Cell Death Discovery

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lipid reactive oxygen species induced membrane damage, is mechanistically uncharacterized in its execution process. Here, we investigated ferroptosis execution in mesenchymal-like ovarian cancer cells treated with ferroptosis inducers ML162 and erastin. We showed that YVAD (a pyroptosis-associated inflammatory caspase inhibitor) and disulfiram (preventing gasdermin pore formation on plasma membrane) deterred ferroptotic cell death. Moreover, we also observed LDH release and IL-1{beta} secretion from ferroptotic cells, suggesting that ferroptosis involves a pore-forming process. Intriguingly, ferroptosis is independent of the canonical inflammasome pathway because caspase-1 is dispensable and not activated upon ferroptosis induction. In contrast, we found that caspase-5 was activated while caspase-4 was not during ferroptosis. In addition, depletion of caspase-5 rendered cells not responding to ferroptosis inducers. Also intriguingly, GSDMD, the well-established caspase-5 substrate, was not involved in ferroptosis. We instead detected GSDME cleavage upon ferroptosis induction and knockdown of GSDME reduced cell death induced by ferroptosis inducers. As caspase-5 activity was necessary for ferroptosis and caspase-5 directly cleaved GSDME, we conclude that the axis of caspase-5/GSDME executes ferroptosis in ovarian cancer cells.

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

Bacterial genotoxins, Cytolethal Distending Toxin (CDT) and colibactin, cause DNA damage in intoxicated epithelial cells of the host. DNA damage influences {beta}-catenin signaling, altering its stability and nuclear translocation, potentially contributing to cancer development. Using non-transformed hepatocytes and cancer-derived intestinal and hepatic epithelial cell lines, we showed that CDT/CdtB induces the phosphorylation of {beta}-catenin at serine 552, along with the loss of {beta}-catenin from adherens junctions. This leads to the subsequent cytoplasmic accumulation and nuclear translocation of {beta}-catenin, ultimately driving TCF/LEF transcription, the crucial downstream event of Wnt/{beta}-catenin signaling, as well as the transcription of some {beta}-catenin target genes. Colibactin induces similar effects. MK-2206, a direct AKT inhibitor, and metformin, an AMP-activated protein kinase activator that indirectly inhibits AKT, both protected cells against various effects induced by CdtB exposure. These effects include {beta}-catenin phosphorylation at Ser552, the disassembly of cell-cell junctions and the subsequent nuclear accumulation of phosphorylated {beta}-catenin, leading to reduced TCF/LEF-mediated transcription. Additionally, MK-2206 and metformin protected from CdtB-induced epithelial-mesenchymal transition (EMT), including increased nuclear accumulation of SNAIL, enhanced matrix degradation and motility. Overall, these data show that infection with genotoxin-producing bacteria controls some EMT features through {beta}-catenin and AKT-dependent signaling.

Colorectal cancer (CRC) poses a severe global health threat with high incidence, mortality, and poor 5-year survival rates for advanced cases despite existing treatments. This study aims to explore the role of STRIP2 in CRC progression and its underlying mechanisms. Impact of STRIP2 on CRC in vitro was investigated via CRC cell proliferation, migration, invasion, and apoptosis. The in vivo impact was investigated via nude mice models. The role of STRIP2 in CRC was investigated via transcriptomic analysis, Western blot, Co-immunoprecipitation assays and ferroptosis validations. STRIP2 is overexpressed in CRC, driving malignant phenotypes in vitro and in vivo. Mechanically, STRIP2 stabilizes the IL17 downstream effector LCN2 by blocking its K48-linked ubiquitination and degradation, enhances anti-ferroptosis of CRC cells. Oe-STRIP2 suppresses ferroptosis, boosting proliferation and reducing oxidative stress; while si-STRIP2 induces the opposite effect. This study suggests STRIP2-mediated stabilization of LCN2 and enhances CRC cells ferroptosis resistance, thus promoting CRC cell survival and mediates malignant progression in CRC, which provides a novel link between STRIP2 and ferroptosis regulation in CRC. HighlightO_LISTRIP2 is overexpressed in CRC tissues and cells C_LIO_LISTRIP2 blocks LCN2 Ubiquitination and stabilizes LCN2 C_LIO_LISTRIP2 suppresses CRC ferroptosis C_LIO_LISTRIP2 drives CRC malignant phenotypes both in vitro & in vivo C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/725308v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1baf7baorg.highwire.dtl.DTLVardef@1de15d9org.highwire.dtl.DTLVardef@16c8078org.highwire.dtl.DTLVardef@667840_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

ADAM17 sheds cell surface molecules such as TNF-, IL-6R and L-selectin. This activity requires either iRhom1 or iRhom2 as adapter molecules. Since iRhom2 is predominantly expressed in leukocytes and upregulated in tissue cells during inflammation, it represents a potential anti-inflammatory target. We therefore investigated the effects of iRhom2 deficiency in mice using in vivo, ex vivo, and in vitro models of acute inflammation. In an in vivo model of LPS-induced lung inflammation, iRhom2 knockout mice showed reduced neutrophil recruitment into the bronchoalveolar space. Notably, the few recruited neutrophils remained L-selectin positive, whereas most neutrophils in wildtype mice were L-selectin negative, confirming that L-selectin shedding depends on the iRhom2/ADAM17 axis. Furthermore, it suggests that impaired shedding is associated with decreased neutrophil recruitment. Additionally, ADAM17-dependent release of TNF- and IL-6R into the alveolar space was diminished in the absence of iRhom2, accompanied by reduced expression of inflammatory mediators. In isolated perfused lungs challenged with LPS, iRhom2 deficiency similarly reduced inflammatory mediator production, indicating a role for iRhom2 in resident lung tissue cells during the initiation of inflammation. To specifically assess immune cell responses, we further examined macrophages, the sole resident immune cells in the lung. In vitro, LPS-stimulated bone marrow derived macrophages lacking iRhom2 showed decreased shedding of TNF- and IL-6R and reduced induction of secondary inflammatory mediators. Thus, targeting iRhom2 effectively suppresses ADAM17-mediated inflammatory responses in the lung, while preserving basal ADAM17 activity through iRhom1, offering a more selective therapeutic strategy with fewer side effects. HighlightsO_LIThe ADAM17 adapter molecule iRhom2 is required for the acute lung inflammation of mice in vivo including cytokine response and neutrophil recruitment. C_LIO_LIIn resident lung tissue cells iRhom2 promotes the LPS induced inflammatory response at the alveolar interface. C_LIO_LIIn macrophages iRhom2 is required for an effective ADAM17 dependent inflammatory response to LPS. C_LIO_LIThus, iRhom2 targeting can serve to suppress inflammatory activities of ADAM17 in the lung. C_LI Graphical AbstractSchematic overview depicting the role of the iRhom2-ADAM17 axis in mediating neutrophil infiltration and cytokine release during induced pulmonary inflammation, serving as a model for acute lung injury (ALI). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/730404v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@e972d9org.highwire.dtl.DTLVardef@7326e0org.highwire.dtl.DTLVardef@1fd73bforg.highwire.dtl.DTLVardef@ba4031_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mammalian single-stranded DNA binding protein 1 (SSB1) has been established as an essential component of genome stability in both human cells and mice. Moreover, SSB1 was recently implicated in cytoplasmic stress response by its involvement in Ras GTPase-activating protein-binding protein 1 (G3BP1)-containing cytoplasmic stress granules (SGs) upon various forms of stress. Here, we generated and analyzed human cellular knockout and rodent ischemia-reperfusion (I/R) models to define SSB1s roles in cytoplasmic stress response. Analysis of wild-type as well as SSB1 and G3BP1 knockout human retinal pigment epithelial (RPE-1) cells shows stress-specific incorporation of SSB1 into SGs and a negative regulator role for SSB1 in SG dynamics under sublethal stress conditions. We find that SSB1 knockout measurably increases cellular sensitivity to oxidative stress but does not alter cell proliferation following mild acute stress. Moreover, we detect SSB1 efflux from the nucleus upon stress that is dependent upon the presence of G3BP1 in a stress-specific manner. In addition, using mouse and rat models we observe significant upregulation and robust cytoplasmic granulation of SSB1 upon renal ischemia-reperfusion stress, establishing SSB1s involvement in complex organismal stress response in vivo. Together, our data demonstrate active involvement of SSB1 in cytoplasmic response to cellular stress and acute kidney injury, with implications for targeting stress response functions in cancerous versus non-cancerous contexts. HIGHLIGHTSO_LISSB1 is incorporated into cytoplasmic stress granules and negatively regulates stress granule assembly under sublethal stress conditions C_LIO_LISSB1 shows stress- and G3BP1-dependent nuclear efflux C_LIO_LISSB1 is upregulated and undergoes apical granulation in renal epithelial cells during renal ischemia-reperfusion injury C_LI

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic {beta}-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures--a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H2O2-induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process. Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope-mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in {beta}-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727587v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4f63b5org.highwire.dtl.DTLVardef@1b3dc8org.highwire.dtl.DTLVardef@7527fcorg.highwire.dtl.DTLVardef@1944f2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

PurposeCisplatin (CDDP) is the most active chemotherapy for ovarian cancer; primary or acquired resistance signals a poor prognosis. Nelfinavir (NFV), an HIV protease inhibitor, has demonstrated anti-tumor activity in multiple cancer models, but its interaction with CDDP in ovarian cancer has yet to be demonstrated. In this work, we addressed whether the combination of CDDP and NFV provides treatment advantage in platinum (Pt)-resistant ovarian cancer cells. MethodsDrug synergy between NFV and CDDP was assessed using cell vitality assays and Loewe additivity modelling. Apoptotic and pyroptotic signalling were evaluated by immunoblotting, mitochondrial membrane potential analysis, and lactate dehydrogenase (LDH) release, and caspase inhibition. Transcriptomic changes were assessed by bulk mRNA sequencing followed by differential gene expression analysis and gene set enrichment analysis. ResultsNFV synergized with CDDP to reduce the viability of Pt-resistant ovarian cancer cells, promoting a regulated lytic cell death phenotype involving apoptotic and pyroptotic features. Combination treatment induced caspase-8 and caspase-3 activation, and downstream gasdermin E (GSDME) processing. Inhibition of caspase-3 significantly attenuated cell death, and caspase-8 inhibition rescued viability and prevented Bid cleavage, caspase-3 activation, and GSDME cleavage. These effects occurred in the context of enhanced endoplasmic reticulum stress, increased DNA damage with reduced DNA repair, and impaired Akt-driven survival signalling. ConclusionsOur findings establish that NFV synergizes with CDDP in killing Pt-resistant ovarian cancer cells by promoting a caspase-8-dependent apoptotic-to-secondary pyroptotic response, supporting further investigation of NFV as a potential drug to be repurposed to increase the efficacy of Pt-based therapy.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

Autosomal recessive Stargardt disease type 1 (STGD1) is the most prevalent inherited juvenile macular degeneration, ultimately leading to irreversible blindness through photoreceptor cell loss, yet the underlying cell death mechanisms remain poorly defined. Receptor-interacting protein kinase 3 (RIPK3) is a well-established mediator of necroptosis and, under certain circumstances, apoptosis downstream of TNF family ligands, and it was found to be progressively upregulated in the retina of a STGD1 abca4-/-rdh8-/- (DKO) mouse model coinciding with the onset and progression of photoreceptor degeneration. Despite elevated RIPK3 expression, necroptosis was not detectable in this model, as evidenced by the absence of phosphorylated MLKL and unaltered photoreceptor degeneration in abca4-/- rdh8-/-mlkl-/- mice. Intriguingly, genetic ablation of Ripk3 exacerbated photoreceptor loss in DKO mice in both chronic (age-dependent) and acute (light-induced) retinal degeneration paradigms. This detrimental effect was partially ameliorated by pan-caspase inhibition in the acute degeneration model, indicating caspase-dependent apoptosis as the primary executioner. Mechanistically, we demonstrated that RIPK3 suppressed extrinsic apoptosis by attenuating caspase-8 activation downstream of TNF family ligands. Collectively, our findings reveal a non-canonical, protective role of RIPK3 in photoreceptors, as a brake on apoptotic signaling rather than a necroptotic executor, in the context of STGD1. These findings redefine the role of RIPK3 in retinal degeneration and emphasize the contextual plasticity of cell death regulators in neurodegenerative diseases.

Structural AbstractO_ST_ABSBackgroundC_ST_ABSTriple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressiveness, heterogeneity and limited availability of effective targeted therapy. We investigated whether LRP-1, a multifunctional cell-surface endocytic and signaling receptor, contributes to TNBC progression. MethodsUsing CRISPR-Cas9, LRP-1-deficient murine 4T1 and human HS578-T TNBC cells were used. Functional consequences were assessed through migration, invasion, and 3D spheroid assays, imaging of focal adhesions and actin organization, atomic force microscopy, and plasmin activity assays. Global molecular reprogramming was analyzed by label-free quantitative proteomics and secretomics. LRP-1-deficient or proficient 4T1 cells were implanted orthotopically in immunocompetent mice; tumor progression was monitored longitudinally while peritumoral collagen architecture and immune microenvironment composition were characterized by second harmonic generation imaging and immunohistochemistry. ResultsWe show that LRP-1 loss reduces TNBC aggressiveness, as reflected by decreased migration and invasive capacity, reduced spheroid evasion, and significant morphological changes in focal adhesion and actin structure. LRP-1-deficient cells became stiffer and showed lower LOXL-4 levels, while pericellular proteolytic activity remained unchanged, suggesting other proteases mechanism. Multi-omic analysis revealed alterations in extracellular matrix (ECM), epithelial-mesenchymal transition, and inflammatory pathways. In vivo, LRP-1-deficiency reduced tumor progression and peritumoral collagen deposition, while increasing CD8+ T and Natural Killer cell infiltration, together with a cytokine profiling compatible with a more immune-permissive microenvironment. ConclusionsLRP-1 act as a key contributor in TNBC progression through matrix remodeling, mechano-adaptation, and immune exclusion. Positioning it as a candidate biomarker for TNBC patients who are likely to benefit from stroma-targeting therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/732906v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b595c2org.highwire.dtl.DTLVardef@7b208aorg.highwire.dtl.DTLVardef@1956e54org.highwire.dtl.DTLVardef@17e55d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

Epigenetic regulation, particularly histone acetylation, plays a critical role in skeletal muscle differentiation by modulating gene expression programs without altering DNA sequence. Histone deacetylases (HDACs) tightly regulate myogenesis by controlling the timing of differentiation. Pharmacological inhibition of HDACs has shown context-dependent effects on muscle cells. We investigated the effects of 1 {micro}M SAHA (suberoylanilide hydroxamic acid) on C2C12 and L6 myoblasts during differentiation using morphological, immunofluorescence, transcriptomic, and proteomic analyses. SAHA delayed early differentiation, reducing myotube formation with partial recovery at later stages. Transcriptomic analysis revealed time-dependent changes in pathways related to cytoskeleton, cell cycle, and chromatin regulation. Proteomics showed increased mitochondrial metabolism and reduced cytoskeletal components in C2C12 cells, while L6 cells displayed alterations in muscle structural and extracellular matrix proteins. SAHA induces stage- and model-dependent reprogramming of myogenesis, highlighting the importance of timing and cellular context in HDAC-targeted therapies.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.

Cell polarity complexes are essential for embryogenesis, but their regulatory mechanisms during early developmental transitions remain incompletely understood. Here, we individually deleted the Crumbs, Par, and Scrib polarity complexes in mouse embryonic stem cells (mESCs). While loss of any single complex did not affect pluripotency or proliferation, deletion of Par complex disrupted the naive-to-primed transition and impaired subsequent differentiation, particularly lumen formation in neural tube organoids. Mechanistically, Par complex deficiency led to hyperphosphorylation of focal adhesion kinase (FAK) at the primed stage, driving a morphological shift from flat monolayer clusters to dome-shaped colonies. FAK inhibition rescued the aberrant morphology. Upstream, Par complex loss increased AKT phosphorylation, which remodeled extracellular matrix (ECM) and regulated integrin signaling via FURIN-LEFTY, ultimately modulating FAK activity. In addition, conditioned medium from wild-type cells partially rescued differentiation defects in Par knockout cells in a LEFTY-dependent manner. These phenotypes were consistently observed in naive-to-primed transition, neural stem cell differentiation, embryoid body formation, teratoma assays, and neural tube organoid differentiation. Together, these findings establish a Par complex-AKT-FURIN-LEFTY-ECM-integrin-FAK signaling cascade that links apical-basal polarity to early lineage specification and morphogenesis, providing a mechanistic framework for how polarity cues are translated into developmental outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722465v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1c62c46org.highwire.dtl.DTLVardef@184d5b5org.highwire.dtl.DTLVardef@1ea9017org.highwire.dtl.DTLVardef@9a0318_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementThis study elucidates the molecular mechanism by which the Par complex regulates the establishment of cell polarity. The authors demonstrate that the Par complex promotes the expression of the protein convertase FURIN via AKT signaling, thereby enhancing the maturation and secretion of LEFTY protein. This process remodels the ECM and modulates integrin signaling, ultimately regulating FAK activity and controlling the establishment of cell polarity. These findings reveal how polarity cues govern early lineage specification and morphogenesis, with implications across multiple developmental contexts.

BackgroundAntibody-mediated blockade of innate receptor-MHC-I interactions represents a promising strategy to enhance anti-tumor immunity, particularly against metastatic cancers resistant to conventional checkpoint inhibitors. In this study, we investigated the effects of the pan anti-MHC-I monoclonal antibody M1/42, which targets MHC-I interactions with Ly49, selectively expressed on murine NK cell subsets. MethodsWe administered M1/42 to mice and assayed the proliferation and activation immune cells. Anti-tumor activity of growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcoma (PDAC) and B16F10 melanoma were assessed, complemented by extensive cellular phenotypic and RNA expression analysis. Binding and cryo-electron microscopic (cryo-EM) and X-ray crystallographic structural studies of M1/42 complexed with the mouse MHC-I molecule, H2-Dd, examined the Ab interaction site in comparison with those of Ly49 inhibitory receptors. ResultsM1/42 administration in mice robustly unleashed the proliferation and activation of natural killer (NK) cells, memory CD4+ and CD8+ T cells, dendritic cells, and macrophages in both lymphoid and non-lymphoid tissues, independent of Fc{gamma} receptors. M1/42 significantly restricted the growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcinoma (PDAC) and B16F10 melanoma in the liver and lungs, accompanied by increased tumor infiltration of effector CD8+ T cells, reduction of T regulatory cells, and a pro-inflammatory cytokine milieu. The anti-tumor effects of M1/42 depend on NK cells and are associated with upregulation of genes involved in antigen processing, interferon gamma responsiveness, and Th1 cytokine production, while downregulating inhibitory PD1/11 signaling. Structural analysis indicated that the effect of M1/42 on Ly49/MHC-I interactions was not due to direct steric competition. ConclusionsCollectively, these findings demonstrate that M1/42 unleashes coordinated innate and adaptive immune responses, overcoming tumor-induced immunosuppression and resistance to checkpoint blockade. This approach represents a paradigm shift in cancer immunotherapy, offering potential for more effective treatment of metastatic cancers that evade immune surveillance through MHC-I modulation. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSA pan anti-mouse MHC-I mAb (M1/42) blocks interaction with several NK inhibitory receptors (Ly49A or Ly49C) resulting in NK cell activation and anti-viral and anti-tumor responses in vitro and in vivo. Other pan anti-human MHC-I mAbs (DX17 and W6/32) function similarly, blocking LILRB inhibitory receptor interaction of myeloid cells and NK cells. These stimulate human immune cells in humanized mouse models. What this study addsThis study analyzes the effects of the pan anti-mouse MHC-I mAb on NK and myeloid cell activation in detail, in the absence of T or B cells, and independent of FcR interaction. Additionally we analyze several mouse models of metastatic tumor progression, indicative of the progressive activation not only of the innate immune response, but also adaptive responses. The molecular mechanism of the mAb blocking of inhibitory receptors is revealed by cryo-EM and X-ray structures of M1/42 Fab/MHC-I (H2-Dd) complexes. How this study might affect research, practice, or policyElucidation of the details of the inhibitory effects of the mouse pan anti-mouse MHC-I mAb provides not only a more advanced understanding of the murine model system, but suggests additional functional avenues to be explored using the parallel an anti-human MHC-I mAbs.

BackgroundCDH1 (E-cadherin) is a key epithelial adhesion molecule traditionally associated with tumor suppression and epithelial-mesenchymal transition (EMT). However, its roles across cancers remain incompletely understood, particularly within multilayer regulatory contexts involving genomic, epigenetic, transcriptional, and immune mechanisms. MethodsCDH1 expression, survival associations, EMT-correlated gene profiles (VIM, SNAI1, ZEB1), immune infiltration patterns, immune checkpoint correlations (PDCD1, CD274, CTLA4), promoter methylation, and genomic alterations were assessed across five epithelial cancers, breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), ovarian cancer (OV), and stomach adenocarcinoma (STAD). Cross-platform validation was performed using TCGA/GDC datasets, GEPIA2, UALCAN, TIMER, KM Plotter, cBioPortal, and g:Profiler. ResultsCDH1 was overexpressed but showed variable prognostic significance; higher expression predicted better survival in COAD, LUAD and STAD, worse survival in BRCA and had no impact in OV. Classic inverse relationships between CDH1 and VIM or ZEB1 were evident only in STAD, and SNAI1 showed no consistent association. Immune infiltration patterns were tumor-specific, ranging from cytotoxic T-cell dominance in LUAD to macrophage-rich profiles in OV; immune checkpoint correlations were similarly context-dependent. Co-expressed genes were enriched for endomembrane transport rather than adhesion pathways. Promoter methylation patterns varied by cancer, whereas genomic alterations of CDH1 were rare. ConclusionsCDH1 does not function as a universal epithelial or EMT marker across epithelial cancers. Instead, its associations with EMT, immune contexture, methylation, and prognosis are context-dependent, supporting a model of CDH1 as a heterogeneous regulator of epithelial plasticity. These findings challenge single-function interpretations and support cancer-specific CDH1 evaluation in translational research.