bims-cagime 2023-11-19 papers

bims-cagime

Biomed News

on Cancer, aging and metabolism

Issue of 2023–11–19
thirty-six papers selected by
Kıvanç Görgülü, Technical University of Munich

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming.
Autophagy regulates tumor growth and metastasis.
Cytoplasmic zoning by protein phase transition after membrane permeabilization.
Generation and ex vivo culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants.
CELLULAR, A Cell Autophagy Imaging Dataset.
Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest.
Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest.
CD201+ fascia progenitors choreograph injury repair.
Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma.
Lineage Plasticity: the New Cancer Hallmark on the Block.
Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.
Tumor Explants Elucidate a Cascade of Paracrine SHH, WNT, and VEGF Signals Driving Pancreatic Cancer Angiosuppression.
Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair.
Stress granules plug and stabilize damaged endolysosomal membranes.
The extracellular matrix dictates regional competence for tumour initiation.
Local membrane source gathering by p62 body drives autophagosome formation.
Genome homeostasis defects drive enlarged cells into senescence.
A nanobody-based strategy for rapid and scalable purification of human protein complexes.
Cancer takes many paths through G1/S.
Cell volume expansion and local contractility drive collective invasion of the basement membrane in breast cancer.
One-carbon unit supplementation fuels tumor-infiltrating T cells and augments checkpoint blockade.
CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.
Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.
Mechanical imbalance between normal and cancer cells drives epithelial defense against cancer.
Refeeding-associated AMPKγ1 complex activity is a hallmark of health and longevity.
FAK family proteins regulate in vivo breast cancer metastasis via distinct mechanisms.
Assessing mechanical agency during apical apoptotic cell extrusion.
Body composition analysis using convolutional neural network in predicting postoperative pancreatic fistula and survival after pancreatoduodenectomy for pancreatic cancer.
Protocol to study human mitochondrial ribosome using quantitative density gradient analysis by mass spectrometry and complexome profiling analysis.
The GET insertase exhibits conformational plasticity and induces membrane thinning.
Cancer Cells Adapt to SWI/SNF Inhibition through Compensatory EP400 Function.
Targeted perturbation of signaling-driven condensates.
Anomalous lateral diffusion of lipids during the fluid/gel phase transition of a lipid membrane.
Prediction of Adjuvant Gemcitabine Sensitivity in Resectable Pancreatic Adenocarcinoma Using the GemPred RNA Signature: An Ancillary Study of the PRODIGE-24/CCTG PA6 Clinical Trial.
Daily briefing: We finally know what causes fainting.
Immunogenic cell stress and death in the treatment of cancer.

bioRxiv. 2023 Oct 27. pii: 2023.10.24.563862. [Epub ahead of print]

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming.

Noelle R J Thielman, Vanessa Funes, Sanjana Davuluri, Hector E Ibanez, Wei-Chih Sun, Juan Fu, Keyu Li, Stephen Muth, Xingyi Pan, Kenji Fujiwara, Dwayne Thomas, MacKenzie Henderson, Selina Shiqing Teh, Qingfeng Zhu, Elizabeth Thompson, Elizabeth M Jaffee, Alex Kolodkin, Fengxi Meng, Lei Zheng.

Axon guidance molecules were found to be the gene family most frequently altered in pancreatic ductal adenocarcinoma (PDA) through mutations and copy number changes. However, the exact molecular mechanism regarding PDA development remained unclear. Using genetically engineered mouse models to examine one of the axon guidance molecules, semaphorin 3D (SEMA3D), we found a dual role for tumor-derived SEMA3D in malignant transformation of pancreatic epithelial cells and a role for nerve-derived SEMA3D in PDA development. This was demonstrated by the pancreatic-specific knockout of the SEMA3D gene from the KRAS G12D and TP53 R 172 H mutation knock-in, PDX1-Cre (KPC) mouse model which demonstrated a delayed tumor initiation and growth comparing to the original KPC mouse model. Our results showed that SEMA3D knockout skews the macrophages in the pancreas away from M2 polarization, providing a potential mechanistic role of tumor-derived SEMA3D in PDA development. The KPC mice with the SEMA3D knockout remained metastasis-free, however, died from primary tumor growth. We then tested the hypothesis that a potential compensation mechanism could result from SEMA3D which is naturally expressed by the intratumoral nerves. Our study further revealed that nerve-derived SEMA3D does not reprogram macrophages directly, but reprograms macrophages indirectly through ARF6 signaling and lactate production in PDA tumor cells. SEMA3D increases tumor-secreted lactate which is sensed by GPCR132 on macrophages and subsequently stimulates pro-tumorigenic M2 polarization in vivo. Tumor intrinsic- and extrinsic-SEMA3D induced ARF6 signaling through its receptor Plexin D1 in a mutant KRAS-dependent manner. Consistently, RNA sequencing database analysis revealed an association of higher KRAS MUT expression with an increase in SEMA3D and ARF6 expression in human PDAs. Moreover, multiplex immunohistochemistry analysis showed an increased number of M2-polarized macrophages proximal to nerves in human PDA tissue expressing SEMA3D. Thus, this study suggests altered expression of SEMA3D in tumor cells lead to acquisition of cancer-promoting functions and the axon guidance signaling originating from nerves is "hijacked" by tumor cells to support their growth. Other axon guidance and neuronal development molecules may play a similar dual role which is worth further investigation.
One sentence summary: Tumor- and nerve-derived SEMA3D promotes tumor progression and metastasis through macrophage reprogramming in the tumor microenvironment.
STATEMENT OF SIGNIFICANCE: This study established the dual role of axon guidance molecule, SEMA3D, in the malignant transformation of pancreatic epithelial cells and of nerve-derived SEMA3D in PDA progression and metastasis. It revealed macrophage reprogramming as the mechanism underlying bothroles. Together, this research elucidated how inflammatory responses promote invasive PDA progression and metastasis through an oncogenic process.

DOI: https://doi.org/10.1101/2023.10.24.563862
bioRxiv. 2023 Nov 03. pii: 2023.10.31.564991. [Epub ahead of print]

Autophagy regulates tumor growth and metastasis.

Lei Qiang, Baozhong Zhao, Mei Ming, Ning Wang, Tong-Chuan He, Seungmin Hwang, Andrew Thorburn, Yu-Ying He.

The role of autophagy in tumorigenesis and tumor metastasis remains poorly understood. Here we show that inhibition of autophagy stabilizes the transcription factor Twist1 through Sequestosome-1 (SQSTM1, also known as p62) and thus increases cell proliferation, migration, and epithelial-mesenchymal transition (EMT) in tumor development and metastasis. Inhibition of autophagy or p62 overexpression blocks Twist1 protein degradation in the proteasomes, while p62 inhibition enhances it. SQSTM1/p62 interacts with Twist1 via the UBA domain of p62, in a Twist1-ubiquitination-dependent manner. Lysine 175 in Twist1 is critical for Twist1 ubiquitination, degradation, and SQSTM1/p62 interaction. For squamous skin cancer and melanoma cells that express Twist1, SQSTM1/p62 increases tumor growth and metastasis in mice. Together, our results identified Twist1 as a key downstream protein for autophagy and suggest a critical role of the autophagy/p62/Twist1 axis in cancer development and metastasis.

DOI: https://doi.org/10.1101/2023.10.31.564991
J Biochem. 2023 Nov 16. pii: mvad094. [Epub ahead of print]

Cytoplasmic zoning by protein phase transition after membrane permeabilization.

Shinju Sugiyama, Kojiro Suda, Keiko Kono.

  Biological membranes, including plasma membrane (PM) and organelle membranes, restrict the flux of ions, molecules, and organelles. However, the barrier function of biological membranes is frequently compromised by various perturbations, including physical membrane damage and protein- or chemical-induced pore formation. Recent evidence suggests that, upon PM damage, protein gelation and solid condensation are utilized to restrict ion/molecule/organelle flux across the damaged membranes by zoning the cytoplasm. In addition, membrane permeabilization dramatically alters intramembrane and extramembrane ion/molecule concentrations via the flux across the permeabilized membrane. The changes in ion/molecule concentration and their downstream pathways induce protein phase transition to form zones for biological processes or protein sequestration. Here, we review the mechanisms and functions of protein phase transition after biological membrane permeabilization.

Keywords:  cellular wound healing; cytoplasmic zoning; liquid-liquid phase separation; membrane permeabilization; phase transition

DOI:  https://doi.org/10.1093/jb/mvad094
STAR Protoc. 2023 Nov 10. pii: S2666-1667(23)00678-0. [Epub ahead of print]4(4): 102711

Generation and ex vivo culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants.

Amanda R Decker-Farrell, Alice Ma, Fangda Li, Alexander Muir, Kenneth P Olive.

  Traditional 2D/3D co-culture models typically do not reflect the cellular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) tumors, while in vivo models can be slow and ill-suited to mechanistic investigations. Here, we present a protocol for culturing murine PDAC explants and a corresponding human PDAC model using tissue slice explants. We describe steps for sponge production, preparation of media and materials, tissue collection, and sectioning. We then detail procedures for explant plating, daily culture, and collection of samples.

Keywords:  Biotechnology and bioenginee; Cancer; Cell culture; Model Organisms; Tissue Engineering

DOI:  https://doi.org/10.1016/j.xpro.2023.102711
Sci Data. 2023 Nov 16. 10(1): 806

CELLULAR, A Cell Autophagy Imaging Dataset.

Amani Al Outa, Steven Hicks, Vajira Thambawita, Siri Andresen, Jorrit M Enserink, Pål Halvorsen, Michael A Riegler, Helene Knævelsrud.

Cells in living organisms are dynamic compartments that continuously respond to changes in their environment to maintain physiological homeostasis. While basal autophagy exists in cells to aid in the regular turnover of intracellular material, autophagy is also a critical cellular response to stress, such as nutritional depletion. Conversely, the deregulation of autophagy is linked to several diseases, such as cancer, and hence, autophagy constitutes a potential therapeutic target. Image analysis to follow autophagy in cells, especially on high-content screens, has proven to be a bottleneck. Machine learning (ML) algorithms have recently emerged as crucial in analyzing images to efficiently extract information, thus contributing to a better understanding of the questions at hand. This paper presents CELLULAR, an open dataset consisting of images of cells expressing the autophagy reporter mRFP-EGFP-Atg8a with cell-specific segmentation masks. Each cell is annotated into either basal autophagy, activated autophagy, or unknown. Furthermore, we introduce some preliminary experiments using the dataset that can be used as a baseline for future research.

DOI: https://doi.org/10.1038/s41597-023-02687-x
Cell Chem Biol. 2023 Nov 09. pii: S2451-9456(23)00372-0. [Epub ahead of print]

Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest.

Jason Rodencal, Nathan Kim, Andrew He, Veronica L Li, Mike Lange, Jianping He, Amy Tarangelo, Zachary T Schafer, James A Olzmann, Jonathan Z Long, Julien Sage, Scott J Dixon.

  Ferroptosis is a non-apoptotic form of cell death that can be triggered by inhibiting the system xc- cystine/glutamate antiporter or the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). We have investigated how cell cycle arrest caused by stabilization of p53 or inhibition of cyclin-dependent kinase 4/6 (CDK4/6) impacts ferroptosis sensitivity. Here, we show that cell cycle arrest can enhance sensitivity to ferroptosis induced by covalent GPX4 inhibitors (GPX4i) but not system xc- inhibitors. Greater sensitivity to GPX4i is associated with increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids (PUFA-PLs). Higher PUFA-PL abundance upon cell cycle arrest involves reduced expression of membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) and epithelial membrane protein 2 (EMP2). A candidate orally bioavailable GPX4 inhibitor increases lipid peroxidation and shrinks tumor volumes when combined with a CDK4/6 inhibitor. Thus, cell cycle arrest may make certain cancer cells more susceptible to ferroptosis in vivo.

Keywords:  CDK4/6; EMP2; MBOAT1; MUFA; PUFA; ferroptosis; iron; lipid peroxidation; p53; palbociclib

DOI:  https://doi.org/10.1016/j.chembiol.2023.10.011
Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00857-2. [Epub ahead of print]83(22): 4047-4061.e6

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest.

Reece Foy, Lisa Crozier, Aanchal U Pareri, Juan Manuel Valverde, Ben Ho Park, Tony Ly, Adrian T Saurin.

  CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

Keywords:  CDK4/6; breast cancer; cell cycle; cell growth; chemotherapy; growth factors; oncogenes; p21; p53; replication stress

DOI:  https://doi.org/10.1016/j.molcel.2023.10.020
Nature. 2023 Nov 15.

CD201+ fascia progenitors choreograph injury repair.

Donovan Correa-Gallegos, Haifeng Ye, Bikram Dasgupta, Aydan Sardogan, Safwen Kadri, Ravinder Kandi, Ruoxuan Dai, Yue Lin, Robert Kopplin, Disha Shantaram Shenai, Juliane Wannemacher, Ryo Ichijo, Dongsheng Jiang, Maximilian Strunz, Meshal Ansari, Illias Angelidis, Herbert B Schiller, Thomas Voltz, Hans-Günther Machens, Yuval Rinkevich.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

DOI: https://doi.org/10.1038/s41586-023-06725-x
bioRxiv. 2023 Oct 27. pii: 2023.10.24.563848. [Epub ahead of print]

Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma.

Diogo Maia-Silva, Allison C Schier, Damianos Skopelitis, Vahag Kechejian, Aktan Alpsoy, Jynelle Liverpool, Dylan J Taatjes, Christopher R Vakoc.

The presence of basal lineage characteristics signifies hyper-aggressive human adenocarcinomas of the breast, bladder, and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor p63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to classical PDAC. Taken together, our comprehensive genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.

DOI: https://doi.org/10.1101/2023.10.24.563848
Cancer Res. 2023 Nov 14.

Lineage Plasticity: the New Cancer Hallmark on the Block.

Arnav Mehta, Ben Z Stanger.

Plasticity refers to the ability of cells to adopt a spectrum of states or phenotypes. In cancer, it is a critical contributor to tumor initiation, progression, invasiveness, and therapy resistance, and it has recently been recognized as an emerging cancer hallmark. Plasticity can occur as a result of cell-intrinsic factors (e.g. genetic, transcriptional or epigenetic fluctuations), or through cell-extrinsic cues (e.g. signaling from components of the tumor microenvironment (TME) or selective pressure from therapy). Over the past decade, technological advances, analysis of patient samples, and studies in mouse model systems have led to a deeper understanding of how such plastic states come about. In this review, we discuss: (i) the definition of plasticity; (ii) methods to measure and quantify plasticity; (iii) the clinical relevance of plasticity; and (iv) therapeutic hypotheses to modulate plasticity in the clinic.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-1067
Mol Oncol. 2023 Nov 16.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Nayela N Chowdhury, Yi Yang, Ananya Dutta, Michelle Luo, Zimu Wei, Sara R Abrahams, Alexey S Revenko, Fenil Shah, Lindsey A Miles, Robert J Parmer, Bas de Laat, Alisa S Wolberg, James P Luyendyk, Melissa L Fishel, Matthew J Flick.

  Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg-) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Keywords:  PDAC; metastasis; mouse model; plasminogen

DOI:  https://doi.org/10.1002/1878-0261.13552
Cancer Discov. 2023 Nov 15.

Tumor Explants Elucidate a Cascade of Paracrine SHH, WNT, and VEGF Signals Driving Pancreatic Cancer Angiosuppression.

Marie C Hasselluhn, Amanda R Decker-Farrell, Lukas Vlahos, Dafydd H Thomas, Alvaro Curiel-Garcia, H Carlo Maurer, Urszula N Wasko, Lorenzo Tomassoni, Stephen A Sastra, Carmine F Palermo, Tanner C Dalton, Alice Ma, Fangda Li, Ezequiel J Tolosa, Hanina Hibshoosh, Martin E Fernandez-Zapico, Alexander Muir, Andrea Califano, Kenneth P Olive.

The sparse vascularity of Pancreatic Ductal Adenocarcinoma (PDAC) presents a mystery: what prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of non-canonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues.

DOI: https://doi.org/10.1158/2159-8290.CD-23-0240
Nat Metab. 2023 Nov 16.

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair.

Marta Kovatcheva, Elena Melendez, Dafni Chondronasiou, Federico Pietrocola, Raquel Bernad, Adrià Caballe, Alexandra Junza, Jordi Capellades, Adrián Holguín-Horcajo, Neus Prats, Sylvere Durand, Meritxell Rovira, Oscar Yanes, Camille Stephan-Otto Attolini, Guido Kroemer, Manuel Serrano.

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

DOI: https://doi.org/10.1038/s42255-023-00916-6
Nature. 2023 Nov 15.

Stress granules plug and stabilize damaged endolysosomal membranes.

Claudio Bussi, Agustín Mangiarotti, Christian Vanhille-Campos, Beren Aylan, Enrica Pellegrino, Natalia Athanasiadi, Antony Fearns, Angela Rodgers, Titus M Franzmann, Anđela Šarić, Rumiana Dimova, Maximiliano G Gutierrez.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

DOI: https://doi.org/10.1038/s41586-023-06726-w
Nature. 2023 Nov 15.

The extracellular matrix dictates regional competence for tumour initiation.

Nordin Bansaccal, Pauline Vieugue, Rahul Sarate, Yura Song, Esmeralda Minguijon, Yekaterina A Miroshnikova, Dagmar Zeuschner, Amandine Collin, Justine Allard, Dan Engelman, Anne-Lise Delaunois, Mélanie Liagre, Leona de Groote, Evy Timmerman, Delphi Van Haver, Francis Impens, Isabelle Salmon, Sara A Wickström, Alejandro Sifrim, Cédric Blanpain.

The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.

DOI: https://doi.org/10.1038/s41586-023-06740-y
Nat Commun. 2023 Nov 13. 14(1): 7338

Local membrane source gathering by p62 body drives autophagosome formation.

Xuezhao Feng, Daxiao Sun, Yanchang Li, Jinpei Zhang, Shiyu Liu, Dachuan Zhang, Jingxiang Zheng, Qing Xi, Haisha Liang, Wenkang Zhao, Ying Li, Mengbo Xu, Jiayu He, Tong Liu, Ayshamgul Hasim, Meisheng Ma, Ping Xu, Na Mi.

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

DOI: https://doi.org/10.1038/s41467-023-42829-8
Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00855-9. [Epub ahead of print]83(22): 4032-4046.e6

Genome homeostasis defects drive enlarged cells into senescence.

Sandhya Manohar, Marianna E Estrada, Federico Uliana, Karla Vuina, Patricia Moyano Alvarez, Robertus A M de Bruin, Gabriel E Neurohr.

  Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with increased cell size, we used reversible cell-cycle arrests combined with growth rate modulation to study how excessive growth affects proliferation. We find that enlarged cells upregulate p21, which limits cell-cycle progression. Cells that re-enter the cell cycle encounter replication stress that is well tolerated in physiologically sized cells but causes severe DNA damage in enlarged cells, ultimately resulting in mitotic failure and permanent cell-cycle withdrawal. We demonstrate that enlarged cells fail to recruit 53BP1 and other non-homologous end joining (NHEJ) machinery to DNA damage sites and fail to robustly initiate DNA damage-dependent p53 signaling, rendering them highly sensitive to genotoxic stress. We propose that an impaired DNA damage response primes enlarged cells for persistent replication-acquired damage, ultimately leading to cell division failure and permanent cell-cycle exit.

Keywords:  DNA damage; cell cycle; cell growth; cell size; senescence

DOI:  https://doi.org/10.1016/j.molcel.2023.10.018
Nat Protoc. 2023 Nov 16.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

Taylor Anthony Stevens, Giovani Pinton Tomaleri, Masami Hazu, Sophia Wei, Vy N Nguyen, Charlene DeKalb, Rebecca M Voorhees, Tino Pleiner.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

DOI: https://doi.org/10.1038/s41596-023-00904-w
Trends Cell Biol. 2023 Nov 10. pii: S0962-8924(23)00211-8. [Epub ahead of print]

Cancer takes many paths through G1/S.

Erik S Knudsen, Agnieszka K Witkiewicz, Seth M Rubin.

  In the commonly accepted paradigm for control of the mammalian cell cycle, sequential cyclin-dependent kinase (CDK) and cyclin activities drive the orderly transition from G1 to S phase. However, recent studies using different technological approaches and examining a broad range of cancer cell types are challenging this established paradigm. An alternative model is evolving in which cell cycles utilize different drivers and take different trajectories through the G1/S transition. We are discovering that cancer cells in particular can adapt their drivers and trajectories, which has important implications for antiproliferative therapies. These studies have helped to refine an understanding of how CDK inhibition impinges on proliferation and have significance for understanding fundamental features of cell biology and cancer.

Keywords:  CDK2 inhibitors; CDK4/6 inhibitors; CDKN2A-p16; cyclin D1; cyclin E; cyclin-dependent kinase; retinoblastoma pathway

DOI:  https://doi.org/10.1016/j.tcb.2023.10.007
Nat Mater. 2023 Nov 13.

Cell volume expansion and local contractility drive collective invasion of the basement membrane in breast cancer.

Julie Chang, Aashrith Saraswathibhatla, Zhaoqiang Song, Sushama Varma, Colline Sanchez, Naomi Hassan Kahtan Alyafei, Dhiraj Indana, Raleigh Slyman, Sucheta Srivastava, Katherine Liu, Michael C Bassik, M Peter Marinkovich, Louis Hodgson, Vivek Shenoy, Robert B West, Ovijit Chaudhuri.

Breast cancer becomes invasive when carcinoma cells invade through the basement membrane (BM)-a nanoporous layer of matrix that physically separates the primary tumour from the stroma. Single cells can invade through nanoporous three-dimensional matrices due to protease-mediated degradation or force-mediated widening of pores via invadopodial protrusions. However, how multiple cells collectively invade through the physiological BM, as they do during breast cancer progression, remains unclear. Here we developed a three-dimensional in vitro model of collective invasion of the BM during breast cancer. We show that cells utilize both proteases and forces-but not invadopodia-to breach the BM. Forces are generated from a combination of global cell volume expansion, which stretches the BM, and local contractile forces that act in the plane of the BM to breach it, allowing invasion. These results uncover a mechanism by which cells collectively interact to overcome a critical barrier to metastasis.

DOI: https://doi.org/10.1038/s41563-023-01716-9
bioRxiv. 2023 Nov 03. pii: 2023.11.01.565193. [Epub ahead of print]

One-carbon unit supplementation fuels tumor-infiltrating T cells and augments checkpoint blockade.

Xincheng Xu, Zihong Chen, Caroline R Bartman, Xi Xing, Kellen Olszewski, Joshua D Rabinowitz.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate the contributions to purine nucleotides of salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic T cells) synthesize purines de novo . Purine synthesis requires two 1C units, which come from serine catabolism and circulating formate. Shortage of 1C units is a potential bottleneck for anti-tumor immunity. Elevating circulating formate drives its usage by tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling control of formate-production kinetics. In MC38 tumors, safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.
Statement of significance: Checkpoint blockade has revolutionized cancer therapy. Durable tumor control, however, is achieved in only a minority of patients. We show that the efficacy of anti-PD-1 blockade can be enhanced by metabolic supplementation with one-carbon donors. Such donors support nucleotide synthesis in tumor-infiltrating T cells and merit future clinical evaluation.

DOI: https://doi.org/10.1101/2023.11.01.565193
Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00853-5. [Epub ahead of print]83(22): 4062-4077.e5

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Lisa Crozier, Reece Foy, Rozita Adib, Ananya Kar, Jordan A Holt, Aanchal U Pareri, Juan M Valverde, Rene Rivera, William A Weston, Rona Wilson, Clement Regnault, Phil Whitfield, Mihaly Badonyi, Laura G Bennett, Ellen G Vernon, Amelia Gamble, Joseph A Marsh, Christopher J Staples, Adrian T Saurin, Alexis R Barr, Tony Ly.

  Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Keywords:  DNA damage; cell cycle; cell growth; cell size; mTOR; p21(Cip1/Waf1); p38MAPK; p53; palbociclib; rapamycin

DOI:  https://doi.org/10.1016/j.molcel.2023.10.016
Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00854-7. [Epub ahead of print]83(22): 4078-4092.e6

Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.

Gemma A Wilson, Karla Vuina, Georgina Sava, Caroline Huard, Leticia Meneguello, Jasmin Coulombe-Huntington, Thierry Bertomeu, Rory J Maizels, Josh Lauring, Janos Kriston-Vizi, Mike Tyers, Simak Ali, Cosetta Bertoli, Robertus A M de Bruin.

  Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

Keywords:  CDK inhibition; CDK7 inhibitor; cancer treatment; cell cycle; cell size; cell-cycle arrest; cellular growth; mTOR singaling; proliferation; samuraciclib; senescence

DOI:  https://doi.org/10.1016/j.molcel.2023.10.017
bioRxiv. 2023 Nov 03. pii: 2023.09.27.559723. [Epub ahead of print]

Mechanical imbalance between normal and cancer cells drives epithelial defense against cancer.

Praver Gupta, Sayantani Kayal, Shilpa P Pothapragada, Harish K Senapati, Padmashree Devendran, Dapeng Bi, Tamal Das.

Cell competition enables normal wildtype cells of epithelial tissue to eliminate mutant cells expressing activated oncoproteins such as HRas V12 . However, the driving force behind this fundamental epithelial defense against cancer remains enigmatic. Here, we employ tissue stress microscopy and theoretical modeling and invent a new collective compressibility measurement technique called gel compression microscopy to unveil the mechanism governing cell competition. Stress microscopy reveals unique compressive stress experienced by the mutant cells, contrasting with predominantly tensile stress experienced by normal cells. A cell-based computer simulation then predicts that this compressive stress arises out of a mechanical imbalance between two competing populations due to a difference in their collective compressibility and rigidity. Gel compression microscopy empirically confirms the prediction and elucidates a three-fold higher compressibility of the mutant population than the normal population. Mechanistically, this difference stems from the reduced abundance and coupling of junctional E-cadherin molecules in the mutant cells, which weakens cell-cell adhesions and renders the mutant population more compressible. Taken together, our study elucidates both the physical principle and the underlying molecular mechanism driving cell competition in epithelial defense against cancer and opens new directions for mechanomedicine in cancer.

DOI: https://doi.org/10.1101/2023.09.27.559723
Nat Aging. 2023 Nov 13.

Refeeding-associated AMPKγ1 complex activity is a hallmark of health and longevity.

Roberto Ripa, Eugen Ballhysa, Joachim D Steiner, Raymond Laboy, Andrea Annibal, Nadine Hochhard, Christian Latza, Luca Dolfi, Chiara Calabrese, Anna M Meyer, Maria Cristina Polidori, Roman-Ulrich Müller, Adam Antebi.

Late-life-initiated dietary interventions show limited efficacy in extending longevity or mitigating frailty, yet the underlying causes remain unclear. Here we studied the age-related fasting response of the short-lived killifish Nothobranchius furzeri. Transcriptomic analysis uncovered the existence of a fasting-like transcriptional program in the adipose tissue of old fish that overrides the feeding response, setting the tissue in persistent metabolic quiescence. The fasting-refeeding cycle triggers an inverse oscillatory expression of genes encoding the AMP-activated protein kinase (AMPK) regulatory subunits Prkag1 (γ1) and Prkag2 (γ2) in young individuals. Aging blunts such regulation, resulting in reduced Prkag1 expression. Transgenic fish with sustained AMPKγ1 countered the fasting-like transcriptional program, exhibiting a more youthful feeding and fasting response in older age, improved metabolic health and longevity. Accordingly, Prkag1 expression declines with age in human tissues and is associated with multimorbidity and multidimensional frailty risk. Thus, selective activation of AMPKγ1 prevents metabolic quiescence and preserves healthy aging in vertebrates, offering potential avenues for intervention.

DOI: https://doi.org/10.1038/s43587-023-00521-y
bioRxiv. 2023 Oct 29. pii: 2023.10.27.564212. [Epub ahead of print]

FAK family proteins regulate in vivo breast cancer metastasis via distinct mechanisms.

Alessandro Genna, Joel Alter, Martina Poletti, Tomer Meirson, Tal Sneh, Michal Gendler, Natalia Saleev, George S Karagiannis, Yarong Wang, Dianne Cox, David Entenberg, Maja H Oktay, Tamas Korcsmaros, John S Condeelis, Hava Gil-Henn.

Breast cancer is the most commonly diagnosed malignancy and the major leading cause of tumor-related deaths in women. It is estimated that the majority of breast tumor-related deaths are a consequence of metastasis, to which no cure exists at present. The FAK family proteins Proline-rich tyrosine kinase (PYK2) and focal adhesion kinase (FAK) are highly expressed in breast cancer, but the exact cellular and signaling mechanisms by which they regulate in vivo tumor cell invasiveness and consequent metastatic dissemination are mostly unknown. Using a PYK2 and FAK knockdown xenograft model we show here, for the first time, that ablation of either PYK2 or FAK decreases primary tumor size and significantly reduces Tumor MicroEnvironment of Metastasis (TMEM) doorway activation, leading to decreased intravasation and reduced spontaneous lung metastasis. Intravital imaging analysis further demonstrates that PYK2, but not FAK, regulates a motility phenotype switch between focal adhesion-mediated fast motility and invadopodia-dependent, ECM-degradation associated slow motility within the primary tumor. Furthermore, we validate our in vivo and intravital imaging results with integrated transcriptomic and proteomic data analysis from xenograft knockdown tumors and reveal new and distinct pathways by which these two homologous kinases regulate breast tumor cell invasiveness and consequent metastatic dissemination. Our findings identify PYK2 and FAK as novel mediators of mammary tumor progression and metastasis and as candidate therapeutic targets for breast cancer metastasis.

DOI: https://doi.org/10.1101/2023.10.27.564212
bioRxiv. 2023 Oct 27. pii: 2023.10.26.564227. [Epub ahead of print]

Assessing mechanical agency during apical apoptotic cell extrusion.

Sommer Anjum, Llaran Turner, Youmna Atieh, George T Eisenhoffer, Lance Davidson.

Epithelial tissues maintain homeostasis through the continual addition and removal of cells. Homeostasis is necessary for epithelia to maintain barrier function and prevent the accumulation of defective cells. Unfit, excess, and dying cells can be removed from epithelia by the process of extrusion. Controlled cell death and extrusion in the epithelium of the larval zebrafish tail fin coincides with oscillation of cell area, both in the extruding cell and its neighbors. Both cell-autonomous and non-autonomous factors have been proposed to contribute to extrusion but have been challenging to test by experimental approaches. Here we develop a dynamic cell-based biophysical model that recapitulates the process of oscillatory cell extrusion to test and compare the relative contributions of these factors. Our model incorporates the mechanical properties of individual epithelial cells in a two-dimensional simulation as repelling active particles. The area of cells destined to extrude oscillates with varying durations or amplitudes, decreasing their mechanical contribution to the epithelium and surrendering their space to surrounding cells. Quantitative variations in cell shape and size during extrusion are visualized by a hybrid weighted Voronoi tessellation technique that renders individual cell mechanical properties directly into an epithelial sheet. To explore the role of autonomous and non-autonomous mechanics, we vary the biophysical properties and behaviors of extruding cells and neighbors such as the period and amplitude of repulsive forces, cell density, and tissue viscosity. Our data suggest that cell autonomous processes are major contributors to the dynamics of extrusion, with the mechanical microenvironment providing a less pronounced contribution. Our computational model based on in vivo data serves as a tool to provide insights into the cellular dynamics and localized changes in mechanics that promote elimination of unwanted cells from epithelia during homeostatic tissue maintenance.

DOI: https://doi.org/10.1101/2023.10.26.564227
Eur J Radiol. 2023 Nov 03. pii: S0720-048X(23)00496-5. [Epub ahead of print]169 111182

Body composition analysis using convolutional neural network in predicting postoperative pancreatic fistula and survival after pancreatoduodenectomy for pancreatic cancer.

Jeongin Yoo, Soon Ho Yoon, Dong Ho Lee, Jin-Young Jang.

   PURPOSE: To evaluate whether body composition measurements acquired using convolutional neural networks (CNNs) from preoperative CT images could predict postoperative pancreatic fistula (POPF) and overall survival (OS) after pancreaticoduodenectomy in patients with pancreatic ductal adenocarcinoma (PDAC).
METHODS: 257 patients (160 men; median age [interquartile range], 67 [60-74]) who underwent pancreaticoduodenectomy for PDAC between January 2013 and December 2017 were included in this retrospective study. Body composition measurements were based on a CNN trained to segment CT images into skeletal muscle area, visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT). Skeletal muscle area, VAT, and SAT were normalized to height square and labeled as skeletal muscle, VAT, and SAT indices, respectively. The independent risk factors for clinically relevant POPF (grade B or C) were determined using a multivariate logistic regression model, and prognostic factors for OS were assessed using Cox proportional hazards regression analyses.
RESULTS: After pancreatioduodenectomy, 27 patients developed POPF grade B or C (10.5 %, 27/257). The VAT index (odds ratio [OR] = 7.43, p < 0.001) was the only independent prognostic factor for POPF grade B or C. During the median follow-up period of 23 months, 205 (79.8 % [205/257]) patients died. For prediction of OS, skeletal muscle index (hazard ratio [HR] = 0.58, p = 0.018) was a significant factor, along with vascular invasion (HR = 1.85, p < 0.001) and neoadjuvant therapy (HR = 0.58, p = 0.011).
CONCLUSIONS: A high VAT index and a low skeletal muscle index can be utilized in predicting the occurrence of POPF grade B or C and poor OS, respectively.

Keywords:  Body composition; Pancreatic fistula; Pancreatic neoplasm; Pancreaticoduodenectomy; Survival analysis

DOI:  https://doi.org/10.1016/j.ejrad.2023.111182
STAR Protoc. 2023 Nov 15. pii: S2666-1667(23)00572-5. [Epub ahead of print]4(4): 102605

Protocol to study human mitochondrial ribosome using quantitative density gradient analysis by mass spectrometry and complexome profiling analysis.

Petra Páleníková, Michal Minczuk, Pedro Rebelo-Guiomar.

  Dynamic macromolecular complexes containing a large number of components are often difficult to study using conventional approaches, such as immunoblotting. Here, we present a protocol for the analysis of macromolecular complexes in near-native conditions using a flexible setup to suit different cellular targets. We describe analysis of human mitochondrial ribosome, composed of 82 proteins, in a standardized way using density gradient ultracentrifugation coupled to quantitative mass spectrometry and subsequent analysis of the generated data (ComPrAn). For complete details on the use and execution of this protocol, please refer to Páleníková et al.1 and Rebelo-Guiomar et al.2.

Keywords:  Bioinformatics; Protein Expression and Purification; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2023.102605
Nat Commun. 2023 Nov 14. 14(1): 7355

The GET insertase exhibits conformational plasticity and induces membrane thinning.

Melanie A McDowell, Michael Heimes, Giray Enkavi, Ákos Farkas, Daniel Saar, Klemens Wild, Blanche Schwappach, Ilpo Vattulainen, Irmgard Sinning.

The eukaryotic guided entry of tail-anchored proteins (GET) pathway mediates the biogenesis of tail-anchored (TA) membrane proteins at the endoplasmic reticulum. In the cytosol, the Get3 chaperone captures the TA protein substrate and delivers it to the Get1/Get2 membrane protein complex (GET insertase), which then inserts the substrate via a membrane-embedded hydrophilic groove. Here, we present structures, atomistic simulations and functional data of human and Chaetomium thermophilum Get1/Get2/Get3. The core fold of the GET insertase is conserved throughout eukaryotes, whilst thinning of the lipid bilayer occurs in the vicinity of the hydrophilic groove to presumably lower the energetic barrier of membrane insertion. We show that the gating interaction between Get2 helix α3' and Get3 drives conformational changes in both Get3 and the Get1/Get2 membrane heterotetramer. Thus, we provide a framework to understand the conformational plasticity of the GET insertase and how it remodels its membrane environment to promote substrate insertion.

DOI: https://doi.org/10.1038/s41467-023-42867-2
Cancer Discov. 2023 Nov 17. OF1

Cancer Cells Adapt to SWI/SNF Inhibition through Compensatory EP400 Function.

Loss of the compensatory chromatin remodeler EP400 sensitizes cancer cells to SWI/SNF inhibition.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2023-183
Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00860-2. [Epub ahead of print]83(22): 4141-4157.e11

Targeted perturbation of signaling-driven condensates.

Tianshu Gui, Cassio Fleming, Caterina Manzato, Benjamin Bourgeois, Nafiseh Sirati, Jasper Heuer, Ioanna Papadionysiou, Daniel I van Montfort, Merel van Gijzen, Lydia M M Smits, Boudewijn M T Burgering, Tobias Madl, Jurian Schuijers.

  Biomolecular condensates have emerged as a major organizational principle in the cell. However, the formation, maintenance, and dissolution of condensates are still poorly understood. Transcriptional machinery partitions into biomolecular condensates at key cell identity genes to activate these. Here, we report a specific perturbation of WNT-activated β-catenin condensates that disrupts oncogenic signaling. We use a live-cell condensate imaging method in human cancer cells to discover FOXO and TCF-derived peptides that specifically inhibit β-catenin condensate formation on DNA, perturb nuclear β-catenin condensates in cells, and inhibit β-catenin-driven transcriptional activation and colorectal cancer cell growth. We show that these peptides compete with homotypic intermolecular interactions that normally drive condensate formation. Using this framework, we derive short peptides that specifically perturb condensates and transcriptional activation of YAP and TAZ in the Hippo pathway. We propose a "monomer saturation" model in which short interacting peptides can be used to specifically inhibit condensate-associated transcription in disease.

Keywords:  NMR; WNT-signaling; biomolecular condensates; signaling inhibition; transcriptional regulation

DOI:  https://doi.org/10.1016/j.molcel.2023.10.023
Phys Chem Chem Phys. 2023 Nov 14.

Anomalous lateral diffusion of lipids during the fluid/gel phase transition of a lipid membrane.

Abhay Kumar, Snehasis Daschakraborty.

A lipid membrane undergoes a phase transition from fluid to gel phase upon changing external thermodynamic conditions, such as decreasing temperature and increasing pressure. Extremophilic organisms face the challenge of preventing this deleterious phase transition. The main focus of their adaptive strategy is to facilitate effective temperature sensing through sensor proteins, relying on the drastic changes in packing density and membrane fluidity during the phase transition. Although the changes in packing density parameters due to the fluid/gel phase transition are studied in detail, the impact on membrane fluidity is less explored in the literature. Understanding the lateral diffusive dynamics of lipids in response to temperature, particularly during the fluid/gel phase transition, is albeit crucial. Here we have simulated the phase transition of a single component lipid membrane composed of dipalmitoylphosphatidylcholine (DPPC) lipids using a coarse-grained (CG) model and studied the changes of the structural and dynamical properties. It is observed that near the phase transition point, both fluid and gel phase domains coexist together. The dynamics remains highly non-Gaussian for a long time even when the mean square displacement reaches the Fickian regime at a much earlier time. This Fickian yet non-Gaussian diffusion (FnGD) is a characteristic of a highly heterogeneous system, previously observed for the lateral diffusion of lipids in raft mimetic membranes having liquid-ordered and liquid-disordered phases co-existing together. We have analyzed the molecular trajectories and calculated the jump-diffusion of the lipids, stemming from sudden jump translations, using a translational jump-diffusion (TJD) approach. An overwhelming contribution of the jump-diffusion of the lipids is observed suggesting anomalous diffusion of lipids during fluid/gel phase transition of the membrane. These results are important in unravelling the intricate nature of lipid diffusion during the phase transition of the membrane and open up a new possibility of investigating the most significant change of membrane properties during phase transition, which can be effectively sensed by proteins.

DOI: https://doi.org/10.1039/d3cp04081j
J Clin Oncol. 2023 Nov 14. JCO2202668

Prediction of Adjuvant Gemcitabine Sensitivity in Resectable Pancreatic Adenocarcinoma Using the GemPred RNA Signature: An Ancillary Study of the PRODIGE-24/CCTG PA6 Clinical Trial.

Rémy Nicolle, Jean-Baptiste Bachet, Alexandre Harlé, Juan Iovanna, Pascal Hammel, Vinciane Rebours, Anthony Turpin, Meher Ben Abdelghani, Alice Wei, Emmanuel Mitry, Anthony Lopez, James Biagi, Eric François, Pascal Artru, Aurélien Lambert, Daniel J Renouf, Laure Monard, Marjorie Mauduit, Nelson Dusetti, Thierry Conroy, Jérome Cros.

PURPOSE: GemPred, a transcriptomic signature predictive of the efficacy of adjuvant gemcitabine (GEM), was developed from cell lines and organoids and validated retrospectively. The phase III PRODIGE-24/CCTG PA6 trial has demonstrated the superiority of modified folinic acid, fluorouracil, irinotecan, and oxaliplatin (mFOLFIRINOX) over GEM as adjuvant therapy in patients with resected pancreatic ductal adenocarcinoma at the expense of higher toxicity. We evaluated the potential predictive value of GemPred in this population.
PATIENTS AND METHODS: Routine formalin-fixed paraffin-embedded surgical specimens of 350 patients were retrieved for RNA sequencing and GemPred prediction (167 in the GEM arm and 183 in the mFOLFIRINOX [mFFX] arm). Survival analyses were stratified by resection margins, lymph node status, and cancer antigen 19-9 level.
RESULTS: Eighty-nine patients' tumors (25.5%) were GemPred+ and were thus predicted to be gemcitabine-sensitive. In the GEM arm, GemPred+ patients (n = 50, 30%) had a significantly longer disease-free survival (DFS) than GemPred- patients (n = 117, 70%; median 27.3 v 10.2 months, hazard ratio [HR], 0.43 [95% CI, 0.29 to 0.65]; P < .001) and cancer-specific survival (CSS; median 68.4 v 28.6 months, HR, 0.42 [95% CI, 0.27 to 0.66]; P < .001). GemPred had no prognostic value in the mFFX arm. DFS and CSS were similar in GemPred+ patients who received adjuvant GEM and mFFX (median 27.3 v 24.0 months, and 68.4 v 51.4 months, respectively). The statistical interaction between GEM and GemPred+ status was significant for DFS (P = .008) and CSS (P = .004). GemPred+ patients had significantly more adverse events of grade ≥3 in the mFFX arm (76%) compared with those in the GEM arm (40%; P = .001).
CONCLUSION: This ancillary study of a phase III randomized trial demonstrates that among the quarter of patients with a GemPred-positive transcriptomic signature, survival was comparable with that of mFOLFIRINOX, whereas those receiving adjuvant gemcitabine had fewer adverse events.

DOI: https://doi.org/10.1200/JCO.22.02668
Nature. 2023 Nov 02.

Daily briefing: We finally know what causes fainting.

Flora Graham.

DOI: https://doi.org/10.1038/d41586-023-03466-9
Semin Cell Dev Biol. 2023 Nov 15. pii: S1084-9521(23)00204-5. [Epub ahead of print]156 11-21

Immunogenic cell stress and death in the treatment of cancer.

Hui Pan, Peng Liu, Liwei Zhao, Yuhong Pan, Misha Mao, Guido Kroemer, Oliver Kepp.

The successful treatment of oncological malignancies which results in long-term disease control or the complete eradication of cancerous cells necessitates the onset of adaptive immune responses targeting tumor-specific antigens. Such desirable anticancer immunity can be triggered via the induction of immunogenic cell death (ICD) of cancer cells, thus converting malignant cells into an in situ vaccine that elicits T cell mediated adaptive immune responses and establishes durable immunological memory. The exploration of ICD for cancer treatment has been subject to extensive research. However, functional heterogeneity among ICD activating therapies in many cases requires specific co-medications to achieve full-blown efficacy. Here, we described the hallmarks of ICD and classify ICD activators into three distinct functional categories namely, according to their mode of action: (i) ICD inducers, which increase the immunogenicity of malignant cells, (ii) ICD sensitizers, which prime cellular circuitries for ICD induction by conventional cytotoxic agents, and (iii) ICD enhancers, which improve the perception of ICD signals by antigen presenting dendritic cells. Altogether, ICD induction, sensitization and enhancement offer the possibility to convert well-established conventional anticancer therapies into immunotherapeutic approaches that activate T cell-mediated anticancer immunity.

DOI: https://doi.org/10.1016/j.semcdb.2023.10.007