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PURPOSE: To determine if inhibition of Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein, using novel MARCKS inhibitor peptides, will reduce the severity of endotoxin-induced uveitis (EIU) in rats. METHODS: EIU was induced in Lewis rats using subcutaneous administration of lipopolysaccharide. In the first phase of the study, 3 different novel MARCKS inhibitor peptides that mimic the N-terminal region of MARCKS (BIO-11006, or lower molecular weight analogs BIO-91201 or BIO-91202; Biomarck Pharmaceuticals, Ltd., Newtown, PA) were administered intravitreally (IVT) at 50 and 100 μM. In the second phase, BIO-91201 was administered IVT at 10, 50, and 100 μM and topically at the 100 μM concentration. The efficacy of MARCKS inhibitor peptides was assessed by clinical examination using slit lamp biomicroscopy, optical coherence tomography (OCT) anterior chamber cell counts, histopathology, and aqueous humor cytokine analysis. RESULTS: Clinical scores were significantly reduced 24 h following uveitis induction in the first phase of the study in the following treatment groups: BIO-11006 50 μM IVT and 100 μM IVT, BIO-91201 50 μM IVT, and BIO-91202 100 μM IVT (P

Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-022-00517-3.

Background: The dysregulation of circular RNAs (circRNAs) has been shown to be correlated with the aggressiveness of renal cell carcinoma (RCC). Hence, this study investigated the role and mechanism of circ_0039569 in RCC progression. Methods: The levels of circ_0039569, miR-133b, and MARCKS (myristoylated alanine-rich protein kinase C substrate) were detected using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by using 5-ethynyl-2′-deoxyuridine assay, colony-formation assay, Transwell assay, flow cytometry, and Western blot. The direct interactions between miR-133b and circ_0039569 or MARCKS were verified by using dual-luciferase reporter and pull-down assays. Xenograft mice models were established to conduct in vivo analysis. Results: Circ_0039569 was highly expressed in RCC tissues and cells. Functionally, silencing of circ_0039569 suppressed cell proliferation, migration, and invasion, but induced cell apoptosis in RCC cells in vitro. Moreover, mice subcutaneous xenograft assay suggested that circ_0039569 knockdown impeded tumor growth in vivo. Mechanistically, circ_0039569 acted as a sponge for miR-133b to regulate the expression of its target MARCKS. Importantly, miR-133b inhibition or MARCKS knockdown attenuated the anticancer effects of circ_0039569 knockdown on RCC growth. Conclusion: Diminished circ_0039569 restrains the growth and propagation of RCC cells via miR-133b/MARCKS axis, indicating an underlying effective therapeutic target for RCC patients.

Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer (BC). We previously demonstrated that protein overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein was associated with shorter survival in IBC patients. MARCKS has been associated with the PI3K/AKT pathway. MARCKS inhibitors are in development. Our objective was to investigate MARCKS, expressed preferentially in IBC that non-IBC (nIBC), as a novel potential therapeutic target for IBC. The biologic activity of MPS, a MARCKS peptide inhibitor, on cell proliferation, migration, invasion, and mammosphere formation was evaluated in IBC (SUM149 and SUM190) and nIBC (MDA-MB-231 and MCF7) cell lines, as well as its effects on protein expression in the PTEN/AKT and MAPK pathways. The prognostic relevance of MARCKS and phosphatase and tensin homolog (PTEN) protein expression as a surrogate marker of metastasis-free survival (MFS) was evaluated by immunohistochemistry (IHC) in a retrospective series of archival tumor samples derived from 180 IBC patients and 355 nIBC patients. In vitro MPS impaired cell proliferation, migration and invasion, and mammosphere formation in IBC cells. MARCKS inhibition upregulated PTEN and downregulated pAKT and pMAPK expression in IBC cells, but not in nIBC cells. By IHC, MARCKS expression and PTEN expression were negatively correlated in IBC samples and were associated with shorter MFS and longer MFS, respectively, in multivariate analysis. The combination of MARCKS-/PTEN+ protein status was associated with longer MFS in IBC patient only (p = 8.7 × 10−3), and mirrored the molecular profile (MARCKS-downregulated/PTEN-upregulated) of MPS-treated IBC cell lines. In conclusion, our results uncover a functional role of MARCKS implicated in IBC aggressiveness. Associated with the good-prognosis value of the MARCKS-/PTEN+ protein status that mirrors the molecular profile of MPS-treated IBC cell lines, our results suggest that MARCKS could be a potential therapeutic target in patients with MARCKS-positive IBC. Future preclinical studies using a larger panel of IBC cell lines, animal models and analysis of a larger series of clinical samples are warranted in order to validate our results.

Aim and Objective: Cell death is a main pathological change in brain ischemia. Astragalus membranaceus (Ast) and ligustrazine (Lig), as traditional Chinese herbs, have a protective effect against ischemia-reperfusion injury. We aim to find whether the underlying protective mechanism of Astragalus membranaceus and ligustrazine against Oxygen–glucose deprivation/reoxygenation (OGD/R) -induced injury in RBMECs is related to PKCδ/MARCKS pathway. Materials and Methods: OGD/R preconditioning was instituted in rat brain microvascular endothelial cells (RBMECs). The survival and apoptosis of RBMECs were detected by a Cell Counting Kit-8 and TUNEL staining; PKCδ/MARCKS and MMP9 expression were examined by immunofluorescence, western blot and quantitative real-time PCR. Results: OGD/R stimulation significantly increased RBMEC apoptosis, whereas Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment evidently reduced cellular apoptosis and increased cell viability (P Conclusions: The administration of Astragalus membranaceus and ligustrazine protected RBMECs against OGD/R-induced apoptosis. PKCδ/MARCKS and MMP9 expression were significantly increased after OGD/R stimulation, while Astragalus membranaceus and ligustrazine treatment evidently suppressed. Collectively, Astragalus membranaceus and ligustrazine play protective effects against OGD/R-induced injury in RBMECs through regulating PKCδ/MARCKS pathway to inhibit MMP9 activation.

Tobacco smoking is a well-known risk factor for both fibrogenesis and fibrotic progression; however, the mechanisms behind these processes remain enigmatic. RTKs (receptor tyrosine kinases) have recently been reported to drive profibrotic phenotypes in fibroblasts during pulmonary fibrosis (PF). Using a phospho-RTK array screen, we identified the RTK AXL as a top upregulated RTK in response to smoke. Both expression and signaling activity of AXL were indeed elevated in lung fibroblasts exposed to tobacco smoke, whereas no significant change to the levels of a canonical AXL ligand, Gas6 (growth arrest–specific 6), was seen upon smoke treatment. Notably, we found that smoke-exposed human lung fibroblasts exhibited highly proliferative and invasive activities and were capable of inducing fibrotic lung lesions in mice. Conversely, genetic suppression of AXL in smoke-exposed fibroblasts cells led to suppression of AXL downstream pathways and aggressive phenotypes. We further demonstrated that AXL interacted with MARCKS (myristoylated alanine-rich C kinase substrate) and cooperated with MARCKS in regulating downstream signaling activity and fibroblast invasiveness. Pharmacological inhibition of AXL with AXL-specific inhibitor R428 showed selectivity for smoke-exposed fibroblasts. In all, our data suggest that AXL is a potential marker for smoke-associated PF and that targeting of the AXL pathway is a potential therapeutic strategy in treating tobacco smoking–related PF.

Epithelial sodium channel (ENaC) is a pivotal regulator of alveolar fluid clearance in the airway epithelium and plays a key role in the treatment of acute lung injury (ALI), which is mainly composed of the three homologous subunits (α, β and γ). The mechanisms of microRNAs in small extracellular vesicles (sEVs) derived from mesenchymal stem cell (MSC-sEVs) on the regulation of lung ion transport are seldom reported. In this study, we aimed at investigating whether miR-34c had an effect on ENaC dysfunction induced by lipopolysaccharide and explored the underlying mechanism in this process. Primarily, the effect of miR-34c on lung edema and histopathology changes in an ALI mouse model was investigated. Then the uptake of PKH26-labeled sEVs was observed in recipient cells, and we observed that the overexpression of miR-34c in MSC-sEVs could upregulate the LPS-inhibited γ-ENaC expression. The dual luciferase reporter gene assay demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS) was one of target genes of miR-34c, the protein expression of which was negatively correlated with miR-34c. Subsequently, either upregulating miR-34c or knocking down MARCKS could increase the protein expression of phospho-phosphatidylinositol 3-kinase (p-PI3K) and phospho-protein kinase B (p-AKT), implying a downstream regulation pathway was involved. All of the above suggest that miR-34c in MSC-sEVs can attenuate edematous lung injury via enhancing γ-ENaC expression, at least partially, through targeting MARCKS and activating the PI3K/AKT signaling pathway subsequently.

Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.

Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.

Purpose Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. Methods CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). Results We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. Conclusions Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

The myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (MARCKSL1) are ubiquitous, highly conserved membrane-associated proteins involved in the structural modulation of the actin cytoskeleton, chemotaxis, motility, cell adhesion, phagocytosis, and exocytosis. MARCKS includes an N-terminal myristoylated domain for membrane binding, a highly conserved MARCKS Homology 2 (MH2) domain, and an effector domain (which is the phosphorylation site). MARCKS can sequester phosphatidylinositol-4, 5-diphosphate (PIP2) at lipid rafts in the plasma membrane of quiescent cells, an action reversed by protein kinase C (PKC), ultimately modulating the immune function. Being expressed mostly in innate immune cells, MARCKS promotes the inflammation-driven migration and adhesion of cells and the secretion of cytokines such as tumor necrosis factor (TNF). From a clinical point of view, MARCKS is overexpressed in patients with schizophrenia and bipolar disorders, while the brain level of MARCKS phosphorylation is associated with Alzheimer’s disease. Furthermore, MARCKS is associated with the development and progression of numerous types of cancers. Data in autoimmune diseases are limited to rheumatoid arthritis models in which a connection between MARCKS and the JAK-STAT pathway is mediated by miRNAs. We provide a comprehensive overview of the structure of MARCKS, its molecular characteristics and functions from a biological and pathogenetic standpoint, and will discuss the clinical implications of this pathway.

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.—Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho-associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline-inducible system in PTEN-null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non-phosphorylatable ED mutant exerted growth-suppressive and radiation-sensitizing effects, while the pseudo-phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein-protein interactions using co-immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS-associated proteins, including importin-β and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

BACKGROUND Accumulating evidence suggests a connection of Myristoylated alanine-rich C-kinase substrate (MARCKS) with several physiological and pathological processes. However, the relevance of MARCKS in gastric cancer (GC) needs to be elucidated. MATERIAL AND METHODS The abundance of MARCKS in GC tissues was assessed using techniques of immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). Moreover, the MARCKS expression profile in the TCGA database was analyzed through an online website analysis. We also investigated MARCKS function using cell wounding and Matrigel invasion assays. RESULTS TCGA analysis and our data suggest that transcript abundance and protein level of MARCKS was higher in GC tumor samples compared with peri-tumor tissues. There was a remarkable association of upregulated MARCKS with the cell differentiation (P

Deep layer III pyramidal cells in the dorsolateral prefrontal cortex (DLPFC) from subjects with schizophrenia and bipolar disorder previously were shown to exhibit dendritic arbor pathology. This study sought to determine whether MARCKS, its regulatory protein dysbindin-1, and two proteins, identified using microarray data, CDC42BPA and ARHGEF6, were associated with dendritic arbor pathology in the DLPFC from schizophrenia and bipolar disorder subjects. Using western blotting, relative protein expression was assessed in the DLPFC (BA 46) grey matter from subjects with schizophrenia (n = 19), bipolar disorder (n = 17) and unaffected control subjects (n = 19). Protein expression data were then correlated with dendritic parameter data obtained previously. MARCKS and dysbindin-1a expression levels did not differ among the three groups. Dysbindin-1b expression was 26% higher in schizophrenia subjects (p = 0.01) and correlated inversely with basilar dendrite length (r = -0.31, p = 0.048) and the number of spines per basilar dendrite (r = -0.31, p = 0.048), but not with dendritic spine density (r = -0.16, p = 0.32). The protein expression of CDC42BPA was 33% higher in schizophrenia subjects (p = 0.03) but, did not correlate with any dendritic parameter (p > 0.05). ARHGEF6 87 kDa isoform expression did not differ among the groups. CDC42BPA expression was not altered in frontal cortex from rats chronically administered haloperidol or clozapine. Dysbindin-1b appears to play a role in dendritic arbor pathology observed previously in the DLPFC in schizophrenia.

Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

BACKGROUND Hepatocellular carcinoma is the fourth most common cause of cancer-related death. However, the cross-talk between tumor immune microenvironment and hepatocellular carcinoma (HCC) remains unclear. MATERIAL AND METHODS We analyzed the expression of miR-143-3p in exosomes from different HCC cell lines. Differentially expressed genes (DEGs) in Tumor-associated macrophages (TAMs) co-cultured with HCC cell lines were overlapped with miR-143-3p target genes. We used the Oncomine, Kaplan-Meier plotter, and The Cancer Genome Atlas (TCGA) databases to assess Myristoylated alanine-rich C-kinase substrate (MARCKS) expression in various types of cancers. The relationship between patient clinicopathological characteristics and MARCKS expression level was identified using the Kaplan-Meier plotter database. Last, we analyzed how MARCKS expression correlated with immune infiltration makers using the TCGA database, Tumor IMmune Estimation Resource (TIMER), and Gene Expression Profiling Interactive Analysis (GEPIA). RESULTS Exosomal miR-143-3p was elevated after IL-6 treatment in the HCC cell line. MARCKS, a target gene of miR-143-3p, was up-regulated in Tumor-associated macrophages co-cultured with high-metastatic-potential HCC cell line. MARCKS expression was identified as significantly correlated with outcome in multiple types of cancer, especially in HCC. High MARCKS expression level was associated with poorer overall survival (OS), Progress-free survival (PFS), and also with patient gender, race, hepatitis virus background, stage, grade, AJCC_T, and vascular invasion. MARCKS was positively associated with levels of T follicular helper cells (TFH) (R = .48, p

Rationale: Cigarette smoking is a major risk factor for lung cancer development and progression; however, the mechanism of how cigarette smoke activates signaling pathways in promoting cancer malignancy remains to be established. Herein, we aimed to determine the contribution of a signaling protein, myristoylated alanine-rich C kinase substrate (MARCKS), in smoke-mediated lung cancer. Methods: We firstly examined the levels of phosphorylated MARCKS (phospho-MARCKS) in smoke-exposed human lung cancer cells and specimens as well as non-human primate airway epithelium. Next, the MARCKS-interactome and its gene networks were identified. We also used genetic and pharmacological approaches to verify the functionality and molecular mechanism of smoke-induced phospho-MARCKS. Results: We observed that MARCKS becomes activated in airway epithelium and lung cancer cells in response to cigarette smoke. Functional proteomics revealed MARCKS protein directly binds to NF-κB-activating protein (NKAP). Following MARCKS phosphorylation at ser159 and ser163, the MARCKS-NKAP interaction was inhibited, leading to the activation of NF-κB signaling. In a screen of two cohorts of lung cancer patients, we confirmed that phospho-MARCKS is positively correlated with phospho-NF-κB (phospho-p65), and poor survival. Surprisingly, smoke-induced phospho-MARCKS upregulated the expression of pro-inflammatory cytokines, epithelial-mesenchymal transition, and stem-like properties. Conversely, targeting of MARCKS phosphorylation with MPS peptide, a specific MARCKS phosphorylation inhibitor, suppressed smoke-mediated NF-κB signaling activity, pro-inflammatory cytokines expression, aggressiveness and stemness of lung cancer cells. Conclusion: Our results suggest that phospho-MARCKS is a novel NF-kB activator in smoke-mediated lung cancer progression and provide a promising molecular model for developing new anticancer strategies.

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease featured by synovial joint inflammation. Increasing evidence has highlighted microRNAs (miRNAs) and histone deacetylase 1 (HDAC1) as active participants in RA progression. Hence, the present study aims to explore the functions of HDAC1 and miR-124 on synovial cell hyperplasia and synovial inflammation in RA.The expression of HDAC1, miR-124 and MARCKS was determined in the synovial tissues collected from 25 RA patients by RT-qPCR and Western blot analysis. Next, a mouse model with collagen-induced arthritis (CIA) was established, from which fibroblast-like synovial cells (FLSs) were isolated. Then the effect of HDAC1, miR-124 and MARCKS on synovial cell hyperplasia and synovial inflammation in CIA mice was evaluated by HE staining, ELISA, and EdU assays. Afterwards, the interaction among HDAC1, miR-124, MARCKS and the JAK/STAT signalling pathway was assessed by ChIP and dual luciferase reporter assay. Finally, the effect of HDAC1 on RA was further verified by establishing a CIA mouse model.HDAC1 was highly expressed and miR-124 and MARCKS were poorly expressed in synovial tissues of CIA. Silencing HDAC1 inhibited synovial cell hyperplasia and synovial inflammation by elevating MARCKS and miR-124 both in vitro and in vivo. Deficiency of HDAC1 promoted H3 and H4 acetylation of miR-124 and MARCKS promoter region. miR-124 alleviated synovial cell hyperplasia and synovial inflammation by repressing the JAK/STAT signalling pathway in CIA.To sum up, silencing HDAC1 mitigates synovial cell hyperplasia and synovial inflammation in mice with CIA by elevating miR-124 and MARCKS expression, thus highlighting a promising competitive new target for RA treatment.

Alzheimer's disease (AD) is the most common neurodegenerative disorder caused by neuronal loss that results in cognitive and functional impairment. Formation of neurofibrillary tangles composed of abnormal hyperphosphorylation of tau protein is one of the major pathological hallmarks of AD. Importantly, several neurodegenerative disorders, including AD, are associated with abnormal protein phosphorylation events. However, little is known thus far on global protein phosphorylation changes in AD. We report a phosphoproteomics study examining the frontal gyrus of people with AD and age-matched cognitively normal subjects, using tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides. We identified 4631 phosphopeptides corresponding to 1821 proteins with liquid chromatography-mass spectrometry (MS)/MS analysis on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Of these, 504 phosphopeptides corresponding to 350 proteins were significantly altered in the AD brain: 389 phosphopeptides increased whereas 115 phosphopeptides decreased phosphorylation. We observed significant changes in phosphorylation of known as well as novel molecules. Using targeted parallel reaction monitoring experiments, we validated the phosphorylation of microtubule-associated protein tau and myristoylated alanine-rich protein kinase C substrate (MARCKS) in control and AD (Control = 6, AD = 11) brain samples. In conclusion, our study provides new evidence on alteration of RNA processing and splicing, neurogenesis and neuronal development, and metabotropic glutamate receptor 5 (GRM5) calcium signaling pathways in the AD brain, and it thus offers new insights to accelerate diagnostics and therapeutics innovation in AD.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an intracellular receptor for polysialic acid. MARCKS supports development, synaptic plasticity, and regeneration after injury. MARCKS binds with its functionally essential effector domain (ED) to polysialic acid. A 25-mer peptide comprising the ED of MARCKS stimulates neuritogenesis of primary hippocampal neurons after addition to the culture. This motivated us to investigate whether ED peptide has similar effects in spinal cord injury. ED peptide supported recovery and regrowth of monoaminergic axons in female, but not in male mice. Sex-specific differences in response to ED peptide application also occurred in cultured neurons. In female but not male neurons, the ED peptide enhanced neurite outgrowth that could be suppressed by inhibitors of the estrogen receptors α and β, fibroblast growth factor receptor-1, protein kinase C, and matrix metalloproteinase 2. In addition, we observed female-specific elevation of phosphorylated MARCKS levels after ED peptide treatment. In male neurons, the ED peptide enhanced neuritogenesis in the presence of an androgen receptor inhibitor to the extent seen in ED peptide-treated female neurons. However, inhibition of androgen receptor did not lead to increased phosphorylation of MARCKS. These results provide insights into the functions of a novel compound contributing to gender-dependent regeneration.

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity., Graphical abstract Unlabelled Image

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks−/− cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

BACKGROUND Male breast cancer (MBC) is a rare and aggressive disease. Thus, identification of new therapeutic targets is crucial. OBJECTIVE Our objective was to evaluate the protein expression of MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in MBC and to investigate its prognostic value. MATERIALS AND METHODS MARCKS protein expression in tumor and stromal cells was analyzed by immunohistochemistry (IHC) in a retrospective series of 96 pre-chemotherapy MBC samples and 80 normal breast samples, from Tunisian patients treated at Salah Azaiez Institute. Correlations were searched between MARCKS expression and clinicopathological features including overall survival (OS). RESULTS MARCKS was overexpressed in epithelial tumor cells in 66% of the MBC samples versus 26% of normal samples (p= 1.40 × 10-7). Such positive MARCKS expression in epithelial tumor cells was associated with positive HER2 status (p= 4.0 × 10-3). It was associated with shorter OS in uni-and multivariate analysis. By contrast, stromal IHC MARCKS expression was correlated only with tumor grade. CONCLUSION MARCKS tumor cell overexpression might in part explain the aggressiveness and the poor prognosis of MBC. MARCKS can represent a potential therapeutic target for MBC.

Overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is implicated in drug resistance and progression of multiple myeloma (MM). The basis for MARCKS induction and impact on MM are not known. Here we show that microRNA-34a (miR-34a), regulates MARCKS translation and is under-expressed in drug-resistant MM cells, leading to increased MARCKS protein level. Over-expression of miR-34a reduces MARCKS expression and sensitizes resistant cells to anti-myeloma drugs. A MARCKS peptide inhibitor (MPS) exerts a dose dependent cytotoxic effect on drug-resistant MM cells with minimal cytotoxicity to normal hematopoietic cells. MPS synergizes with the proteasomal-inhibitor bortezomib to effectively kill drug-resistant MM cells both in vitro and in a xenograft model of MM. While MARCKS inhibition killed MM cells, it also enhanced a pro-survival autophagic pathway that sustained growth following MARCKS inhibition. In accordance, combined treatment with MARCKS antagonists, bortezomib and the autophagy inhibitor, chloroquine, significantly diminished tumor growth in drug-resistant MM cell lines as well as primary MM cells. This study uncovers a mechanism of drug resistance involving miR-34a-MARCKS autoregulatory loop and provides a framework for a potentially new therapeutic strategy to overcome drug resistance in multiple myeloma.

Axons of the corpus callosum (CC), the white matter tract that connects the left and right hemispheres of the brain, receive instruction from a number of chemoattractant and chemorepulsant cues during their initial navigation towards and across the midline. While it has long been known that the CC is malformed in the absence of Myristoylated alanine-rich C-kinase substrate (MARCKS), evidence for a direct role of MARCKS in axon navigation has been lacking. Here, we show that MARCKS is necessary for netrin-1 (NTN1) signaling through the DCC receptor, which is critical for axon guidance decisions. Marcks null (Marcks(−/−)) neurons fail to respond to exogenous NTN1 and are deficient in markers of DCC activation. Without MARCKS, the subcellular distributions of two critical mediators of NTN1-DCC signaling, the tyrosine kinases PTK2 and SRC, are disrupted. Together, this work establishes a novel role for MARCKS in axon dynamics and highlights the necessity of MARCKS as an organizer of DCC signaling at the membrane.

We developed a highly sensitive silicon platform, suitable to assess the molecular organization of protein samples. Prototype platforms were obtained using different electrochemical protocols for the electrodeposition of Ag-nanoparticles onto the hydrogenated silicon surface. A platform with high Surface Enhanced Raman Scattering efficiency was selected based on the surface coverage and the number density of particles size distribution. The performance of the platform was determined by studying the interaction of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein with the substrate according to its molecular organization. The chemical and structural characteristics of MARCKS molecules were examined under two configurations: i) a disordered distribution given by a MARCKS solution drop deposited onto the platform and, ii) a compact monolayer transferred to the platform by the Langmuir-Blodgett method. Raman spectra show vibrational bands of Phenylalanine and Lysine residues specific for the protein effector domain, and evidence the presence of alpha helix structure in both configurations. Moreover, we distinguished the supramolecular order between the compact monolayer and random molecular distribution. The platforms containing Ag-nanoparticles are suitable for studies of protein structure and interactions, advancing a methodological strategy for our long term goal, which is to explore the interaction of proteins with model membranes. Fil: Klug, Joaquín. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina Fil: Torresan, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Lurgo, Florencia Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Borioli, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Lacconi, Gabriela Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina