Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma.

BACKGROUND: MARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. METHODS: Immunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). RESULTS: Elevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. CONCLUSION: These findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.

CircTRIM1 encodes TRIM1-269aa to promote chemoresistance and metastasis of TNBC via enhancing CaM-dependent MARCKS translocation and PI3K/AKT/mTOR activation.

Peptides and proteins encoded by noncanonical open reading frames (ORFs) of circRNAs have recently been recognized to play important roles in disease progression, but the biological functions and mechanisms of these peptides and proteins are largely unknown. Here, we identified a potential coding circular RNA, circTRIM1, that was upregulated in doxorubicin-resistant TNBC cells by intersecting transcriptome and translatome RNA-seq data, and its expression was correlated with clinicopathological characteristics and poor prognosis in patients with TNBC. CircTRIM1 possesses a functional IRES element along with an 810 nt ORF that can be translated into a novel endogenously expressed protein termed TRIM1-269aa. Functionally, we demonstrated that TRIM1-269aa, which is involved in the biological functions of circTRIM1, promoted chemoresistance and metastasis in TNBC cells both in vitro and in vivo. In addition, we found that TRIM1-269aa can be packaged into exosomes and transmitted between TNBC cells. Mechanistically, TRIM1-269aa enhanced the interaction between MARCKS and calmodulin, thus promoting the calmodulin-dependent translocation of MARCKS, which further initiated the activation of the PI3K/AKT/mTOR pathway. Overall, circTRIM1, which encodes TRIM1-269aa, promoted TNBC chemoresistance and metastasis by enhancing MARCKS translocation and PI3K/AKT/mTOR activation. Our investigation has yielded novel insights into the roles of protein-coding circRNAs and supported circTRIM1/TRIM1-269aa as a novel promising prognostic and therapeutic target for patients with TNBC.

Binding-induced lipid domains: Peptide-membrane interactions with PIP2 and PS.

Cell signaling is an important process involving complex interactions between lipids and proteins. The myristoylated alanine-rich C-kinase substrate (MARCKS) has been established as a key signaling regulator, serving a range of biological roles. Its effector domain (ED), which anchors the protein to the plasma membrane, induces domain formation in membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). The mechanisms governing the MARCKS-ED binding to membranes remain elusive. Here, we investigate the composition-dependent affinity and MARCKS-ED-binding-induced changes in interfacial environments using two-dimensional infrared spectroscopy and fluorescence anisotropy. Both negatively charged lipids facilitate the MARCKS-ED binding to lipid vesicles. Although the hydrogen-bonding structure at the lipid-water interface remains comparable across vesicles with varied lipid compositions, the dynamics of interfacial water show divergent patterns due to specific interactions between lipids and peptides. Our findings also reveal that PIP2 becomes sequestered by bound peptides, while the distribution of PS exhibits no discernible change upon peptide binding. Interestingly, PIP2 and PS become colocalized into domains both in the presence and absence of MARCKS-ED. More broadly, this work offers molecular insights into the effects of membrane composition on binding.

Emerging role of microRNAs as regulators of protein kinase C substrate MARCKS and MARCKSL1 in cancer.

MicroRNAs (miRNAs) have emerged as pivotal regulators of gene expression, playing essential roles in diverse cellular processes, including the development and progression of cancer. Among the numerous proteins influenced by miRNAs, the MARCKS/MARCKSL1 protein, a key regulator of cellular cytoskeletal dynamics and membrane-cytosol communication, has garnered significant attention due to its multifaceted involvement in various cancer-related processes, including cell migration, invasion, metastasis, and drug resistance. Motivated by the encouraging early clinical success of peptides targeting MARCKS in several pathological conditions, this review article delves into the intricate interplay between miRNAs and the MARCKS protein in cancer. Herein, we have highlighted the latest findings on specific miRNAs that modulate MARCKS/MARCKSL1 expression, providing a comprehensive overview of their roles in different cancer types. We have underscored the need for in-depth investigations into the therapeutic feasibility of targeting the miRNA-MARCKS axis in cancer, taking cues from the successes witnessed in related fields. Unlocking the full potential of miRNA-mediated MARCKS regulation could pave the way for innovative and effective therapeutic interventions against various cancer types.

Patent landscape highlighting therapeutic implications of peptides targeting myristoylated alanine-rich protein kinase-C substrate (MARCKS).

INTRODUCTION: MARCKS protein, a protein kinase C (PKC) substrate, is known to be at the intersection of several intracellular signaling pathways and plays a pivotal role in cellular physiology. Unlike PKC inhibitors, MARCKS-targeting drug (BIO-11006) has shown early success in clinical trials involving lung diseases. Recent research investigations have identified two MARCKS-targeting peptides which possess multifaceted implications against asthma, cancer, inflammation, and lung diseases. AREAS COVERED: This review article provides the patent landscape and recent developments on peptides targeting MARCKS for therapeutic purposes. Online free open-access databases were used to fetch out the patent information, and research articles were fetched using PubMed. EXPERT OPINION: Research studies highlighting the intriguing role of MARCKS in human disease and physiology have dramatically increased in recent years. A similar increasing trend in the number of patents has also been observed related to the MARCKS-targeting peptides. Thus, there is a need to amalgamate and translate such a trend into therapeutic intervention. Our review article provides an overview of such recent advances, and we believe that our compilation will fetch the interest of researchers around the globe to develop MARCKS-targeting peptides in future for human diseases.

Small Extracellular Vesicles Containing miR-34c Derived from Bone Marrow Mesenchymal Stem Cells Regulates Epithelial Sodium Channel via Targeting MARCKS.

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, ß 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.

Peptide Inhibitors of MARCKS Suppress Endotoxin Induced Uveitis in Rats.

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 < 0.05). OCT anterior chamber cell counts were significantly reduced in the first phase of the study in all treatment groups (P < 0.001). OCT anterior chamber cell counts and histopathology scores were significantly reduced in the second phase of the study in the BIO-91201 50 µM IVT group (P < 0.05). No effect was seen with topical administration. Conclusion: MARCKS inhibitor peptides were effective in reducing the severity of ocular inflammation and cellular influx in EIU.

Circ_0039569 Competes with MARCKS for miR-133b Binding Sites to Promote the Progression of Renal Cell Carcinoma.

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.

MARCKS on Tumor-Associated Macrophages is Correlated with Immune Infiltrates and Poor Prognosis in Hepatocellular Carcinoma.

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 < .001), T helper type 2 (Th2) cells (R = .47, p < .001), macrophages (R = .41, p ≤ .001), T helper cells (R = .40, p < .001), T helper type 1 (Th1) cells (R = .38, p < .001), T cells (R = .34, p < .001), NK CD56bright cells (R = .34, p < .001) and immature DC (iDC) (R = .33, p < .001), and negatively associated with levels of T helper 17 (Th17) cells. Also, MARCKS may influence the M2 polarization and immune escape. CONCLUSION: The present study suggests that MARCKS on TAMs is associated with poor prognosis and immune cell infiltration in HCC.

Targeting the AXL Receptor in Combating Smoking-related Pulmonary Fibrosis.

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.

MARCKS affects cell motility and response to BTK inhibitors in CLL.

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.

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer.

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.

Myristoylated alanine-rich C-kinase substrate effector domain peptide improves sex-specific recovery and axonal regrowth after spinal cord injury.

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 ß, 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.

MicroRNA-142-3p attenuates hepatic ischemia/reperfusion injury via targeting of myristoylated alanine-rich C-kinase substrate.

MiR-142-3p as one key molecule in oncogenesis and inflammation plays crucial roles in hepatic fibrosis, hepatocellular carcinoma and other liver disease. However, there have no literatures to report its effects on hepatic ischemia-reperfusion (HI/R) injury. In the present work, hypoxia reoxygenation (H/R) models on AML12 and HepG2 cells, and ischemia/reperfusion model in mice were established. The methods of real-time PCR, dual luciferase reporter, mimic, inhibitor, agomir, antagomir and siRNA transfection assays were used. The expression levels of miR-142-3p were decreased in model groups in vitro and in vivo compared with control group or Sham group, which directly targeted MARCKS to regulate its expression. Then, MARCKS activated p38/JNK signal, up-regulated NF-κB expression to accelerate inflammation, and inhibited PI3K/AKT signal to promote apoptosis. Moreover, miR-142-3p mimic in vitro and agomir in vivo lowered the expression levels of MARCKS, thereby alleviating apoptosis and inflammation to relieve HI/R injury. Furthermore, miR-142- 3p inhibitor in vitro and antagomir in vivo up-regulated the expression levels of MARCKS to aggravate HI/R damage via promoting inflammation and apoptosis. Consistently, MARCKS siRNA markedly inhibited HI/R injury by restraining apoptosis and inflamm- ation in mice. MiR-142-3p played a considerable part in adjusting HI/R injury by targeting MARCKS, and miR-142-3p/MARCKS should be a new therapeutic target for HI/R injury.

Restoration of MARCKS enhances chemosensitivity in cancer.

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.

MARCKS protein overexpression is associated with poor prognosis in male breast cancer.

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.

Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

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.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

OBJECTIVE: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 µM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol­3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine­rich C­kinase 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 Rho­associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline­inducible system in PTEN­null 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 non­phosphorylatable ED mutant exerted growth­suppressive and radiation­sensitizing effects, while the pseudo­phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein­protein interactions using co­immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS­associated proteins, including importin­ß 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.

Myristoylated alanine rich protein kinase C substrate is a potential cancer prognostic factor that regulates cell migration and invasion in glioblastoma.

Myristoylated alanine­rich C­kinase substrate (MARCKS) serves an important role in various pathological processes in several malignancies. However, little is known about the specific role and molecular mechanism of MARCKS in glioblastoma (GBM). In the present study, it was found that the expression of MARCKS was significantly upregulated in GBM, and was associated with a poor clinical outcome in patients with GBM. Knockdown of MARCKS suppressed the migration and invasion of GBM cells in vitro. Western blotting showed that the knockdown of MARCKS reduced the expression of phosphorylated phosphoinositide 3­kinase and protein kinase B, as well as zinc finger protein SNAI1 expression, thereby modulating the expression of its downstream epithelial­mesenchymal transition (EMT)­associated factors, including E­cadherin, vimentin, N­cadherin and ß­catenin in GBM cells. These results indicate that MARCKS functioned in the migration and invasion of GBM, and therefore may provide a potential therapeutic target in GBM therapy.