Alanine-rich C-kinase Substrate Research Articles

Stromal Expression of MARCKS Protein in Ovarian Carcinomas Has Unfavorable Prognostic Value.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 × 10−3). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 × 10−9). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.

Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers.

Emerging evidence implicates myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of protein kinase C (PKC), in a critical role for cancer development and progression. MARCKS is tethered to the plasma membrane but can shuttle between the cytosol and plasma membrane via the myristoyl-electrostatic switch. Phosphorylation of MARCKS by PKC leads to its translocation from the plasma membrane to the cytosol where it functions in actin cytoskeletal remodeling, Ca2+ signaling through binding to calmodulin, and regulation of exocytic vesicle release in secretory cells such as neurons and airway goblet cells. Although the contribution of MARCKS to various cellular processes has been extensively studied, its roles in neoplastic disease have been conflicting. This review highlights the molecular and functional differences of MARCKS that exist between normal and tumor cells. We also discuss the recent advances in the potential roles of MARCKS in tumorigenesis, metastasis, and resistance to anti-cancer therapies, with a focus on addressing the inconsistent results regarding the function of MARCKS as a promoter or inhibitor of oncogenesis.

Inhibition of AKT1 signaling promotes invasion and metastasis of non-small cell lung cancer cells with K-RAS or EGFR mutations

Accumulating evidence supports a role of the PI3K-AKT pathway in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote metastasis, however under certain circumstances, it also shows an inhibitory activity on metastatic processes, and the cause of such conflicting results is largely unclear. Here we found that AKT1 is an important regulator of metastasis and down-regulation of its activity is associated with increased metastatic potential of A549 cells. Inhibition of AKT1 enhanced migration and invasion in KRAS- or EGFR-mutant non-small cell lung cancer (NSCLC) cells. The allosteric AKT inhibitor MK-2206 promoted metastasis of KRAS-mutated A549 cells in vivo. We next identified that the phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and LAMC2 protein level were increased with AKT1 inhibition, and MARCKS or LAMC2 knockdown abrogated migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in the NSCLC cells with KRAS or EGFR mutations, and establishes an AKT1-MARCKS-LAMC2 feedback loop in this regulation.

CSF-Based Analysis for Identification of Potential Serum Biomarkers of Neural Tube Defects.

The protein composition of cerebrospinal fluid (CSF) in neural tube defects (NTDs) remains unknown. We investigated the protein composition of CSF from 9 infants with NTDs using isobaric tags for relative and absolute quantitation (iTRAQ). We identified 568 proteins in the CSF of infants with spina bifida, which is the most common type of NTD. Among these, 18 proteins were associated with neural tube closure in the CSF during human embryonic neurulation and 5 were involved in NTDs. Based on these results, an animal model was further utilized to investigate early serum biomarkers for NTDs. We found that the myristoylated alanine-rich C-kinase substrate, Kunitz-type protease inhibitor 2, and apolipoprotein B-100 protein levels were decreased in both embryos and the sera of pregnant Sprague-Dawley rats carrying embryos with NTDs. CSF proteins may be useful in the discovery of potential serum biomarkers for NTDs.

Abstract 4893: AKT1 signaling negatively regulates invasion and metastasis of non-small cell lung cancer cells carrying mutations in KRAS or EGFR

Abstract Signaling driven by the phosphoinositide-3 kinase (PI3K) and AKT/protein kinase B (PKB) pathway plays an important role in cancer development and progression, and thus this pathway is an attractive therapeutic target. Although many different inhibitors targeting either PI3K or AKT have been developed, the therapeutic benefit of these inhibitors in the clinic remains elusive, in part because of the perplexing role of AKT signaling in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote migration, invasion and metastasis; however under certain circumstances and different cellular systems, it also exhibits an inhibitory activity on these cellular processes and the underlying cause of these conflicting results is unclear. Through analysis of the Reverse Phase Protein Array (RPPA) performed on a series of non-small cell lung cancer (NSCLC) brain metastasis models generated via intracardiac injection in mice, we identified AKT1 (one of three AKT family members) as a critical negative regulator of A549 tumor metastasis. AKT1 tyrosine phosphorylation (but not AKT2 or AKT3) inversely correlated with the metastatic potential of A549 cells which carry a KRAS mutation. Using AKT1 siRNAs, we demonstrated that inhibition of AKT1 significantly enhanced migration and invasion of KRAS- or EGFR-mutant NSCLC cells, but not wild type KRAS and EGFR cells. Similarly, the AKT inhibitor MK-2206 also enhanced the migration and invasion of KRAS- or EGFR-mutant NSCLC cells in vitro, and promoted metastasis of KRAS-mutant A549 cells in vivo. The PI3K-AKT pathway is downstream of both EGFR and KRAS, whose constitutive activation caused by mutations likely alter the signal output of PI3K-AKT pathway and thus the activity on cell invasiveness. Therefore, our results provide a sound explanation that under certain genetic backgrounds (as we demonstrated here with KRAS or EGFR mutations) the AKT signaling may switch from being a promoter to an inhibitor of cell migration, invasion and metastasis. This observation may have strong clinical implications for the development of inhibitors of this pathway for cancer treatment. Furthermore, we found that AKT1 blockade induced phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and elevation of Laminin subunit gamma-2 (LAMC2), which in turn inhibited AKT phosphorylation and promoted cell invasiveness. In contrast, MARCKS or LAMC2 knockdown abrogated the activities of migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in NSCLC cells with KRAS or EGFR mutation, and provides mechanistic insights for the understanding of the potentially contrasting roles of AKT signaling in the regulation of cell invasiveness. Citation Format: Guanhua Rao, Mariaelena Pierobon, In-Kyu Kim, Wei-Hsun Hsu, Jianghong Deng, Yong-Wha Moon, Emanuel F. Petricoin, Yu-Wen Zhang, Yisong Wang, Giuseppe Giaccone. AKT1 signaling negatively regulates invasion and metastasis of non-small cell lung cancer cells carrying mutations in KRAS or EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2017-4893

Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

MARCKS is Necessary for Oligodendrocyte Precursor Cell Maturation.

Oligodendrocyte precursor cell (OPC) development into myelinated oligodendrocytes demands vigorous membrane addition. Since myristoylated alanine-rich C-kinase substrate (MARCKS) reportedly contributes to Ras-associated protein (Rab)-10-associated vesicle insertion into neuronal membranes, we investigated the role of MARCKS in OPC maturation. We found that either knockdown of MARCKS or interruption of its interaction with Rab10 would cause a decrease of the cell membrane area during OPC development. Enhanced MARCKS phosphorylation by Nogo66 or myelin debris treatment inhibited OPC maturation, while its dephosphorylation by protein phosphatase 2A activator D-erythro-sphingosine promoted OPC development in the presence of myelin debris. Our results demonstrated that MARCKS is involved in OPC maturation by interacting with Rab10.

MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection.

MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21−/− macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21−/− macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21−/− BMDMs in response to LPS or Listeria. TNF-α was increased in miR-21−/− BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21−/− cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21−/− macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21−/− BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21−/− mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.

Anti-factor Xa activities of zingerone with anti-platelet aggregation activity

Zingerone (ZGR), a phenolic alkanone found in Zingiber officinale, has been reported to have various pharmacological activities such as anti-inflammatory, anti-apoptotic, and protecting myocardial infarction and irritable bowel disorder. The aim was to identify the unreported bioactive anti-factor Xa (FXa) and anti-platelet activities of ZGR. ZGR was evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity and production, and thrombus formation. ZGR reduced activated partial thromboplastin time and it inhibited the catalytic activity of FXa toward its substrate S-2222 in a noncompetitive inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619 (not thrombin). However, ZGR did not prolong bleeding time in mice, as shown by tail clipping. ZGR also inhibited ADP- and U46619- induced phosphorylation of myristolated alanine-rich C-kinase substrate (MARCKS) and the expressions of P-selectin and PAC-1 in platelets. In an animal model of arterial and pulmonary thrombosis, ZGR showed enhanced antithrombotic effects. ZGR also elicited anticoagulant effects in mice. Our results reveal that ZGR is an antithrombotic compound with both FXa inhibitory and anti-platelet aggregation activities. Collectively, these results show that ZGR could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs.

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study

Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases.In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells.In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker.Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. DisclosuresNo relevant conflicts of interest to declare.

Trypsin and MALDI matrix pre-coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry.

Prefabricated surfaces containing α-cyano-4-hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α-cyano-4-hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography-tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine-rich C-kinase substrate (29.8 kDa) and spectrin alpha chain, non-erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre-coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.

Nonalcoholic Steatohepatitis Modulates Membrane Protein Retrieval and Insertion Processes.

Interindividual variability in drug response in nonalcoholic steatohepatitis (NASH) can be mediated by altered regulation of drug metabolizing enzymes and transporters. Among these is the mislocalization of multidrug resistance-associated protein (MRP2)/Mrp2 away from the canalicular membrane, which results in decreased transport of MRP2/Mrp2 substrates. The exact mechanism of this mislocalization is unknown, although increased activation of membrane retrieval processes may be one possibility. The current study measures the activation status of various mediators implicated in the active membrane retrieval or insertion of membrane proteins to identify which processes may be important in rodent methionine and choline deficient diet-induced NASH. The mediators currently known to be associated with transporter mislocalization are stimulated by oxidative stressors and choleretic stimuli, which play a role in the pathogenesis of NASH. The activation of protein kinases PKA, PKCα, PKCδ, and PKCε and substrates radixin, myristoylated alanine-rich C-kinase substrate, and Rab11 were measured by comparing the expression, phosphorylation, and membrane translocation between control and NASH. Many of the mediators exhibited altered activation in NASH rats. Consistent with membrane retrieval of Mrp2, NASH rats exhibited a decreased phosphorylation of radixin and increased membrane localization of PKCδ and PKCε, thought to be mediators of radixin dephosphorylation. Altered activation of PKCδ, PKA, and PKCα may impair the Rab11-mediated active insertion of Mrp2. Overall, these data suggest alterations in membrane retrieval and insertion processes that may contribute to altered localization of membrane proteins in NASH.

Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors.

PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.

Determinants of Curvature-Sensing Behavior for MARCKS-Fragment Peptides

It is increasingly recognized that membrane curvature plays an important role in various cellular activities such as signaling and trafficking, as well as key issues involving health and disease development. Thus, curvature-sensing peptides are essential to the study and detection of highly curved bilayer structures. The effector domain of myristoylated alanine-rich C-kinase substrate (MARCKS-ED) has been demonstrated to have curvature-sensing ability. Research of the MARCKS-ED has further revealed that its Lys and Phe residues play an essential role in how MARCKS-ED detects and binds to curved bilayers. MARCKS-ED has the added property of being a lower-molecular-weight curvature sensor, which offers advantages in production. With that in mind, this work investigates peptide-sequence-related factors that influence curvature sensing and explores whether peptide fragments of even shorter length can function as curvature sensors. Using both experimental and computational methods, we studied the curvature-sensing capabilities of seven fragments of MARCKS-ED. Two of the longer fragments were designed from approximately the two halves of the full-length peptide whereas the five shorter fragments were taken from the central stretch of MARCKS-ED. Fully atomistic molecular dynamics simulations show that the fragments that remain bound to the bilayer exhibit interactions with the bilayer similar to that of the full-length MARCKS-ED peptide. Fluorescence enhancement and anisotropy assays, meanwhile, reveal that five of the MARCKS fragments possess the ability to sense membrane curvature. Based on the sequences of the curvature-sensing fragments, it appears that the ability to sense curvature involves a balance between the numbers of positively charged residues and hydrophobic anchoring residues. Together, these findings help crystallize our understanding of the molecular mechanisms underpinning the curvature-sensing behaviors of peptides, which will prove useful in the design of future curvature sensors.

Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin.

Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvβ3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvβ3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvβ3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis.

MARCKS contributes to stromal cancer-associated fibroblast activation and facilitates ovarian cancer metastasis.

The Cancer Genome Atlas network has revealed that the ‘mesenchymal’ epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation

AbstractPlatelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a “molecular switch,” binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).

Single Molecule Diffusion of Phosphatidylinositol Bisphosphate (PIP2) Lipids on Asymmetric Lipid Bilayers under the Influence of Polycationic Macromolecules

Proteins binding with PIP2 lipids in the plasma membrane initiate and regulate many cell signaling events. Previous research suggests that proteins interact with PIP2 lipids through electrostatic interactions between polycationic protein motifs and anionic lipid head groups, leading to potential sequestration of multiple PIP2 lipids. The sequestration of PIP2 lipids will cause a change in the lipid mobility and the spatial distribution of PIP2 in the plasma membrane. We set out to understand the molecular structure and dynamics of the protein-PIP2 interface at single molecule level. In this study, a polycationic polymer, quaternized polyvinylpyridine (QPVP), and Myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were chosen to mimic the membrane binding proteins. The behavior of PIP2 lipids in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid.

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine.Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays.Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment.Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. DisclosuresKastelowitz:University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.