Youngwook Ahn, Manfred Kraus, Anand Giddabasappa.

KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

DOI: https://doi.org/10.1158/1535-7163.MCT-22-0810
Methods Mol Biol. 2023 ;2645 189-209

Serum-Free Production of Human Stem Cell-Derived Liver Spheres for Cancer Metastasis Research.

Alvile Kasarinaite, James Drew, Mantas Jonaitis, Elaine Ma, Laura M Machesky, David C Hay.

  Renewable and scalable human liver tissue platforms are a powerful tool to study organ physiology and model diseases, such as cancer. Stem cell-derived models provide an alternative to cell lines, which can display limited relevance to primary cells and tissue. Historically, two-dimensional (2D) cultures have been used to model liver biology as they are easy to scale and deploy. However, 2D liver models lack functional diversity and phenotypic stability in long-term culture. To address those issues, protocols for generating the three-dimensional (3D) tissue aggregates have been developed. Here, we describe a methodology to generate 3D liver spheres from pluripotent stem cells. Liver spheres are composed of three key liver cell types (hepatic progenitor cells, endothelial cells, and hepatic stellate cells) and have been used to study human cancer cell metastasis.

Keywords:  3D cell culture; Cancer; Cell differentiation; Endothelial cell; Hepatic stellate cell; Hepatocyte; Liver; Metabolism; Metastasis; Pluripotent stem cells; Tissue engineering

DOI:  https://doi.org/10.1007/978-1-0716-3056-3_11
J Magn Reson Imaging. 2023 May 17.

Single Breath-Hold MR Elastography for Fast Biomechanical Probing of Pancreatic Stiffness.

Anne-Sophie van Schelt, Lukas M Gottwald, Nienke P M Wassenaar, Jurgen H Runge, Ralph Sinkus, Jaap Stoker, Aart J Nederveen, Eric M Schrauben.

  BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) stromal disposition is thought to influence chemotherapy efficacy and increase tissue stiffness, which could be quantified noninvasively via MR elastography (MRE). Current methods cause position-based errors in pancreas location over time, hampering accuracy. It would be beneficial to have a single breath-hold acquisition.PURPOSE: To develop and test a single breath-hold three-dimensional MRE technique utilizing prospective undersampling and a compressed sensing reconstruction (CS-MRE).
STUDY TYPE: Prospective.
POPULATION: A total of 30 healthy volunteers (HV) (31 ± 9 years; 33% male) and five patients with PDAC (69 ± 5 years; 80% male).
FIELD STRENGTH/SEQUENCE: 3-T, GRE Ristretto MRE.
ASSESSMENT: First, optimization of multi breath-hold MRE was done in 10 HV using four combinations of vibration frequency, number of measured wave-phase offsets, and TE and looking at MRE quality measures in the pancreas head. Second, viscoelastic parameters delineated in the pancreas head or tumor of CS-MRE were compared against (I) 2D and (II) 3D four breath-hold acquisitions in HV (N = 20) and PDAC patients. Intrasession repeatability was assessed for CS-MRE in a subgroup of healthy volunteers (N = 15).
STATISTICAL TESTS: Tests include repeated measures analysis of variance (ANOVA), Bland-Altman analysis, and coefficients of variation (CoVs). A P-value <.05 was considered statistically significant.
RESULTS: Optimization of the four breath-hold acquisitions resulted in 40 Hz vibration frequency, five wave-phases, and echo time (TE) = 6.9 msec as the preferred method (4BH-MRE). CS-MRE quantitative results did not differ from 4BH-MRE. Shear wave speed (SWS) and phase angle differed significantly between HV and PDAC patients using 4BH-MRE or CS-MRE. The limits of agreement for SWS were [-0.09, 0.10] m/second and the within-subject CoV was 4.8% for CS-MRE.
DATA CONCLUSION: CS-MRE might allow a single breath-hold MRE acquisition with comparable SWS and phase angle as 4BH-MRE, and it may still enable to differentiate between HV and PDAC.
LEVEL OF EVIDENCE: 2 Technical Efficacy Stage: 2.

Keywords:  compressed sensing; magnetic resonance elastography; pancreatic ductal adenocarcinoma; pancreatic magnetic resonance elastography; single breath-hold magnetic resonance elastography; visco-elastic properties of the pancreas

DOI:  https://doi.org/10.1002/jmri.28773
Methods Mol Biol. 2023 ;2660 43-59

Generating Mitochondrial-Nuclear Exchange (MNX) Mice to Identify Mitochondrial Determinants of Cancer Metastasis.

Danny R Welch, Melissa A Larson, Carolyn J Vivian, Jay L Vivian.

  Understanding the contributions of mitochondrial genetics to disease pathogenesis is facilitated by a new and unique model-the mitochondrial-nuclear exchange mouse. Here we report the rationale for their development, the methods used to create them, and a brief summary of how MNX mice have been used to understand the contributions of mitochondrial DNA in multiple diseases, focusing on cancer metastasis. Polymorphisms in mtDNA which distinguish mouse strains exert intrinsic and extrinsic effects on metastasis efficiency by altering epigenetic marks in the nuclear genome, changing production of reactive oxygen species, altering the microbiota, and influencing immune responses to cancer cells. Although the focus of this report is cancer metastasis, MNX mice have proven to be valuable in studying mitochondrial contributions to other diseases as well.

Keywords:  Animal model; Genetics; Mitochondria; Mitochondria-nuclear exchange mice

DOI:  https://doi.org/10.1007/978-1-0716-3163-8_4
Nat Rev Cancer. 2023 May 17.

Nerve-driven metastasis.

Daniela Senft.

DOI: https://doi.org/10.1038/s41568-023-00588-0
J Clin Invest. 2023 May 15. pii: e162260. [Epub ahead of print]133(10):

Partial tumor irradiation plus pembrolizumab in treating large advanced solid tumor metastases.

Mark C Korpics, Benjamin E Onderdonk, Rebekah E Dadey, Jared H Hara, Lilit Karapetyan, Yuanyuan Zha, Theodore G Karrison, Adam C Olson, Gini F Fleming, Ralph R Weichselbaum, Riyue Bao, Steven J Chmura, Jason J Luke.

  BACKGROUNDWe previously demonstrated the safety of stereotactic body radiotherapy followed by pembrolizumab (SBRT+P) in patients with advanced solid tumors. This phase I clinical trial was expanded to study the safety of partial tumor irradiation (partial-Rx). We assessed irradiated local failure (LF) and clinical outcomes with correlations to biomarkers including CD8+ T cell radiomics score (RS) and circulating cytokines.METHODSPatients received SBRT to 2-4 metastases and pembrolizumab for up to 7 days after SBRT. Tumors measuring up to 65 cc received the full radiation dose (complete-Rx), whereas tumors measuring more than 65 cc received partial-Rx. Landmark analysis was used to assess the relationship between tumor response and overall survival (OS). Multivariable analysis was performed for RS and circulating cytokines.RESULTSIn the combined (expansion plus original) cohort, 97 patients (219 metastases) were analyzed and received SBRT+P. Forty-six (47%) patients received at least 1 partial-Rx treatment. There were 7 (7.2%)dose-limiting toxicities (DLTs). 1-year LF was 7.6% overall, and 13.3% and 5.4% for partial-Rx and complete-Rx tumors, respectively (HR 2.32, 95% CI 0.90-5.97, P = 0.08). The overall, unirradiated, and irradiated objective response rates were 22%, 12%, and 34%, respectively. Irradiated tumor response to SBRT+P was associated with prolonged OS; 1-year OS was 71% (responders), 42% (mixed-responders), and 0% (nonresponders) (P < 0.01). High-RS was significantly associated with improved LF, progression-free survival (PFS), and OS. Elevated circulating IL-8 was independently associated with inferior PFS and OS.CONCLUSIONSBRT+P is safe in patients with large, advanced solid tumors. Additional studies are warranted to assess noninferiority of complete versus partial irradiation of tumors in the setting of immunotherapy.TRIAL REGISTRATIONClinicaltrials.gov NCT02608385FUNDINGMerck Investigator Studies Program; Hillman Fellows for Innovative Cancer Research Program; NIH grants UM1CA186690-06, P50CA254865-01A1, P30CA047904-32, and R01DE031729-01A1.

Keywords:  Cancer; Clinical Trials; Immunotherapy; Oncology; Radiation therapy

DOI:  https://doi.org/10.1172/JCI162260
Nature. 2023 May 17.

Lysine catabolism reprograms tumour immunity through histone crotonylation.

Huairui Yuan, Xujia Wu, Qiulian Wu, Adam Chatoff, Emily Megill, Jinjun Gao, Tengfei Huang, Tingting Duan, Kailin Yang, Chunyu Jin, Fanen Yuan, Shuai Wang, Linjie Zhao, Pascal O Zinn, Kalil G Abdullah, Yingming Zhao, Nathaniel W Snyder, Jeremy N Rich.

Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.

DOI: https://doi.org/10.1038/s41586-023-06061-0
J Phys Chem B. 2023 May 19.

Fluorescence-Based Detection of Proteins and Their Interactions in Live Cells.

Michael R Stoneman, Valerică Raicu.

Recent advances in fluorescence-based microscopy techniques, such as single molecule fluorescence, Förster resonance energy transfer (FRET), fluorescence intensity fluctuations analysis, and super-resolution microscopy have expanded our ability to study proteins in greater detail within their native cellular environment and to investigate the roles that protein interactions play in biological functions, such as inter- and intracellular signaling and cargo transport. In this Perspective, we provide an up-to-date overview of the current state of the art in fluorescence-based detection of proteins and their interactions in living cells with an emphasis on recent developments that have facilitated the characterization of the spatial and temporal organization of proteins into oligomeric complexes in the presence and absence of natural and artificial ligands. Further advancements in this field will only deepen our understanding of the underlying mechanisms of biological processes and help develop new therapeutic targets.

DOI: https://doi.org/10.1021/acs.jpcb.3c01419