bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2024–12–22
forty-five papers selected by
Kıvanç Görgülü, Technical University of Munich
  1. Engineered extrachromosomal oncogene amplifications promote tumorigenesis.
  2. Macropinocytosis controls metabolic stress-driven CAF subtype identity in pancreatic cancer.
  3. Measuring Phospholipid Flippase Activity by NBD-Lipid Uptake in Living Yeast Cells.
  4. Beta cells are essential drivers of pancreatic ductal adenocarcinoma development.
  5. Toward clinical applications of spatial-omics in cancer research.
  6. Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization.
  7. Engineered Bacteria Manipulate Cysteine Metabolism to Boost Ferroptosis-Based Pancreatic Ductal Adenocarcinoma Therapy.
  8. HMGA2 Expression Predicts Subtype, Survival, and Treatment Outcome in Pancreatic Ductal Adenocarcinoma.
  9. A hormetic response model for glutamine stress in cancer.
  10. Monitoring Accessible Cholesterol Levels in Immune Cells.
  11. Chaperone-mediated autophagy as a modulator of aging and longevity.
  12. Theoretical framework and emerging challenges of lipid metabolism in cancer.
  13. Ponsegromab for Cancer Cachexia - A New Dawn for an Old Condition?
  14. Molecular Pathway and Immune Profile Analysis of IPMN-Derived Versus PanIN-Derived Pancreatic Ductal Adenocarcinomas.
  15. Methods for Visualizing and Quantifying Cholesterol Distribution in Mammalian Cells Using Filipin and D4 Probes.
  16. Systems Biology of the Cancer Cell.
  17. Pan-cancer analysis of biallelic inactivation in tumor suppressor genes identifies KEAP1 zygosity as a predictive biomarker in lung cancer.
  18. AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity.
  19. Functional heterogeneity of fibroblasts in primary tumors and metastases.
  20. Fatty Acid Synthase-Derived Lipid Stores Support Breast Cancer Metastasis.
  21. Orthotopic gastric cancer mouse model identifies trajectory of lymphatic metastasis.
  22. Mapping mitochondrial aging.
  23. Vitamin D and Pancreatic Ductal Adenocarcinoma-A Review of a Complicated Relationship.
  24. PRRX1 has functional roles in pancreatic acinar to ductal metaplasia and carcinogenesis.
  25. Probe my Pathway (PmP): a portal to explore the chemical coverage of the human Reactome.
  26. Olaparib and Radiotherapy Induce Type I Interferon- and CD8+ T Cell-Dependent Sensitization to Immunotherapy in Pancreatic Cancer.
  27. Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress.
  28. Biomarker Discovery for Early Detection of Pancreatic Ductal Adenocarcinoma (PDAC) Using Multiplex Proteomics Technology.
  29. Selective Labeling of Phosphatidylcholine to Track Their Translocation Between Organelles in Living Cells.
  30. An antibiotic that mediates immune destruction of senescent cancer cells.
  31. Biomolecular condensates: A new lens on cancer biology.
  32. The 2024 generation.
  33. Isolation and characterization of microbiota from human pancreatic tumors and small intestine.
  34. Using GRAM Domain-Based Biosensors to Monitor Accessible Cholesterol Distribution in Live Cells.
  35. Identification of antigen-presenting cell-T cell interactions driving immune responses to food.
  36. Selenoprotein O Promotes Melanoma Metastasis and Regulates Mitochondrial Complex II Activity.
  37. Phase I study of intratumoral injection of talimogene laherparepvec for the treatment of advanced pancreatic cancer.
  38. Mechanosensitive nuclear uptake of chemotherapy.
  39. HistoPlexer: Histopathology-based Protein Multiplex Generation using Deep Learning.
  40. Elevated Expression of Cell Adhesion, Metabolic, and Mucus Secretion Gene Clusters Associated with Tumorigenesis, Metastasis, and Poor Survival in Pancreatic Ductal Adenocarcinoma.
  41. Unraveling the causal relationship between serum minerals and pancreatic cancer: a Mendelian randomization study.
  42. Targeting PRMT1 reduces cancer persistence and tumor relapse in EGFR- and KRAS-mutant lung cancer.
  43. Target cell tension regulates macrophage trogocytosis.
  44. Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.
  45. Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.
  1. Nature. 2024 Dec 18.
    Engineered extrachromosomal oncogene amplifications promote tumorigenesis.
    Davide Pradella, Minsi Zhang, Rui Gao, Melissa A Yao, Katarzyna M Gluchowska, Ylenia Cendon-Florez, Tanmay Mishra, Gaspare La Rocca, Moritz Weigl, Ziqi Jiao, Hieu H M Nguyen, Marta Lisi, Mateusz M Ozimek, Chiara Mastroleo, Kevin Chen, Felix Grimm, Jens Luebeck, Shu Zhang, Andrea Alice Zolli, Eric G Sun, Bhargavi Dameracharla, Zhengqiao Zhao, Yuri Pritykin, Carlie Sigel, Howard Y Chang, Paul S Mischel, Vineet Bafna, Cristina R Antonescu, Andrea Ventura.
      Focal gene amplifications are among the most common cancer-associated mutations1 but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs)2 in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harbouring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumour formation in an autochthonous mouse model of hepatocellular carcinoma. These findings offer insights into the role of ecDNA-mediated gene amplifications in tumorigenesis. We anticipate that this approach will be valuable for investigating further unresolved aspects of ecDNA biology and for developing new preclinical immunocompetent mouse models of human cancers harbouring specific focal gene amplifications.
    DOI:  https://doi.org/10.1038/s41586-024-08318-8
  2. bioRxiv. 2024 Dec 05. pii: 2024.11.29.625709. [Epub ahead of print]
    Macropinocytosis controls metabolic stress-driven CAF subtype identity in pancreatic cancer.
    Yijuan Zhang, Li Ling, Swetha Maganti, Jennifer L Hope, Cheska Marie Galapate, Florent Carrette, Karen Duong-Polk, Anindya Bagchi, David A Scott, Andrew M Lowy, Linda M Bradley, Cosimo Commisso.
      Pancreatic ductal adenocarcinoma (PDAC) tumors are deficient in glutamine, an amino acid that tumor cells and CAFs use to sustain their fitness. In PDAC, both cell types stimulate macropinocytosis as an adaptive response to glutamine depletion. CAFs play a critical role in sculpting the tumor microenvironment, yet how adaptations to metabolic stress impact the stromal architecture remains elusive. In this study, we find that macropinocytosis functions to control CAF subtype identity when glutamine is limiting. Our data demonstrate that metabolic stress leads to an intrinsic inflammatory CAF (iCAF) program driven by MEK/ERK signaling. Utilizing in vivo models, we find that blocking macropinocytosis alters CAF subtypes and reorganizes the tumor stroma. Importantly, these changes in stromal architecture can be exploited to sensitize PDAC to immunotherapy and chemotherapy. Our findings demonstrate that metabolic stress plays a role in shaping the tumor microenvironment, and that this attribute can be harnessed for therapeutic impact.
    Keywords:  CAF heterogeneity; Metabolic stress; drug delivery; macropinocytosis; pancreatic cancer; stromal architecture; tumor microenvironment
    DOI:  https://doi.org/10.1101/2024.11.29.625709
  3. Methods Mol Biol. 2025 ;2888 133-145
    Measuring Phospholipid Flippase Activity by NBD-Lipid Uptake in Living Yeast Cells.
    Bhawik K Jain, Alexander J Mansueto, Todd R Graham.
      Phospholipid flippases in the P4-ATPase family are essential for establishing membrane asymmetry. These ATP-powered pumps translocate specific lipids from the exofacial leaflet to the cytosolic leaflet of the plasma membrane, thereby concentrating substrate lipids, such as phosphatidylserine, in the cytosolic leaflet while non-substrate lipids populate the exofacial leaflet. Here, we describe a method for measuring P4-ATPase transport activity in the yeast plasma membrane by using flow cytometry to quantify the uptake of lipids derivatized with a fluorescent [7-nitro-2-1,3-benzoxadiazol-4-yl)amino] (NBD) group on a short (C6) fatty acyl chain. The NBD-lipid uptake assay quantitatively measures P4-ATPase transport activity and substrate selectivity in the native membrane environment.
    Keywords:  Flippase assay; Flow cytometry; Lipid transporter; NBD-lipids; P4-ATPase; Plasma membrane
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_10
  4. bioRxiv. 2024 Dec 03. pii: 2024.11.29.626079. [Epub ahead of print]
    Beta cells are essential drivers of pancreatic ductal adenocarcinoma development.
    Cathy C Garcia, Aarthi Venkat, Daniel C McQuaid, Sherry Agabiti, Alex Tong, Rebecca L Cardone, Rebecca Starble, Akin Sogunro, Jeremy B Jacox, Christian F Ruiz, Richard G Kibbey, Smita Krishnaswamy, Mandar Deepak Muzumdar.
      Pancreatic endocrine-exocrine crosstalk plays a key role in normal physiology and disease. For instance, endocrine islet beta (β) cell secretion of insulin or cholecystokinin (CCK) promotes progression of pancreatic adenocarcinoma (PDAC), an exocrine cell-derived tumor. However, the cellular and molecular mechanisms that govern endocrine-exocrine signaling in tumorigenesis remain incompletely understood. We find that β cell ablation impedes PDAC development in mice, arguing that the endocrine pancreas is critical for exocrine tumorigenesis. Conversely, obesity induces β cell hormone dysregulation, alters CCK-dependent peri-islet exocrine cell transcriptional states, and enhances islet proximal tumor formation. Single-cell RNA-sequencing, in silico latent-space archetypal and trajectory analysis, and genetic lineage tracing in vivo reveal that obesity stimulates postnatal immature β cell expansion and adaptation towards a pro-tumorigenic CCK+ state via JNK/cJun stress-responsive signaling. These results define endocrine-exocrine signaling as a driver of PDAC development and uncover new avenues to target the endocrine pancreas to subvert exocrine tumorigenesis.
    DOI:  https://doi.org/10.1101/2024.11.29.626079
  5. Nat Cancer. 2024 Dec 17.
    Toward clinical applications of spatial-omics in cancer research.
    Cameron Walker, Michael Angelo.
      
    DOI:  https://doi.org/10.1038/s43018-024-00868-0
  6. FASEB J. 2024 Dec 13. 38(24): e70247
    Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization.
    Theressa Ewa, Neel Panchwagh, Chin-Hsien Tai, Leela Rani Avula, Sarah Joseph, Michael W Rudloff, Nargis Malik, Xianyu Zhang, Christine Alewine.
      Peritoneum is the second most common site of metastasis in patients with pancreatic ductal adenocarcinoma (PDAC). Peritoneal colonization is impaired in PDAC cells with knockout (KO) of the cancer surface antigen mesothelin (MSLN) or by introducing Y318A mutation in MSLN to prevent binding to mucin-16 (MUC-16). MSLN has a membrane-bound form but is also shed to release soluble MSLN (sMSLN). Their individual roles in peritoneal metastasis are unknown. Here, a C-terminal truncated MSLN mutant (∆591) incapable of cell membrane insertion but proficient in secretion was engineered. Expression of ∆591 MSLN failed to rescue peritoneal metastasis in MSLN KO cells and inhibited peritoneal colonization when overexpressed in WT PDAC cells. Exposing PDAC cells to conditioned medium (CM) containing excess sMSLN impaired cancer cell clustering in vitro and in peritoneal fluid in vivo, while CM containing only Y318A sMSLN did not. These data demonstrate that interaction of membrane-bound MSLN with MUC-16 promotes cell clustering that is critical for efficient peritoneal metastasis. However, peritoneal colonization by MSLN KO cells was rescued by expression of ∆591 mutant MSLN bearing Y318A mutation, suggesting that sMSLN also has a MUC-16-independent role in peritoneal spread. Alterations in inflammatory signaling pathways occurred following KO cell exposure to CM containing sMSLN, and CM from cancer cells with intact peritoneal metastasis provoked increased KO cell secretion of IL-1α. While excess sMSLN inhibits cell clustering and peritoneal colonization, sMSLN may also promote PDAC peritoneal metastasis independent of MUC-16.
    Keywords:  IL‐1α; cell clustering; mesothelin; pancreatic cancer; peritoneal metastasis
    DOI:  https://doi.org/10.1096/fj.202400446R
  7. Adv Mater. 2024 Dec 18. e2412982
    Engineered Bacteria Manipulate Cysteine Metabolism to Boost Ferroptosis-Based Pancreatic Ductal Adenocarcinoma Therapy.
    Chaoqiang Qiao, Lexuan Wang, Chuting Huang, Qian Jia, Weier Bao, Peilin Guo, Dengxu Tan, Zhuang Chen, Changhong Shi, Zhiping Rao, Ruili Zhang, Wei Wei, Zhongliang Wang.
      Cysteine metabolism is a key determinant of the defense against ferroptosis in pancreatic ductal adenocarcinoma (PDAC). Blocking cysteine metabolism may trigger potent ferroptosis in PDAC cells by generating lipid peroxides during tumor metabolic processes. However, current methods to limit cysteine availability fall short, failing to efficiently block cysteine metabolism due to inadequate tumor targeting and compensatory cysteine sources. Inspired by sulfur-metabolizing bacteria, synthetic biology to develop an engineered bacterium capable of directly depleting cysteine to block its metabolism is used. Acting as a living drug, these engineered bacteria colonize the tumor and continuously produce engineered cyst(e)inase enzyme (CGL) under the stimulation of tumor hypoxia. The CGL exhausts the substrate cysteine, completely impeding cysteine metabolism. This process dismantles the ferroptosis defense system in PDAC cells, triggers potent ferroptosis, and achieves efficient treatment. The results demonstrate that engineered bacteria designed for cysteine metabolism modulation possess unparalleled advantages in efficacy, persistence, and precision in blocking cysteine metabolism, making them highly suitable for effective ferroptosis treatment of PDAC.
    Keywords:  cysteine metabolism; engineered bacteria; ferroptosis treatment
    DOI:  https://doi.org/10.1002/adma.202412982
  8. Clin Cancer Res. 2024 Dec 16.
    HMGA2 Expression Predicts Subtype, Survival, and Treatment Outcome in Pancreatic Ductal Adenocarcinoma.
    Naomi Yamamoto, Stephanie Dobersch, Ian Loveless, Annie N Samraj, Gun Ho Jang, Miki Haraguchi, Liang-I Kang, Marianna B Ruzinova, Kiran R Vij, Jacqueline L Mudd, Thomas Walsh, Rachael A Safyan, E Gabriela Chiorean, Sunil R Hingorani, Nathan M Bolton, Li Li, Ryan C Fields, David G DeNardo, Faiyaz Notta, Howard C Crawford, Nina G Steele, Sita Kugel.
       PURPOSE: To establish HMGA2 as a marker of basal-like disease in pancreatic ductal adenocarcinoma (PDAC) and explore its use as a biomarker for prognosis and treatment resistance.
    EXPERIMENTAL DESIGN: We identified high expression of HMGA2 in basal PDAC cells in a scRNAseq Atlas of 172 patient samples. We then analyzed HMGA2 expression, along with expression of the classical marker GATA6, in a cohort of 580 PDAC samples with multiplex immunohistochemistry. We further supplemented these data with an additional 30 diverse patient samples and multiple independent single-cell RNAseq databases.
    RESULTS: We found that expression of HMGA2, but not previously described basal markers CK5 or CK17, predicted overall survival in our cohort. Combining HMGA2 and GATA6 status allowed for identification of two key study groups: an HMGA2+/GATA6- cohort with worse survival, low tumor-infiltrating CD8+ T cells, increased FAP+ fibroblasts, and poorer response to gemcitabine-based chemotherapies (n=94, median survival=11.2 months post-surgery); and an HMGA2-/GATA6+ cohort with improved survival, increased CD8+ T-cell infiltrate, decreased FAP+ fibroblasts, and improved survival with gemcitabine-based chemotherapy (n=198, median survival=21.7 months post-surgery). HMGA2 was also prognostic for overall survival in RNA sequencing from an independent cohort.
    CONCLUSIONS: IHC stratification of primary tumors by HMGA2 and GATA6 status in pancreatic cancer is associated with differential outcomes, survival following chemotherapy, and tumor microenvironments. As a nuclear marker for basal disease, HMGA2 complements GATA6 to identify disease subtypes in PDAC.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-24-2200
  9. Trends Cancer. 2024 Dec 16. pii: S2405-8033(24)00274-7. [Epub ahead of print]
    A hormetic response model for glutamine stress in cancer.
    Shea F Grenier, Cosimo Commisso.
      Glutamine metabolism supports the development and progression of many cancers and is considered a therapeutic target. Attempts to inhibit glutamine metabolism have resulted in limited success and have not translated into clinical benefit. The outcomes of these clinical studies, along with preclinical investigations, suggest that cellular stress responses to glutamine deprivation or targeting may be modeled as a biphasic hormetic response. By recognizing the multifaceted aspects of glutamine metabolism inhibition within a more comprehensive biological framework, the adoption of this model may guide future fundamental and translational studies. To achieve clinical efficacy, we posit that as a field we will need to anticipate the hormetic effects of glutamine stress and consider how best to co-target cancer cell adaptive mechanisms.
    Keywords:  cancer; glutamine stress; hormesis; metabolism; targeted therapies; therapeutics
    DOI:  https://doi.org/10.1016/j.trecan.2024.11.008
  10. Methods Mol Biol. 2025 ;2888 83-99
    Monitoring Accessible Cholesterol Levels in Immune Cells.
    Duo H Ma, Neal M Alto, Arun Radhakrishnan.
      Cholesterol is a critical lipid that is present at high concentrations in the plasma membranes of animal cells. Most of the membrane cholesterol is sequestered by other membrane lipids and the transmembrane domains of proteins. Cholesterol in excess of such sequestration forms a pool that is referred to as "accessible cholesterol." This pool of cholesterol plays a crucial role in maintaining lipid homeostasis and in controlling cell growth. The accessible cholesterol pool can also be exploited by bacteria and viruses to promote infection and host immune responses rapidly lower levels of this pool to confer protection. We had previously developed a bacterial toxin sensor called ALOD4 to monitor and quantify accessible cholesterol in cultured cells. Here, we report the characterization of a modified version of ALOD4 that is specialized to detect and monitor accessible cholesterol levels in primary immune cells by flow cytometry analysis.
    Keywords:  Anthrolysin O; Cholesterol pools; ER; Plasma membrane; Splenocytes
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_7
  11. Front Aging. 2024 ;5 1509400
    Chaperone-mediated autophagy as a modulator of aging and longevity.
    S Joseph Endicott.
      Chaperone-mediated autophagy (CMA) is the lysosomal degradation of individually selected proteins, independent of vesicle fusion. CMA is a central part of the proteostasis network in vertebrate cells. However, CMA is also a negative regulator of anabolism, and it degrades enzymes required for glycolysis, de novo lipogenesis, and translation at the cytoplasmic ribosome. Recently, CMA has gained attention as a possible modulator of rodent aging. Two mechanistic models have been proposed to explain the relationship between CMA and aging in mice. Both of these models are backed by experimental data, and they are not mutually exclusionary. Model 1, the "Longevity Model," states that lifespan-extending interventions that decrease signaling through the INS/IGF1 signaling axis also increase CMA, which degrades (and thereby reduces the abundance of) several proteins that negatively regulate vertebrate lifespan, such as MYC, NLRP3, ACLY, and ACSS2. Therefore, enhanced CMA, in early and midlife, is hypothesized to slow the aging process. Model 2, the "Aging Model," states that changes in lysosomal membrane dynamics with age lead to age-related losses in the essential CMA component LAMP2A, which in turn reduces CMA, contributes to age-related proteostasis collapse, and leads to overaccumulation of proteins that contribute to age-related diseases, such as Alzheimer's disease, Parkinson's disease, cancer, atherosclerosis, and sterile inflammation. The objective of this review paper is to comprehensively describe the data in support of both of these explanatory models, and to discuss the strengths and limitations of each.
    Keywords:  aging; autophagy; chaperone-mediated autophagy; longevity; metabolism
    DOI:  https://doi.org/10.3389/fragi.2024.1509400
  12. Semin Cancer Biol. 2024 Dec 12. pii: S1044-579X(24)00096-8. [Epub ahead of print]108 48-70
    Theoretical framework and emerging challenges of lipid metabolism in cancer.
    Qiuying Gu, Yuan Wang, Ping Yi, Chunming Cheng.
      Elevated lipid metabolism is one of hallmarks of malignant tumors. Lipids not only serve as essential structural components of biological membranes but also provide energy and substrates for the proliferation of cancer cells and tumor growth. Cancer cells meet their lipid needs by coordinating the processes of lipid absorption, synthesis, transport, storage, and catabolism. As research in this area continues to deepen, numerous new discoveries have emerged, making it crucial for scientists to stay informed about the developments of cancer lipid metabolism. In this review, we first discuss relevant concepts and theories or assumptions that help us understand the lipid metabolism and -based cancer therapies. We then systematically summarize the latest advancements in lipid metabolism including new mechanisms, novel targets, and up-to-date pre-clinical and clinical investigations of anti-cancer treatment with lipid metabolism targeted drugs. Finally, we emphasize emerging research directions and therapeutic strategies, and discuss future prospective and emerging challenges. This review aims to provide the latest insights and guidance for research in the field of cancer lipid metabolism.
    Keywords:  Cancer; Cancer therapy; Cancer treatment; Cholesterol; Fatty acids; Inhibitors; Lipid metabolism; Lipid synthesis; Theoretical framework; Tumor metabolism
    DOI:  https://doi.org/10.1016/j.semcancer.2024.12.002
  13. N Engl J Med. 2024 Dec 19. 391(24): 2371-2373
    Ponsegromab for Cancer Cachexia - A New Dawn for an Old Condition?
    Barry J A Laird, Richard J E Skipworth.
      
    DOI:  https://doi.org/10.1056/NEJMe2411741
  14. Int J Mol Sci. 2024 Dec 07. pii: 13164. [Epub ahead of print]25(23):
    Molecular Pathway and Immune Profile Analysis of IPMN-Derived Versus PanIN-Derived Pancreatic Ductal Adenocarcinomas.
    Margaret A Park, Kristyn Gumpper-Fedus, Somashekar G Krishna, Maria C Genilo-Delgado, Stephen Brantley, Phil A Hart, Mary E Dillhoff, Maria F Gomez, Toni L Basinski, Shaffer R Mok, Anjuli K Luthra, Jason B Fleming, Amir Mohammadi, Barbara A Centeno, Kun Jiang, Aleksandra Karolak, Daniel Jeong, Dung-Tsa Chen, Paul A Stewart, Jamie K Teer, Zobeida Cruz-Monserrate, Jennifer B Permuth.
      Intraductal papillary mucinous neoplasms (IPMN) are commonly detected pancreatic cysts that may transform into pancreatic ductal adenocarcinoma (PDAC). Predicting which IPMNs will progress to PDAC remains a clinical challenge. Moreover, identifying those clinically evident IPMNs for which a surveillance approach is best is a dire clinical need. Therefore, we aimed to identify molecular signatures that distinguished between PDAC with and without clinical evidence of an IPMN to identify novel molecular pathways related to IPMN-derived PDAC that could help guide biomarker development. Data from the Oncology Research Information Exchange Network (ORIEN) multi-institute sequencing project were utilized to analyze 66 PDAC cases from Moffitt Cancer Center and The Ohio State University Wexner Medical Center, for which tumor whole transcriptome sequencing datasets were generated. Cases were classified based on whether a tumor had originated from an IPMN (n = 16) or presumably through the pancreatic intraepithelial neoplasia (PanIN) pathway (n = 50). We then performed differential expression and pathway analysis using Gene-Set Enrichment Analysis (GSEA) and Pathway Analysis with Down-weighted Genes (PADOG) algorithms. We also analyzed immune profiles using the Tumor-Immune Microenvironment Deconvolution web portal for Bulk Transcriptomics (TIMEx). Both GSEA and TIMEx indicate that PanIN-derived PDAC tumors enrich inflammatory pathways (complement, hedgehog signaling, coagulation, inflammatory response, apical surface, IL-2/STAT5, IL-6/STAT3, EMT, KRAS signaling, apical junction, IFN-gamma, allograft rejection) and are comparatively richer in almost all immune cell types than those from IPMN-derived PDAC. IPMN-derived tumors were enriched for metabolic and energy-generating pathways (oxidative phosphorylation, unfolded protein response, pancreas beta cells, adipogenesis, fatty acid metabolism, protein secretion), and the most significantly upregulated genes (padj < 0.001) included mucin 2 (MUC2) and gastrokine-2 (GKN2). Further, the metabolic-linked gene signature enriched in the IPMN-derived samples is associated with a cluster of early-stage and long-survival (top 4th quartile) PDAC cases from The Cancer Genome Atlas (TCGA) expression database. Our data suggest that IPMN-derived and PanIN-derived PDACs differ in the expression of immune profiles and metabolic pathways. These initial findings warrant validation and follow-up to develop biomarker-based strategies for early PDAC detection and treatment.
    Keywords:  IPMN; PanIN; pancreatic ductal adenocarcinoma; transcriptomics
    DOI:  https://doi.org/10.3390/ijms252313164
  15. Methods Mol Biol. 2025 ;2888 101-118
    Methods for Visualizing and Quantifying Cholesterol Distribution in Mammalian Cells Using Filipin and D4 Probes.
    Julie Eichler, Sophie Huver, Céline J Knorr, Corinne Wendling, Toshihide Kobayashi, Catherine Tomasetto, Fabien Alpy.
      Cholesterol is a key component of biological membranes and, like many cellular lipids, is unevenly distributed among organelles. Disruptions in cholesterol trafficking are associated with various pathologies, including lysosomal lipid storage disorders, often characterized by intracellular cholesterol accumulation. A significant challenge in studying cholesterol trafficking is the lack of easy methods to trace this molecule in situ. Fluorescent probes that specifically bind cholesterol have enabled the visualization and imaging of cholesterol distribution within cells. This chapter details optimized methods for visualizing and quantifying free cholesterol at the plasma membrane and intracellulaly, both in individual cells and in large cell populations. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to monomeric EGFP (mEGFP-D4 and the more sensitive mutant mEGFP-D4H) and the polyene macrolide filipin. We describe robust methods for quantifying plasma membrane cholesterol by flow cytometry and to visualize intracellular cholesterol pools by light microscopy. Furthermore, we introduce a refined filipin staining protocol that enhances intracellular cholesterol detection. For precise quantification, we developed an automated image analysis pipeline. This chapter provides a comprehensive guide for staining and quantifying cellular cholesterol, offering valuable tools for studying cholesterol dynamics in mammalian cells.
    Keywords:  Cholesterol; D4 probe; Filipin; Late endosome; Lysosome; Perfringolysin O; Plasma membrane
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_8
  16. Annu Rev Biomed Eng. 2024 Dec 17.
    Systems Biology of the Cancer Cell.
    Kevin A Janes, Matthew J Lazzara.
      Questions in cancer have engaged systems biologists for decades. During that time, the quantity of molecular data has exploded, but the need for abstractions, formal models, and simplifying insights has remained the same. This review brings together classic breakthroughs and recent findings in the field of cancer systems biology, focusing on cancer-cell pathways for tumorigenesis and therapeutic response. Cancer cells mutate and transduce information from their environment to alter gene expression, metabolism, and phenotypic states. Understanding the molecular architectures that make each of these steps possible is a long-term goal of cancer systems biology pursued by iterating between quantitative models and experiments. We argue that such iteration is the best path to deploying targeted therapies intelligently so that each patient receives the maximum benefit for their cancer.
    DOI:  https://doi.org/10.1146/annurev-bioeng-103122-030552
  17. Cell. 2024 Dec 12. pii: S0092-8674(24)01327-8. [Epub ahead of print]
    Pan-cancer analysis of biallelic inactivation in tumor suppressor genes identifies KEAP1 zygosity as a predictive biomarker in lung cancer.
    Mark Zucker, Maria A Perry, Samuel I Gould, Arielle Elkrief, Anton Safonov, Rohit Thummalapalli, Miika Mehine, Debyani Chakravarty, A Rose Brannon, Marc Ladanyi, Pedram Razavi, Mark T A Donoghue, Yonina R Murciano-Goroff, Kristiana Grigoriadis, Nicholas McGranahan, Mariam Jamal-Hanjani, Charles Swanton, Yuan Chen, Ronglai Shen, Sarat Chandarlapaty, David B Solit, Nikolaus Schultz, Michael F Berger, Jason Chang, Adam J Schoenfeld, Francisco J Sánchez-Rivera, Ed Reznik, Chaitanya Bandlamudi.
      The canonical model of tumor suppressor gene (TSG)-mediated oncogenesis posits that loss of both alleles is necessary for inactivation. Here, through allele-specific analysis of sequencing data from 48,179 cancer patients, we define the prevalence, selective pressure for, and functional consequences of biallelic inactivation across TSGs. TSGs largely assort into distinct classes associated with either pan-cancer (Class 1) or lineage-specific (Class 2) patterns of selection for biallelic loss, although some TSGs are predominantly monoallelically inactivated (Class 3/4). We demonstrate that selection for biallelic inactivation can be utilized to identify driver genes in non-canonical contexts, including among variants of unknown significance (VUSs) of several TSGs such as KEAP1. Genomic, functional, and clinical data collectively indicate that KEAP1 VUSs phenocopy established KEAP1 oncogenic alleles and that zygosity, rather than variant classification, is predictive of therapeutic response. TSG zygosity is therefore a fundamental determinant of disease etiology and therapeutic sensitivity.
    Keywords:  KEAP1; Knudson's two-hit; biallelic inactivation; biallelic loss; cancer genomics; clinical sequencing; lung cancer; pancancer; predictive biomarkers; tumor suppressor genes
    DOI:  https://doi.org/10.1016/j.cell.2024.11.010
  18. Autophagy. 2024 Dec 15. 1-21
    AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity.
    Louise Pierre, Florian Juszczak, Valentine Delmotte, Morgane Decarnoncle, Benjamin Ledoux, Laurent Bultot, Luc Bertrand, Marielle Boonen, Patricia Renard, Thierry Arnould, Anne-Emilie Declèves.
      Renal proximal tubules are a primary site of injury in metabolic diseases. In obese patients and animal models, proximal tubular epithelial cells (PTECs) display dysregulated lipid metabolism, organelle dysfunctions, and oxidative stress that contribute to interstitial inflammation, fibrosis and ultimately end-stage renal failure. Our research group previously pointed out AMP-activated protein kinase (AMPK) decline as a driver of obesity-induced renal disease. Because PTECs display high macroautophagic/autophagic activity and rely heavily on their endo-lysosomal system, we investigated the effect of lipid stress on autophagic flux and lysosomes in these cells. Using a model of highly differentiated primary PTECs challenged with palmitate, our data placed lysosomes at the cornerstone of the lipotoxic phenotype. As soon as 6 h after palmitate exposure, cells displayed impaired lysosomal acidification subsequently leading to autophagosome accumulation and activation of lysosomal biogenesis. We also showed the inability of lysosomal quality control to restore acidic pH which finally drove PTECs dedifferentiation. When palmitate-induced AMPK activity decline was prevented by AMPK activators, lysosomal acidification and the differentiation profile of PTECs were preserved. Our work provided key insights on the importance of lysosomes in PTECs homeostasis and lipotoxicity and demonstrated the potential of AMPK in protecting the organelle from lipid stress.Abbreviation: ACAC: acetyl-CoA carboxylase; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK: AMP-activated protein kinase; APQ1: aquaporin 1 (Colton blood group); BSA: bovine serum albumin; CDH16: cadherin 16; CKD: chronic kidney disease; CTSB: cathepsin B; CTSD: cathepsin D; EPB41L5: erythrocyte membrane protein band 4.1 like 5; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EMT: epithelial-to-mesenchymal transition; FA: fatty acid; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; GUSB: glucuronidase beta; HEXB: hexosaminidase subunit beta; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; LMP: lysosomal membrane permeabilization; LRP2: LDL receptor related protein 2; LSD: lysosomal storage disorder; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TRP cation channel 1; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MmPTECs: Mus musculus (mouse) proximal tubular epithelial cells; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleate; PA: palmitate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PTs: proximal tubules; PTECs: proximal tubular epithelial cells; PRKAA: protein kinase AMP-activated catalytic subunit alpha; RFP: red fluorescent protein; RPS6KB: ribosomal protein S6 kinase B; SLC5A2: solute carrier family 5 member 2; SOX9: SRY-box transcription factor 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; VIM: vimentin.
    Keywords:  AMPK; Autophagy; chronic kidney disease; lipid accumulation; obesity; proximal tubules
    DOI:  https://doi.org/10.1080/15548627.2024.2435238
  19. Trends Cancer. 2024 Dec 13. pii: S2405-8033(24)00260-7. [Epub ahead of print]
    Functional heterogeneity of fibroblasts in primary tumors and metastases.
    Priscilla S W Cheng, Marta Zaccaria, Giulia Biffi.
      Cancer-associated fibroblasts (CAFs) are abundant components of the tumor microenvironment (TME) of most solid malignancies and have emerged as key regulators of cancer progression and therapy response. Although recent technological advances have uncovered substantial CAF molecular heterogeneity at the single-cell level, defining functional roles for most described CAF populations remains challenging. With the aim of bridging CAF molecular and functional heterogeneity, this review focuses on recently identified functional interactions of CAF subtypes with malignant cells, immune cells, and other stromal cells in primary tumors and metastases. Dissecting the heterogeneous functional crosstalk of specific CAF populations with other components is starting to uncover candidate combinatorial strategies for therapeutically targeting the TME and cancer progression.
    Keywords:  cancer-associated fibroblasts; fibroblast heterogeneity; metastases; primary tumors; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.trecan.2024.11.005
  20. Res Sq. 2024 Dec 05. pii: rs.3.rs-5510550. [Epub ahead of print]
    Fatty Acid Synthase-Derived Lipid Stores Support Breast Cancer Metastasis.
    Chaylen Andolino, Eylem Kulkoyluoglu Cotul, Zilin Xianyu, Yun Li, Divya Bhat, Mitchell Ayers, Kimberly K Buhman, Stephen D Hursting, Michael K Wendt, Dorothy Teegarden.
      Lipid accumulation is associated with breast cancer metastasis. However, the mechanisms underlying how breast cancer cells increase lipid stores and their functional role in disease progression remain incompletely understood. Herein we quantified changes in lipid metabolism and characterized cytoplasmic lipid droplets in metastatic versus non-metastatic breast cancer cells. 14 C-labeled palmitate was used to determine differences in fatty acid (FA) uptake and oxidation. Despite similar levels of palmitate uptake, metastatic cells increase lipid accumulation and oxidation of endogenous FAs compared to non-metastatic cells. Isotope tracing also demonstrated that metastatic cells support increased de novo lipogenesis by converting higher levels of glutamine and glucose into the FA precursor, citrate. Consistent with this, metastatic cells displayed increased levels of fatty acid synthase (FASN) and de novo lipogenesis. Genetic depletion or pharmacologic inhibition of FASN reduced cell migration, survival in anoikis assays, and in vivo metastasis. Finally, global proteomic analysis indicated that proteins involved in proteasome function, mitotic cell cycle, and intracellular protein transport were reduced following FASN inhibition of metastatic cells. Overall, these studies demonstrate that breast cancer metastases accumulate FAs by increasing de novo lipogenesis, storing TAG as cytoplasmic lipid droplets, and catabolizing these stores to drive several FAO-dependent steps in metastasis.
    DOI:  https://doi.org/10.21203/rs.3.rs-5510550/v1
  21. JCI Insight. 2024 Dec 17. pii: e186565. [Epub ahead of print]
    Orthotopic gastric cancer mouse model identifies trajectory of lymphatic metastasis.
    Huafeng Fu, Qinbo Cai, Zhijun Zhou, Yulong He, Min Li, DongJie Yang.
      
    Keywords:  Cancer; Gastric cancer; Gastroenterology; Mouse models; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.186565
  22. Elife. 2024 Dec 20. pii: e105191. [Epub ahead of print]13
    Mapping mitochondrial aging.
    Alessandro Bitto.
      Measuring mitochondrial respiration in frozen tissue samples provides the first comprehensive atlas of how aging affects mitochondrial function in mice.
    Keywords:  aging; cellular respiration; computational biology; mitochondria; mouse; respiration atlas; sex; systems biology
    DOI:  https://doi.org/10.7554/eLife.105191
  23. Nutrients. 2024 Nov 27. pii: 4085. [Epub ahead of print]16(23):
    Vitamin D and Pancreatic Ductal Adenocarcinoma-A Review of a Complicated Relationship.
    Iustina Grosu, Alexandru Constantinescu, Mihaela Daniela Balta, Ovidiu Băjenaru, Cătălina Nuța, Christopher Pavel, Vasile Sandru, Mihai Munteanu, Octavian Andronic.
       INTRODUCTION: From the observation of a negative relationship between UV-B exposure and cancer rates, we hypothesized that vitamin D (VD) may play a protective role in oncogenesis. Moreover, repurposing a well-known and relatively safe drug for conditions with dismal prospects, such as pancreatic ductal adenocarcinoma (PDAC), is a tempting idea. Thus, we aimed to summarize the current knowledge regarding the role of VD in the prevention and treatment of PDAC.
    METHODS: We conducted a systematic review of VD and PDAC using Medline-indexed studies accessed through PubMed as the primary data source. This study aimed to identify articles focusing on the role of VD as a risk and prognostic factor for PDAC, mechanistic studies evaluating the effects of VD or vitamin D analogs (VDAs) in PDAC models, and clinical trials on VDAs in PDAC. After the screening, 97 studies were included in the final manuscript.
    CONCLUSION: Even though the results from epidemiologic studies were contradictory, basic research has demonstrated that VD can act on PDAC cells either directly, inhibiting proliferation, apoptosis, EMT, migration, invasion, and stemness, or indirectly, through stromal remodeling. A better understanding of the consequences of VD-induced tumor-stroma cross-talk alterations is needed to determine whether VD/VDAs can be used to our own advantage in the treatment of PDAC.
    Keywords:  cancer associated fibroblasts; epithelial-to-mesenchymal transition; pancreatic ductal adenocarcinoma; stromal remodeling; tumor microenvironment; vitamin D; vitamin D receptor
    DOI:  https://doi.org/10.3390/nu16234085
  24. Cell Mol Gastroenterol Hepatol. 2024 Dec 16. pii: S2352-345X(24)00197-8. [Epub ahead of print] 101442
    PRRX1 has functional roles in pancreatic acinar to ductal metaplasia and carcinogenesis.
    Noriyuki Nishiwaki, Kensuke Sugiura, Kensuke Suzuki, Alina L Li, Constanza Tapia Contreras, Gizem Efe, Alice E Shin, Dorsay Sadeghian, Jun Zhao, Anirban Maitra, Jason R Pitarresi, Peter A Sims, Rohit Chandwani, Anil K Rustgi.
      
    Keywords:  PRRX1; acinar; acinar-ductal metaplasia; ductal; pancreas
    DOI:  https://doi.org/10.1016/j.jcmgh.2024.101442
  25. Database (Oxford). 2024 Dec 05. pii: baae116. [Epub ahead of print]2024
    Probe my Pathway (PmP): a portal to explore the chemical coverage of the human Reactome.
    Haejin Angela Kwak, Lihua Liu, Matthieu Schapira.
      Deciphering pathway-phenotype associations is critical for a system-wide understanding of cells and the chemistry of life. An approach to reach this goal is to systematically modulate pathways pharmacologically. The targeted and controlled regulation of an increasing number of proteins is becoming possible, thanks to the growing list of chemical probes and chemogenomic compounds available to cell biologists, but no resource is available that directly maps these chemical tools on cellular pathways. To fill this gap, we developed Probe my Pathway (PmP), a database where high-quality chemical probes and well-characterized sets of chemogenomic compounds are mapped on all the human pathways of the Reactome database. The web interface allows users to browse the data via icicle charts or search the data for compounds, proteins, or pathways. Chemists can rapidly find pathways with low chemical coverage or explore the structural chemistry of ligands targeting specific cellular machineries. Cell biologists can look for chemical probes targeting different proteins in the same pathway or find which pathways are targeted by chemical probes of interest. PmP is updated annually and will grow with the expanding chemical tool kit produced by Target 2035 and other efforts. Database URL: https://apps.thesgc.org/pmp/.
    DOI:  https://doi.org/10.1093/database/baae116
  26. Mol Cancer Ther. 2024 Dec 17. OF1-OF16
    Olaparib and Radiotherapy Induce Type I Interferon- and CD8+ T Cell-Dependent Sensitization to Immunotherapy in Pancreatic Cancer.
    Victoria M Valvo, Qiang Zhang, Long Jiang, Erin A Holcomb, Ashley N Pearson, Anna G Edmunds, Hailey G Faulkner, Jadyn G James, Akshay Tate, Amanda K Huber, Zhuwen Wang, Yupei Guo, David Karnak, Leslie A Parsels, Joshua D Parsels, Yu L Lei, Alnawaz Rehemtulla, Heng Lin, Eileen S Carpenter, Daniel R Wahl, Vaibhav Sahai, Theodore S Lawrence, Michael D Green, Meredith A Morgan.
      PARP inhibitors sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation by inducing DNA damage and replication stress. These mechanisms also have the potential to enhance radiation-induced type I interferon (T1IFN)-mediated antitumoral immune responses. We hypothesized that the PARP inhibitor olaparib would also potentiate radiation-induced T1IFN to promote antitumor immune responses and sensitization of otherwise resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and sensitivity to αPD-L1 immunotherapy, as well as on the tumor microenvironment by single-cell RNA sequencing. We found that olaparib enhanced T1IFN production after radiation and had superior therapeutic efficacy in immunocompetent models. Olaparib and radiation treatment sensitized PDAC tumors to αPD-L1, resulting in decreased tumor burden and a 33% complete response rate. Combination treatment provided durable immune responses as shown by tumor rejection upon tumor rechallenge of previously cured mice. Furthermore, single-cell RNA sequencing analysis revealed that combination treatment induced an immunogenic tumor microenvironment characterized by interferon (IFN) responses in both PDAC and myeloid cell populations, macrophage polarization, and increased CD8+ terminal effector T-cell frequency and activity, findings which were confirmed by IHC and flow cytometry. Furthermore, CD8+ T cells and T1IFN signaling were required for therapeutic efficacy as host depletion of CD8+ T cells or the T1IFN receptor diminished treatment responses. Overall, our results indicate that olaparib enhances radiation-induced T1IFN-mediated immune signaling and subsequently an adaptive immune response, thus sensitizing pancreatic cancer to αPD-L1 therapy, supporting an ongoing clinical trial of this therapy in patients with PDAC.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-24-0210
  27. Elife. 2024 Dec 19. pii: RP96284. [Epub ahead of print]13
    Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress.
    Showkat Ahmad Dar, Sulochan Malla, Vlastimil Martinek, Matthew John Payea, Christopher Tai-Yi Lee, Jessica Martin, Aditya Jignesh Khandeshi, Jennifer L Martindale, Cedric Belair, Manolis Maragkakis.
      Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.
    Keywords:  RNA decay; cell biology; cell line; genetics; genomics; human; mouse; stress response
    DOI:  https://doi.org/10.7554/eLife.96284
  28. J Proteome Res. 2024 Dec 19.
    Biomarker Discovery for Early Detection of Pancreatic Ductal Adenocarcinoma (PDAC) Using Multiplex Proteomics Technology.
    Alcibiade Athanasiou, Natasha Kureshi, Anja Wittig, Maria Sterner, Ramy Huber, Norma A Palma, Thomas King, Ralph Schiess.
      Early detection of pancreatic ductal adenocarcinoma (PDAC) can improve survival but is hampered by the absence of early disease symptoms. Imaging remains key for surveillance but is cumbersome and may lack sensitivity to detect small tumors. CA19-9, the only FDA-approved blood biomarker for PDAC, is insufficiently sensitive and specific to be recommended for surveillance. We aimed to discover a blood-based protein signature to improve PDAC detection in our main target population consisting of stage I or II PDAC patients (n = 75) and various controls including healthy controls (n = 50), individuals at high risk (genetic and familial) for PDAC (n = 47), or those under surveillance for an intraductal papillary mucinous neoplasm (n = 36). Roughly 3000 proteins were measured using Olink multiplex technology and conventional immunoassays. Machine learning combined biomarker candidates into 4- to 6-plex signatures. These signatures significantly (p < 0.001) outperformed CA19-9 with 84% sensitivity at 95% specificity, compared to CA19-9's sensitivity of 53% in the target population. Exploratory analysis was performed in new-onset diabetes (n = 81) and chronic pancreatitis (n = 50) patients. In conclusion, 41 promising biomarker candidates across multiple signatures were identified using proteomics technology and will be further tested in an independent cohort.
    Keywords:  Olink; PDAC; pancreatic cancer; protein biomarker; quantitative proteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00752
  29. Methods Mol Biol. 2025 ;2888 1-11
    Selective Labeling of Phosphatidylcholine to Track Their Translocation Between Organelles in Living Cells.
    Tomonori Tamura, Itaru Hamachi.
      Phosphatidylcholine (PC) is a major phospholipid that forms biological membranes in eukaryotes. PC is mainly synthesized in the endoplasmic reticulum (ER) and Golgi apparatus and then transported to other organelle membranes via multiple mechanisms. Such interorganelle lipid transport is thought to play an important role in the maintenance of cell morphology, organelle functions, and homeostasis, though the details of this process have not yet been well investigated. This chapter describes a technology for the selective labeling and fluorescence imaging of PC in target organelles. This approach involves the metabolic incorporation of azidocholine, followed by a spatially restricted bioorthogonal click reaction that enables the visualization and quantitative analysis of interorganelle PC transport in live cells using confocal microscopy.
    Keywords:  Endoplasmic reticulum; Interorganelle lipid transport; Live-cell imaging; Mitochondria; Organelle-selective click reaction; Phosphatidylcholine
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_1
  30. Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2417724121
    An antibiotic that mediates immune destruction of senescent cancer cells.
    Gabriele Casagrande Raffi, Jian Chen, Xuezhao Feng, Zhen Chen, Cor Lieftink, Shuang Deng, Jinzhe Mo, Chuting Zeng, Marit Steur, Jing Wang, Onno B Bleijerveld, Liesbeth Hoekman, Nicole van der Wel, Feng Wang, Roderick Beijersbergen, Jian Zheng, Rene Bernards, Liqin Wang.
      Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).
    Keywords:  pyroptosis; senescence; senolytics
    DOI:  https://doi.org/10.1073/pnas.2417724121
  31. Biochim Biophys Acta Rev Cancer. 2024 Dec 13. pii: S0304-419X(24)00176-8. [Epub ahead of print]1880(1): 189245
    Biomolecular condensates: A new lens on cancer biology.
    Lifei Jiang, Yibin Kang.
      Cells are compartmentalized into different organelles to ensure precise spatial temporal control and efficient operation of cellular processes. Membraneless organelles, also known as biomolecular condensates, are emerging as previously underappreciated ways of organizing cellular functions. Condensates allow local concentration of protein, RNA, or DNA molecules with shared functions, thus facilitating spatiotemporal control of biochemical reactions spanning a range of cellular processes. Studies discussed herein have shown that aberrant formation of condensates is associated with various diseases such as cancers. Here, we summarize how condensates mechanistically contribute to malignancy-related cellular processes, including genomic instability, epigenetic rewiring, oncogenic transcriptional activation, and signaling. An improved understanding of condensate formation and dissolution will enable development of new cancer therapies. Finally, we address the remaining challenges in the field and suggest future efforts to better integrate condensates into cancer research.
    Keywords:  Biomolecular condensates; Cancer; Cell biology; Phase separation; Signaling
    DOI:  https://doi.org/10.1016/j.bbcan.2024.189245
  32. Nat Cancer. 2024 Dec 17.
    The 2024 generation.
    Shruthy Suresh Aggarwal, Cristina Andreani, Zihou Deng, Julia Frede, Polina Kameneva, Marta Kovatcheva, Goran Micevic, Fay Nicolson, Jens Puschhof, Morgan Roberts, Gang Wang, Dionysios C Watson.
      
    DOI:  https://doi.org/10.1038/s43018-024-00866-2
  33. bioRxiv. 2024 Dec 03. pii: 2024.11.27.625762. [Epub ahead of print]
    Isolation and characterization of microbiota from human pancreatic tumors and small intestine.
    Dominik Awad, Holly Attebury, Ryan Hong, Kwi Kim, Li Zhang, Allison Bischoff, Aaron deDekker, Matthew Hoostal, Joseph A Nieto Carrion, Noah S Nelson, Chris Strayhorn, Timothy Frankel, Marina Pasca di Magliano, Costas A Lyssiotis, Thomas M Schmidt, Donnele Daley.
      Pancreatic ductal adenocarcinoma has a unique tumor microbiome and the systemic depletion of bacteria or fungi using antibiotic/antifungal cocktails leads to a decrease in pancreatic tumor burden in mice. However, functional studies remain rare due to the limited availability of clinically relevant microbiota. Here, we describe in detail the isolation of bacteria and fungi from the small intestine and tumor of pancreatic cancer patients at the Rogel Cancer Center. We then further characterized the impact of a newly isolated Klebsiella oxytoca strain ( UMKO1 ) on the pancreatic tumor microenvironment using bacterial genome sequencing, untargeted and targeted metabolomics, as well as an ex vivo tumor transplant system. We found that UMKO1 possesses a gene for the long form of cytidine deaminase, which can inactivate the standard PDAC chemotherapeutic agent gemcitabine. In addition, we found that UMKO1 can produce several indoles when grown in tumor-like conditions, metabolites that can lead to an immune suppressive environment and interfere with therapy outcome. To test this in detail, we assessed changes in immune populations in pancreatic tumor explants upon exposure to the supernatant of UMKO1 and other isolated bacteria grown in tumor Interstitial fluid media (TIFM). We found that while none of the bacterial supernatants changed the abundance of CD8 T cells, granzyme B positive CD8 T cells were the lowest in tumor explants exposed to UMKO1 , and not other isolated Klebsiella species or the non-pathogenic laboratory strain E. coli K12 . In summary, the isolated collection of bacteria and fungi from this study are a valuable toolbox to study the impact of microbiota on pancreatic cancer.
    DOI:  https://doi.org/10.1101/2024.11.27.625762
  34. Methods Mol Biol. 2025 ;2888 67-82
    Using GRAM Domain-Based Biosensors to Monitor Accessible Cholesterol Distribution in Live Cells.
    Tomoki Naito, Minyoung Na, Dylan Hong Zheng Koh, Yasunori Saheki.
      Cholesterol is a vital component of cellular membranes, playing a crucial role in membrane integrity and cell signaling. It is unevenly distributed among different cellular compartments, with the highest enrichment in the plasma membrane (PM). Dysregulation of cellular cholesterol distribution is linked to various disorders, including cardiovascular and neurological diseases. Regulated transport of a specific pool of cholesterol, known as accessible cholesterol, is essential for maintaining cellular cholesterol distribution, yet visualization tools for accessible cholesterol in live cells are limited. Here, we introduce methods for visualizing accessible cholesterol using biosensors based on the GRAM domain of GRAMD1s/Asters (GRAMD1a, GRAMD1b, and GRAMD1c). We present two variants of the GRAM domain of GRAMD1b, GRAM-H, and GRAM-W, with different affinities for cholesterol, enabling the detection of both increase and decrease of accessible cholesterol in the PM. Additionally, we demonstrate the successful coupling of dimerization-dependent fluorescent protein (ddFP) with these biosensors to monitor accessible PM cholesterol levels in cell populations using flow cytometry. These GRAM domain-based biosensors complement existing tools, providing new insights into the molecular mechanisms underlying accessible cholesterol distribution.
    Keywords:  Cholesterol; Flow cytometry; GRAMD1; Lipid transfer protein; Live cell imaging
    DOI:  https://doi.org/10.1007/978-1-0716-4318-1_6
  35. Science. 2024 Dec 19. eado5088
    Identification of antigen-presenting cell-T cell interactions driving immune responses to food.
    Maria C C Canesso, Tiago B R Castro, Sandra Nakandakari-Higa, Ainsley Lockhart, Julia Luehr, Juliana Bortolatto, Roham Parsa, Daria Esterházy, Mengze Lyu, Tian-Tian Liu, Kenneth M Murphy, Gregory F Sonnenberg, Bernardo S Reis, Gabriel D Victora, Daniel Mucida.
      The intestinal immune system must concomitantly tolerate food and commensals and protect against pathogens. Antigen-presenting cells (APCs) orchestrate these immune responses by presenting luminal antigens to CD4+ T cells and inducing their differentiation into regulatory (pTreg) or inflammatory (Th) subsets. We used a proximity labeling method (LIPSTIC) to identify APCs that presented dietary antigens under tolerizing and inflammatory conditions and understand cellular mechanisms by which tolerance to food is induced and can be disrupted by infection. Helminth infections disrupted tolerance induction proportionally to the reduction in the ratio between tolerogenic APCs, including migratory dendritic cells (cDC1s) and Rorγt+ APCs, and inflammatory APCs, that were primarily cDC2s. These inflammatory cDC2s expanded by helminth infection did not present dietary antigens, thus avoiding diet-specific Th2 responses.
    DOI:  https://doi.org/10.1126/science.ado5088
  36. Cancer Res. 2024 Dec 19.
    Selenoprotein O Promotes Melanoma Metastasis and Regulates Mitochondrial Complex II Activity.
    Luiza Martins Nascentes Melo, Marie Sabatier, Vijayashree Ramesh, Krystina J Szylo, Cameron S Fraser, Alexandra Pon, Evann C Mitchell, Kelly A Servage, Gabriele Allies, Isa V Westedt, Feyza Cansiz, Jonathan Krystkiewicz, Andrea Kutritz, Dirk Schadendorf, Sean J Morrison, Jessalyn M Ubellacker, Anju Sreelatha, Alpaslan Tasdogan.
      Evolutionarily conserved selenoprotein O (SELENOO) catalyzes a post-translational protein modification known as AMPylation that is essential for the oxidative stress response in bacteria and yeast. Given that oxidative stress experienced in the blood limits survival of metastasizing melanoma cells, SELENOO might be able to impact metastatic potential. However, further work is needed to elucidate the substrates and functional relevance of the mammalian homologue of SELENOO. Here, we revealed that SELENOO promotes cancer metastasis and identified substrates of SELENOO in mammalian mitochondria. In patients with melanoma, high SELENOO expression was correlated with metastasis and poor overall survival. In a murine model of spontaneous melanoma metastasis, SELENOO deficiency significantly reduced metastasis to distant visceral organs, which could be rescued by treatment with the antioxidant N-acetylcysteine. Mechanistically, SELENOO AMPylated multiple mitochondrial substrates, including succinate dehydrogenase subunit A, one of the four key subunits of mitochondrial complex II. Consistently, SELENOO-deficient cells featured increased mitochondrial complex II activity. Together, these findings demonstrate that SELENOO deficiency limits melanoma metastasis by modulating mitochondrial function and oxidative stress.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-2194
  37. Oncologist. 2024 Dec 02. pii: oyae200. [Epub ahead of print]
    Phase I study of intratumoral injection of talimogene laherparepvec for the treatment of advanced pancreatic cancer.
    Karie Runcie, Yadriel Bracero, Avishai Samouha, Gulam Manji, Helen E Remotti, Tamas A Gonda, Yvonne Saenger.
       BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) presents a redoubtable challenge due to late-stage diagnosis and limited treatment options, necessitating innovative therapeutic strategies.
    METHODS: Here, we report our results investigating the safety and efficacy of talimogene laherparepvec (T-VEC), an FDA-approved oncolytic herpes simplex virus type 1, in patients with advanced PDAC. Nine patients with treatment-refractory advanced PDAC received escalating doses of T-VEC via endoscopic injection.
    RESULTS: While no objective responses were observed, stable disease was achieved in 44% of patients, with a median overall survival of 7.8 months, including one patient who survived 28 months. Adverse events were manageable, with the maximum tolerated dose determined to be 108 PFU/mL.
    CONCLUSION: Our findings underscore the feasibility of intratumoral T-VEC administration in advanced PDAC. Further investigation, particularly in combination with immunotherapies administered systemically is warranted to more optimally assess T-VEC in this challenging malignancy.ClinicalTrials.gov Identifier: NCT03086642.
    Keywords:  T-VEC; endoscopic treatment; immunotherapy; pancreatic cancer; talimogene laherparepvec
    DOI:  https://doi.org/10.1093/oncolo/oyae200
  38. Sci Adv. 2024 Dec 20. 10(51): eadr5947
    Mechanosensitive nuclear uptake of chemotherapy.
    Nicholas R Scott, Sowon Kang, Sapun H Parekh.
      The nucleus is at the nexus of mechanotransduction and the final barrier for most first line chemotherapeutics. Here, we study the intersection between nuclear-cytoskeletal coupling and chemotherapy nuclear internalization. We find that chronic and acute modulation of intracellular filaments changes nuclear influx of doxorubicin (DOX). Rapid changes in cell strain by disruption of cytoskeletal and nuclear filaments sensitize nuclei to DOX, whereas chronic reduction of cell strain desensitize nuclei to DOX. Extracted nuclei from invasive cancer cells lines from different tissues have distinct nuclear permeability to DOX. Last, we show that mechano-priming of cells by paclitaxel markedly improves DOX nuclear internalization, rationalizing the observed drug synergies. Our findings reveal that nuclear uptake is a critical, previously unquantified aspect of drug resistance. With nuclear permeability to chemotherapy being tunable via modulation of nuclear mechanotransduction, mechano-priming may be useful to help overcome drug resistance in the future.
    DOI:  https://doi.org/10.1126/sciadv.adr5947
  39. medRxiv. 2024 Dec 07. pii: 2024.01.26.24301803. [Epub ahead of print]
    HistoPlexer: Histopathology-based Protein Multiplex Generation using Deep Learning.
    Tumor Profiler Consortium
      Multiplexed imaging technologies provide crucial insights into interactions between tumors and their surrounding tumor microenvironment (TME), but their widespread adoption is limited by cost, time, and tissue availability. We introduce HistoPlexer, a deep learning (DL) framework that generates spatially-resolved protein multiplexes directly from histopathology images. HistoPlexer employs the conditional generative adversarial networks with custom loss functions that mitigate slice-to-slice variations and preserve spatial protein correlations. In a comprehensive evaluation on metastatic melanoma samples, HistoPlexer consistently outperforms existing approaches, achieving superior Multiscale Structural Similarity Index and Peak Signal-to-Noise Ratio. Qualitative evaluation by domain experts demonstrates that the generated protein multiplexes closely resemble the real ones, evidenced by Human Eye Perceptual Evaluation error rates exceeding the 50% threshold for perceived realism. Importantly, HistoPlexer preserves crucial biological relationships, accurately capturing spatial co-localization patterns among proteins. In addition, the spatial distribution of cell types derived from HistoPlexer-generated protein multiplex enables effective stratification of tumors into immune hot versus cold subtypes. When applied to an independent cohort, incorporating additional features from HistoPlexer-generated multiplexes enhances the performance of the DL model for survival prediction and immune subtyping, outperforming the model reliant solely on Hematoxylin & Eosin (H&E) image features. By enabling the generation of whole-slide protein multiplex from the H&E image, HistoPlexer offers a cost- and time-effective approach to understanding the TME, and holds promise for advancing precision oncology.
    DOI:  https://doi.org/10.1101/2024.01.26.24301803
  40. Cancers (Basel). 2024 Dec 03. pii: 4049. [Epub ahead of print]16(23):
    Elevated Expression of Cell Adhesion, Metabolic, and Mucus Secretion Gene Clusters Associated with Tumorigenesis, Metastasis, and Poor Survival in Pancreatic Ductal Adenocarcinoma.
    Karthik Balakrishnan, Yi Xiao, Yuanhong Chen, Jixin Dong.
       OBJECTIVES: Technological advances in identifying gene expression profiles are being applied to study an array of cancers. The goal of this study was to identify differentially expressed genes in pancreatic ductal adenocarcinoma (PDAC) and examine their potential role in tumorigenesis and metastasis.
    METHODS: The transcriptomic profiles of PDAC and non-tumorous tissue samples were derived from the gene expression omnibus (GEO), which is a public repository. The GEO2R tool was used to further derive differentially expressed genes from those profiles.
    RESULTS: In this study, a total of 68 genes were derived from upregulated PDAC genes in three or more transcriptomic profiles and were considered PDAC gene sets. The identified PDAC gene sets were examined in the molecular signatures database (MSigDB) for ontological investigation, which revealed that these genes were involved in the extracellular matrix and associated with the cell adhesion process in PDAC tumorigenesis. The gene set enrichment analysis showed greater enrichment scores for the gene sets. Moreover, the identified gene sets were examined for protein-protein interaction using the STRING database. Based on functional k-means clustering, the following three functional cluster groups were identified in this study: extracellular matrix/cell adhesion, metabolic, and mucus secretion-related protein groups. The receiver operating characteristic (ROC) curve revealed greater specificity and sensitivity for these cluster genes in predicting PDAC tumorigenesis and metastases. In addition, the expression of the cluster genes affects the overall survival rate of PDAC patients. Using the cancer genome atlas (TCGA) database, the associations between expression levels and clinicopathological features were validated.
    CONCLUSIONS: Overall, the genes identified in this study appear to be critical in PDAC development and can serve as potential diagnostic and prognostic targets for pancreatic cancer treatment.
    Keywords:  PDAC; differentially expressed genes; gene cluster; metastasis; survival; tumorigenesis
    DOI:  https://doi.org/10.3390/cancers16234049
  41. Discov Oncol. 2024 Dec 18. 15(1): 788
    Unraveling the causal relationship between serum minerals and pancreatic cancer: a Mendelian randomization study.
    Cong Liu, Huajun Wu, Dongdong Cai, Xin Yu.
       BACKGROUND: Pancreatic cancer is among the most lethal malignancies, characterized by a poor prognosis and limited modifiable factors. Emerging evidence indicates that serum mineral levels may influence the likelihood of developing pancreatic cancer. However, the causal relationship between serum minerals and pancreatic cancer remains unclear and warrants further investigation.
    METHODS: This Mendelian randomization (MR) study was conducted to explore the causal effects of serum mineral levels on pancreatic cancer risk. Genetic variants associated with serum mineral levels, including calcium, iron, magnesium, zinc, selenium, and copper, were selected as instrumental variables (IVs) from large-scale genome-wide association study (GWAS) data. Multiple methods, including inverse variance weighting (IVW), MR-Egger, weighted median, weight methods, were employed to perform MR analysis. The effect sizes from the MR analysis, using two independent GWAS summary datasets related to pancreatic cancer, were combined through meta-analysis. The Cochrane Q test, MR-Egger intercept test, MR-PRESSO global test, and leave-one-out test were conducted for sensitivity tests.
    RESULTS: Our MR analysis demonstrated a significant causal effect of genetically predicted serum calcium levels on increased pancreatic cancer risk [OR = 1.64, 95% CI 1.05-2.57, P = 0.029 (discovery cohort); OR = 1.52, 95% CI 1.07-2.15, P = 0.019 (validation cohort)], while no significant associations were found for other serum minerals (P > 0.05). Additional meta-analysis reinforces and substantiates this conclusion (pooled OR = 1.56, 95% CI 1.19-2.06, P = 0.001). No evidence of pleiotropy or heterogeneity was detected across multiple sensitivity tests (P > 0.05).
    CONCLUSION: This study provides new evidence supporting the causal role of certain serum minerals, particularly calcium, in the development of pancreatic cancer. These findings may help inform future research into preventive strategies or therapies aimed at modulating mineral levels in patients at high risk of pancreatic cancer.
    Keywords:  Genome-wide association study; Mendelian randomization analysis; Meta-analysis; Minerals; Pancreatic neoplasms
    DOI:  https://doi.org/10.1007/s12672-024-01695-6
  42. Cancer Res Commun. 2024 Dec 19.
    Targeting PRMT1 reduces cancer persistence and tumor relapse in EGFR- and KRAS-mutant lung cancer.
    Xiaoxiao Sun, Karl Kumbier, Savitha Gayathri, Veronica Steri, Lani F Wu, Steven J Altschuler.
      Incomplete killing of cancer cells undermines oncogene-targeting therapies and drives disease relapse. Eliminating cancer cells that persist during treatment is crucial for improving treatment outcomes. Here, we discovered that a specific isoform of type I protein arginine methyltransferases (PRMTs), namely PRMT1, enables lung cancer cells with EGFR or KRASG12C driver mutations and high STAT1 activity to persist through targeted drug treatments. PRMT1 knockdown, combined with EGFR or KRASG12C inhibitors, decreased persistence and delayed cancer cell regrowth across cell line models and significantly prolonged tumor regression in xenograft models. In contrast, we found that knockdown of two other type I PRMT isoforms, PRMT4 and PRMT6, increased persistence. Finally, we found that targeting PRMT1 to reduce persistence is more effective in lung cancer models with intact vs. deleted chromosome 5q31.1, a region enriched with JAK-STAT pathway genes, suggesting a potential stratification criterion. Together, our study pinpoints the PRMT1 isoform as a critical vulnerability of cancer persistence in EGFR- or KRASG12C-targeted therapies.
    DOI:  https://doi.org/10.1158/2767-9764.CRC-24-0389
  43. bioRxiv. 2024 Dec 05. pii: 2024.12.02.626490. [Epub ahead of print]
    Target cell tension regulates macrophage trogocytosis.
    Caitlin E Cornell, Aymeric Chorlay, Deepak Krishnamurthy, Nicholas R Martin, Lucia Baldauf, Daniel A Fletcher.
      Macrophages are known to engulf small membrane fragments, or trogocytose, target cells and pathogens, rather than fully phagocytose them. However, little is known about what causes macrophages to choose trogocytosis versus phagocytosis. Here, we report that cortical tension of target cells is a key regulator of macrophage trogocytosis. At low tension, macrophages will preferentially trogocytose antibody-opsonized cells, while at high tension they tend towards phagocytosis. Using model vesicles, we demonstrate that macrophages will rapidly switch from trogocytosis to phagocytosis when membrane tension is increased. Stiffening the cortex of target cells also biases macrophages to phagocytose them, a trend that can be countered by increasing antibody surface density and is captured in a mechanical model of trogocytosis. This work suggests that a distinct molecular pathway for trogocytosis is not required to explain differences in trogocytosis among target cell types and points to a mechanism for target cells to modulate trogocytosis.
    DOI:  https://doi.org/10.1101/2024.12.02.626490
  44. Autophagy. 2024 Dec 18.
    Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.
    Van Bui, Xinwen Liang, Yansheng Ye, William Giang, Fang Tian, Yoshinori Takahashi, Hong-Gang Wang.
      Macroautophagy/autophagy, an evolutionarily conserved cellular degradation pathway, involves phagophores that sequester cytoplasmic constituents and mature into autophagosomes for subsequent lysosomal delivery. The ATG8 gene family, comprising the MAP1LC3/LC3 and GABARAP/GBR subfamilies in mammals, encodes ubiquitin-like proteins that are conjugated to phagophore membranes during autophagosome biogenesis. A central question in the field is how Atg8-family proteins are precisely involved in autophagosome formation, which remains controversial and challenging, at least in part due to the short lifespan of phagophores. In this study, we depleted the autophagosome closure regulator VPS37A to arrest autophagy at the vesicle completion step and determined the roles of mammalian Atg8-family proteins (mATG8s) in nutrient starvation-induced autophagosome biogenesis. Our investigation revealed that LC3 loss hinders phagophore formation, while GBR loss impedes both phagophore formation and expansion. The defect in membrane expansion by GBR loss appears to be attributed to compromised recruitment of ATG proteins containing an LC3-interacting region (LIR), including ULK1 and ATG3. Moreover, a combined deficiency of both LC3 and GBR subfamilies nearly completely inhibits phagophore formation, highlighting their redundant regulation of this process. Consequently, cells lacking all mATG8 members exhibit defects in downstream events such as ESCRT recruitment and autophagic flux. Collectively, these findings underscore the critical roles of mammalian Atg8-family proteins in phagophore formation and expansion during autophagy.
    Keywords:  Autophagosome closure; ESCRT; LIR; The mammalian Atg8 family of proteins; membrane expansion; phagophore formation
    DOI:  https://doi.org/10.1080/15548627.2024.2443300
  45. Autophagy. 2024 Dec 15.
    Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.
    Isabel Gordaliza-Alaguero, Paula Sànchez-Fernàndez-de-Landa, Dragana Radivojevikj, Laura Villarreal, Gianluca Arauz-Garofalo, Marina Gay, Marta Martinez-Vicente, Jorge Seco, Pau Martin-Malpartida, Marta Vilaseca, María J Macías, Manuel Palacin, Saška Ivanova, Antonio Zorzano.
      MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.
    Keywords:  Autophagosomes; endosomes; mitochondria; mitochondria-endoplasmic reticulum contact sites; mitochondrial dynamics; nutrient deprivation
    DOI:  https://doi.org/10.1080/15548627.2024.2440843
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