Exploring the Enigma: The Role of the Epithelial Protein Lost in Neoplasm in Normal Physiology and Cancer Pathogenesis.

The cytoskeleton plays a pivotal role in maintaining the epithelial phenotype and is vital to several hallmark processes of cancer. Over the past decades, researchers have identified the epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) as a key regulator of cytoskeletal dynamics, cytoskeletal organization, motility, as well as cell growth and metabolism. Dysregulation of EPLIN is implicated in various aspects of cancer progression, such as tumor growth, invasion, metastasis, and therapeutic resistance. Its altered expression levels or activity can disrupt cytoskeletal dynamics, leading to aberrant cell motility and invasiveness characteristic of malignant cells. Moreover, the involvement of EPLIN in cell growth and metabolism underscores its significance in orchestrating key processes essential for cancer cell survival and proliferation. This review provides a comprehensive exploration of the intricate roles of EPLIN across diverse cellular processes in both normal physiology and cancer pathogenesis. Additionally, this review discusses the possibility of EPLIN as a potential target for anticancer therapy in future studies.

Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes.

Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells even over long distances and promoting the transport of different cellular cargos. NTs are also involved in the intercellular spread of different pathogens and the deterioration of some neurological disorders. Transport processes via NTs may be controlled by cytoskeletal elements. NTs are frequently observed membrane projections in numerous mammalian cell lines, including various immune cells, but their functional significance in the 'antibody factory' B cells is poorly elucidated. Here, we report that as active channels, NTs of B-lymphoma cells can mediate bidirectional mitochondrial transport, promoted by the cooperation of two different cytoskeletal motor proteins, kinesin along microtubules and myosin VI along actin, and bidirectional transport processes are also supported by the heterogeneous arrangement of the main cytoskeletal filament systems of the NTs. We revealed that despite NTs and axons being different cell extensions, the mitochondrial transport they mediate may exhibit significant similarities. Furthermore, we found that microtubules may improve the stability and lifespan of B-lymphoma-cell NTs, while F-actin strengthens NTs by providing a structural framework for them. Our results may contribute to a better understanding of the regulation of the major cells of humoral immune response to infections.

Anisotropic power-law viscoelasticity of living cells is dominated by cytoskeletal network structure.

During physiological and pathological processes, cells experience significant morphological alterations with the re-arrangement of cytoskeletal filaments, resulting in anisotropic viscoelasticity. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. We investigate how cell shape affects its creep responses in longitudinal and perpendicular directions. It is shown that cells exhibit power-law rheological behavior in both longitudinal and perpendicular directions under step stress, with a more solid-like behavior along the longitudinal direction. We reveal that the cell volume and cytoskeletal filament orientation, which have been neglected in most existing models, play a critical role in regulating cellular anisotropic viscoelasticity. The stiffness of the cell in both directions increases linearly with increasing its aspect ratio, due to the decrease of cell volume. Moreover, the increase in the cell's aspect ratio produces the aggregation of cytoskeletal filaments along the longitudinal direction, resulting in higher stiffness in this direction. It is also shown that the increase in cell's aspect ratio corresponds to a process of cellular ordering, which can be quantitatively characterized by the orientational entropy of cytoskeletal filaments. In addition, we present a simple yet robust method to establish the relationship between cell's aspect ratio and cell volume, thus providing a theoretical framework to capture the anisotropic viscoelastic behavior of cells. This study suggests that the structure-based cell models may be further developed to investigate cellular rheological responses to external mechanical stimuli and may be extended to the tissue scale. STATEMENT OF SIGNIFICANCE: The viscoelastic behaviors of cells hold significant importance in comprehending the roles of mechanical forces in embryo development, invasion, and metastasis of cancer cells. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. Our study highlights the crucial role of previously neglected factors, such as cell volume and cytoskeletal filament orientation, in regulating cellular anisotropic viscoelasticity. We further propose an orientational entropy of cytoskeletal filaments to quantitatively characterize the ordering process of cells with increasing aspect ratios. Moreover, we derived the analytical interrelationships between cell aspect ratio, cell stiffness, cell volume, and cytoskeletal fiber orientation. This study provides a theoretical framework to describe the anisotropic viscoelastic mechanical behavior of cells.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases.

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors.

Multifaceted roles of PDCD6 both within and outside the cell.

Programmed cell death protein 6 (PDCD6) is an evolutionarily conserved Ca2+-binding protein. PDCD6 is involved in regulating multifaceted and pleiotropic cellular processes in different cellular compartments. For instance, nuclear PDCD6 regulates apoptosis and alternative splicing. PDCD6 is required for coat protein complex II-dependent endoplasmic reticulum-to-Golgi apparatus vesicular transport in the cytoplasm. Recent advances suggest that cytoplasmic PDCD6 is involved in the regulation of cytoskeletal dynamics and innate immune responses. Additionally, membranous PDCD6 participates in membrane repair through endosomal sorting complex required for transport complex-dependent membrane budding. Interestingly, extracellular vesicles are rich in PDCD6. Moreover, abnormal expression of PDCD6 is closely associated with many diseases, especially cancer. PDCD6 is therefore a multifaceted but pivotal protein in vivo. To gain a more comprehensive understanding of PDCD6 functions and to focus and stimulate PDCD6 research, this review summarizes key developments in its role in different subcellular compartments, processes, and pathologies.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Two-component macrophage model for active phagocytosis with pseudopod formation.

Macrophage phagocytosis is critical for the immune response, homeostasis regulation, and tissue repair. This intricate process involves complex changes in cell morphology, cytoskeletal reorganization, and various receptor-ligand interactions controlled by mechanical constraints. However, there is a lack of comprehensive theoretical and computational models that investigate the mechanical process of phagocytosis in the context of cytoskeletal rearrangement. To address this issue, we propose a novel coarse-grained mesoscopic model that integrates a fluid-like cell membrane and a cytoskeletal network to study the dynamic phagocytosis process. The growth of actin filaments results in the formation of long and thin pseudopods, and the initial cytoskeleton can be disassembled upon target entry and reconstructed after phagocytosis. Through dynamic changes in the cytoskeleton, our macrophage model achieves active phagocytosis by forming a phagocytic cup utilizing pseudopods in two distinct ways. We have developed a new algorithm for modifying membrane area to prevent membrane rupture and ensure sufficient surface area during phagocytosis. In addition, the bending modulus, shear stiffness, and cortical tension of the macrophage model are investigated through computation of the axial force for the tubular structure and micropipette aspiration. With this model, we simulate active phagocytosis at the cytoskeletal level and investigate the mechanical process during the dynamic interplay between macrophage and target particles.

Type III intermediate filaments in redox interplay: key role of the conserved cysteine residue.

Intermediate filaments (IFs) are cytoskeletal elements involved in mechanotransduction and in the integration of cellular responses. They are versatile structures and their assembly and organization are finely tuned by posttranslational modifications. Among them, type III IFs, mainly vimentin, have been identified as targets of multiple oxidative and electrophilic modifications. A characteristic of most type III IF proteins is the presence in their sequence of a single, conserved cysteine residue (C328 in vimentin), that is a hot spot for these modifications and appears to play a key role in the ability of the filament network to respond to oxidative stress. Current structural models and experimental evidence indicate that this cysteine residue may occupy a strategic position in the filaments in such a way that perturbations at this site, due to chemical modification or mutation, impact filament assembly or organization in a structure-dependent manner. Cysteine-dependent regulation of vimentin can be modulated by interaction with divalent cations, such as zinc, and by pH. Importantly, vimentin remodeling induced by C328 modification may affect its interaction with cellular organelles, as well as the cross-talk between cytoskeletal networks, as seems to be the case for the reorganization of actin filaments in response to oxidants and electrophiles. In summary, the evidence herein reviewed delineates a complex interplay in which type III IFs emerge both as targets and modulators of redox signaling.

A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.

Effect of LDL Extracted from Human Plasma on Membrane Stiffness in Living Endothelial Cells and Macrophages via Scanning Ion Conductance Microscopy.

Mechanical properties of living cells play a crucial role in a wide range of biological functions and pathologies, including atherosclerosis. We used low-stress Scanning Ion-Conductance Microscopy (SICM) correlated with confocal imaging and demonstrated the topographical changes and mechanical properties alterations in EA.hy926 and THP-1 exposed to LDL extracted from CVD patients' blood samples. We show that the cells stiffened in the presence of LDL, which also triggered caveolae formation. Endothelial cells accumulated less cholesterol in the form of lipid droplets in comparison to THP-1 cells based on fluorescence intensity data and biochemical analysis; however, the effect on Young's modulus is higher. The cell stiffness is closely connected to the distribution of lipid droplets along the z-axis. In conclusion, we show that the sensitivity of endothelial cells to LDL is higher compared to that of THP-1, triggering changes in the cytoskeleton and membrane stiffness which may result in the increased permeability of the intima layer due to loss of intercellular connections and adhesion.

Structural analysis of PTPN21 reveals a dominant-negative effect of the FERM domain on its phosphatase activity.

PTPN21 belongs to the four-point-one, ezrin, radixin, moesin (FERM) domain-containing protein tyrosine phosphatases (PTP) and plays important roles in cytoskeleton-associated cellular processes like cell adhesion, motility, and cargo transport. Because of the presence of a WPE loop instead of a WPD loop in the phosphatase domain, it is often considered to lack phosphatase activity. However, many of PTPN21's biological functions require its catalytic activity. To reconcile these findings, we have determined the structures of individual PTPN21 FERM, PTP domains, and a complex between FERM-PTP. Combined with biochemical analysis, we have found that PTPN21 PTP is weakly active and is autoinhibited by association with its FERM domain. Disruption of FERM-PTP interaction results in enhanced ERK activation. The oncogenic HPV18 E7 protein binds to PTP at the same location as PTPN21 FERM, indicating that it may act by displacing the FERM domain from PTP. Our results provide mechanistic insight into PTPN21 and benefit functional studies of PTPN21-mediated processes.

Keratin Expression in Podocytopathies, ANCA-Associated Vasculitis and IgA Nephropathy.

Keratins are the main components of the cell cytoskeleton of epithelial cells. Epithelial cells under stressful stimuli react by modifying their keratin expression pattern. Glomerular diseases are pathological conditions that may lead to loss of kidney function if not timely diagnosed and treated properly. This study aims to examine glomerular and tubular keratin expression in podocytopathies, ANCA-associated vasculitis, and IgA nephropathy and how this expression correlates to clinical outcomes. We included 45 patients with podocytopathies (minimal change disease and focal segmental glomerulosclerosis), ANCA-associated vasculitis, and IgA nephropathy, with or without crescentic lesions, and healthy controls. All tissues were assessed by photon microscopy and immunohistochemistry. Biopsy sections were examined for keratins 7, 8, 18, and 19 expression in the glomerular and tubulointerstitial areas separately. Moreover, we examined how keratin expression was correlated with long-term kidney function outcomes. All four studied keratins had significantly increased glomerular expression in patients with ANCA vasculitis compared to controls and MCD patients. Tubular expression of keratins 7, 8, and 19 was related to kidney outcome in all groups. Patients with crescents had higher expression of all keratins in both glomeruli and tubulointerstitium. The presence of tubular atrophy, interstitial fibrosis, mesangial hyperplasia, and interstitial inflammation did not affect keratin expression. Keratins, an abundant component of renal epithelial cells, have the potential to be featured as a biomarker for kidney function prognosis in patients with glomerular diseases.

Myosin F controls actin organization and dynamics in Toxoplasma gondii.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.

Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells.

The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule interaction.

Tauopathies such as Alzheimer's disease are characterized by aggregation and increased phosphorylation of the microtubule-associated protein tau. Tau's pathological changes are closely linked to neurodegeneration, making tau a prime candidate for intervention. We developed an approach to monitor pathological changes of aggregation-prone human tau in living neurons. We identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that interact with tau and modulate tau kinases. We found that PHOX15 inhibits tau aggregation, restores tau's physiological microtubule interaction, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and suggest that PHOX15 binding reduces the protofilament's ability to adopt a PHF-like conformation by modifying a key glycine triad. Our data demonstrate that live-cell imaging of a tauopathy model enables screening of compounds that modulate tau-microtubule interaction and allows identification of a promising polypharmacological drug candidate that simultaneously inhibits tau aggregation and reduces tau phosphorylation.

Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

HKDC1 promotes tumor immune evasion in hepatocellular carcinoma by coupling cytoskeleton to STAT1 activation and PD-L1 expression.

Immune checkpoint blockade (ICB) has shown considerable promise for treating various malignancies, but only a subset of cancer patients benefit from immune checkpoint inhibitor therapy because of immune evasion and immune-related adverse events (irAEs). The mechanisms underlying how tumor cells regulate immune cell response remain largely unknown. Here we show that hexokinase domain component 1 (HKDC1) promotes tumor immune evasion in a CD8+ T cell-dependent manner by activating STAT1/PD-L1 in tumor cells. Mechanistically, HKDC1 binds to and presents cytosolic STAT1 to IFNGR1 on the plasma membrane following IFNγ-stimulation by associating with cytoskeleton protein ACTA2, resulting in STAT1 phosphorylation and nuclear translocation. HKDC1 inhibition in combination with anti-PD-1/PD-L1 enhances in vivo T cell antitumor response in liver cancer models in male mice. Clinical sample analysis indicates a correlation among HKDC1 expression, STAT1 phosphorylation, and survival in patients with hepatocellular carcinoma treated with atezolizumab (anti-PD-L1). These findings reveal a role for HKDC1 in regulating immune evasion by coupling cytoskeleton with STAT1 activation, providing a potential combination strategy to enhance antitumor immune responses.