CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed.

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.

CircRNA SEPT9 contributes to malignant behaviors of glioma cells via miR-432-5p-mediated regulation of LASP1.

BACKGROUND: Circular RNA (circRNA) septin 9 (circSEPT9; hsa_circ_0005320) has been reported to be abnormally up-regulated in glioma. However, the exact role and working mechanism of circSEPT9 in glioma progression are barely known. METHODS: RNA and protein levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and flow cytometry. Cell apoptosis was evaluated by flow cytometry. Cell motility was analyzed by transwell assays. Cell glycolytic metabolism was analyzed using commercial kits. Dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay were conducted to verify the intermolecular interactions. Xenograft mice model was utilized to assess the role of circSEPT9 in vivo. RESULTS: CircSEPT9 was highly expressed in glioma tissues and cell lines. CircSEPT9 interference inhibited the proliferation, migration, invasion and glycolytic metabolism and triggered the apoptosis of glioma cells. MicroRNA-432-5p (miR-432-5p) was a target of circSEPT9, and circSEPT9 silencing-mediated effects in glioma cells were largely alleviated by the addition of anti-miR-432-5p. MiR-432-5p bound to the 3' untranslated region (3'UTR) of LIM and SH3 protein 1 (LASP1), and LASP1 overexpression largely overturned miR-432-5p-induced effects in glioma cells. CircSEPT9 up-regulated LASP1 expression by acting as miR-432-5p sponge. CircSEPT9 silencing suppressed xenograft tumor growth in vivo. CONCLUSION: CircSEPT9 exerted an oncogenic role to enhance the malignant behaviors of glioma cells by binding to miR-432-5p to induce LASP1 expression.

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.

Bile duct ligation causes opposite impacts on the expression and function of BCRP and P-gp in rat brain partly via affecting membrane expression of ezrin/radixin/moesin proteins.

Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are co-located at blood-brain barrier (BBB) cells, preventing their substrates from entering brain. Accumulating evidence demonstrates that liver failure impairs P-gp and BCRP expression and function in the brain. In the current study, we investigated how liver failure influenced the expression and function of brain BCRP and P-gp in rats subjected to bile duct ligation (BDL). The function of BCRP, P-gp and BBB integrity was assessed using distribution of prazosin, rhodamine 123 and fluorescein, respectively. We showed that BDL significantly decreased BCRP function, but increased P-gp function without affecting BBB integrity. Furthermore, we found that BDL significantly downregulated the expression of membrane BCRP and upregulated the expression of membrane P-gp protein in the cortex and hippocampus. In human cerebral microvascular endothelial cells, NH4Cl plus unconjugated bilirubin significantly decreased BCRP function and expression of membrane BCRP protein, but upregulated P-gp function and expression of membrane P-gp protein. The decreased expression of membrane BCRP protein was linked to the decreased expression of membrane radixin protein, while the increased expression of membrane P-gp protein was related to the increased location of membrane ezrin protein. Silencing ezrin impaired membrane location of P-gp, whereas silencing radixin impaired membrane location of BCRP protein. BDL rats showed the increased expression of membrane ezrin protein and decreased expression of membrane radixin protein in the brain. We conclude that BDL causes opposite effects on the expression and function of brain BCRP and P-gp, attributing to the altered expression of membrane radixin and ezrin protein, respectively, due to hyperbilirubinemia and hyperammonemia.

Expression of lipoma preferred partner in mammary and extramammary Paget disease.

BACKGOUND: This study aims to identify the expression of lipoma preferred partner (LPP) in Paget disease (PD) and to further understand the pathogenesis of PD. METHODS: Tissue microarray was used to evaluate the expression of LPP by immunohistochemistry in 40 PD patients. The results of LPP expression were combined with clinical and histopathological characteristics. Patient files were analyzed retrospectively. RESULTS: Twenty-one cases were mammary Paget disease (MPD) and 19 extramammary Paget disease (EMPD) involving the vulva, scrotum, and penis. LPP was expressed in PD and this expression was significantly greater in MPD versus EMPD (P = .031). The expression of LPP in MPD was significantly related with age (P = .009) and expression of Ki-67 (P = .011). No statistically significant differences were observed in LPP expression as related to sex, body location, and time of PD diagnosis. CONCLUSIONS: While LPP is expressed in both MPD and EMPD, the intensity of this expression is greater in MPD. LPP expression is positively correlated with Ki-67 and is more prevalent in middle-aged versus senior MPD patients. Further research is needed to determine its potential role in tumorigenesis and distribution.

Down-Regulation of AHNAK2 Inhibits Cell Proliferation, Migration and Invasion Through Inactivating the MAPK Pathway in Lung Adenocarcinoma.

AHNAK nucleoprotein 2 (AHNAK2) has been emerged as a crucial protein for neuroblast differentiation and cell migration, thereby involving in the development of various cancers. However, the specific molecular mechanism of AHNAK2 in lung adenocarcinoma is inconclusive. By accessing to the Oncomine dataset and GEPIA website, a higher expression level of AHNAK2 was observed in lung adenocarcinoma tissue samples. Overall survival (OS) curve plotted by Kaplan-Meier method showed that up-regulation of AHNAK2 was related with poor prognosis of lung adenocarcinoma patients. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis and western blot were conducted to examine the expression level of genes in lung adenocarcinoma cells. Through functional in vitro experiments, cell proliferation, migration and invasion were all suppressed after AHNAK2 knockdown using Cell counting kit-8 (CCK-8) assay, wound-healing and transwell analysis. Reduction of AHNAK2 decreased the apoptosis rate using flow cytometry analysis. Moreover, the key markers of MAPK pathway, p-MEK, p-ERK and p-P90RSK were decreased due to the transfection of si-AHNAK2 in A549 cells. U0126, a MEK inhibitor, showed the similar effects on MAPK-related protein levels with si-AHNAK2. To sum up, AHNAK2 is significantly increased in lung adenocarcinoma and plays a carcinogenic role by activating the MAPK signaling pathway, providing a novel insight and raising possibility for lung adenocarcinoma treatment.

LINC01503/miR-342-3p facilitates malignancy in non-small-cell lung cancer cells via regulating LASP1.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown. METHODS: Thirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo. RESULTS: We demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1. CONCLUSION: These results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.

Interleukin-6 affects pacsin3, ephrinA4 expression and cytoskeletal proteins in differentiating primary skeletal myoblasts through transcriptional and post-transcriptional mechanisms.

Interleukin (IL)-6 is a proinflammatory cytokine released in injured and contracting skeletal muscles. In this study, we examined cellular expression of proteins associated with cytoskeleton organization and cell migration, chosen on the basis of microRNA profiling, in rat primary skeletal muscle cells (RSkMC) treated with IL-6 (1 ng/ml) for 11 days. MiRNA microarray analysis and qRT-PCR revealed increased expression of miR-154-3p and miR-338-3p in muscle cells treated with IL-6. Pacsin3 was downregulated post-transcriptionally by IL-6, but not by IGF-I. Ephrin4A protein was increased both in IL-6- and IGF-I-treated myocytes. IL-6, but not IGF-I, stimulated migratory ability of RSkMC, examined in wound healing assay. Alpha-actinin protein was slightly augmented in RSKMC treated with IL-6, similarly to IGF-I. IL-6, but not IGF-I, upregulated desmin in differentiating RSkMC. IL-6 supplementation caused accumulation of alpha-actinin and desmin in near-nuclear area of muscle cells, which was manifested by increased ratio: mean near-nuclear fluorescence/mean peripheral cytoplasm fluorescence of these proteins. We concluded that IL-6, a known proinflammatory cytokine and a physical activity-associated myokine, acting during differentiation of primary skeletal muscle cells, alters expression of nonmuscle-specific miRNAs. This cytokine causes differential effects on pacsin-3 and ephrinA4, through post-transcriptional inhibition and stimulation, respectively. IL-6-exerted modifications of cytoskeletal proteins in muscle cells include both transcriptional (desmin and dynein heavy chain 5) and post-transcriptional activation (alpha-actinin). Moreover, IL-6 augments near-nuclear distribution of cytoskeletal proteins, alpha-actinin and desmin and promotes migration of myocytes. Such effects suggest that IL-6 plays a role during skeletal muscle regeneration, acting through mechanisms independent of regulation of myogenic program.

Immunohistochemical negative expression of ezrin predicts poor prognosis in clear cell renal cell carcinoma.

PURPOSE: To analyze the immunohistochemical expression of ezrin and moesin in clear cell renal cell carcinoma (ccRCC). These proteins, as part of the ezrin-radixin-moesin complex link the cell membrane to the actin cytoskeleton, affecting such processes as cell adhesion, cell survival, cell motility, and signal transduction. Our aim was to examine the impact of their expression on clinical outcomes and survival rates. PATIENTS AND METHODS: Five hundred seventy-five consecutive patients who had been treated surgically for ccRCC in a single center between 1985 and 2016 were selected. A single pathologist reviewed all cases to perform a uniform reclassification and determined the most representative tumor areas for construction of a tissue microarray. RESULTS: Of all ccRCC specimens, 106 (18.3%) were negative for ezrin, and 469 (81.7%) had positive ezrin expression; 16 (2.8%) were negative and 559 (97.2%) were positive for moesin, respectively. Ezrin expression was associated with pT stage (P < 0.001), clinical stage (P = 0.012), synchronic metastasis (P < 0.001), incidental tumors (P = 0.007), and International Society of Urological Pathology histological grade (P = 0.025). There was a correlation between moesin expression and clinical stage (P = 0.027), pT stage (P = 0.025), and pN stage (P = 0.007). Ezrin expression significantly influenced tumor-related deaths. By multivariate analysis, negative ezrin expression was an independent risk factor for disease-specific survival (HR 1.89; 95% CI 1.11-3.20). CONCLUSIONS: Negativity for ezrin in ccRCC patients significantly impacts survival rates. We encourage further prospective studies to analyze ezrin analysis to evaluate its significance in the prognosis of ccRCC.

Rhododenol Activates Melanocytes and Induces Morphological Alteration at Sub-Cytotoxic Levels.

Rhododenol (RD), a whitening cosmetic ingredient, was withdrawn from the market due to RD-induced leukoderma (RIL). While many attempts have been made to clarify the mechanism underlying RIL, RIL has not been fully understood yet. Indeed, affected subjects showed uneven skin pigmentation, but the features are different from vitiligo, a skin hypopigmentary disorder, alluding to events more complex than simple melanocyte cytotoxicity. Here, we discovered that rhododenol treatment reduced the number of melanocytes in a pigmented 3D human skin model, Melanoderm™, confirming the melanocyte toxicity of RD. Of note, melanocytes that survived in the RD treated tissues exhibited altered morphology, such as extended dendrites and increased cell sizes. Consistently with this, sub-cytotoxic level of RD increased cell size and elongated dendrites in B16 melanoma cells. Morphological changes of B16 cells were further confirmed in the immunocytochemistry of treated cells for actin and tubulin. Even more provoking, RD up-regulated the expression of tyrosinase and TRP1 in the survived B16 cells. Evaluation of mRNA expression of cytoskeletal proteins suggests that RD altered the cytoskeletal dynamic favoring cell size expansion and melanosome maturation. Collectively, these results suggest that RD not only induces cytotoxicity in melanocytes but also can lead to a profound perturbation of melanocyte integrity even at sub-cytotoxic levels.

AHNAK Nucleoprotein 2 Performs a Promoting Role in the Proliferation and Migration of Uveal Melanoma Cells.

AHNAK nucleoprotein 2 (AHNAK2) is supposed to participate in calcium signaling and cytoarchitecture by directly interacting with some proteins. Recently, it was identified as a novel candidate oncogene in human tumors. The author's present study aimed to investigate the expression and biological function of AHNAK2 in uveal melanoma (UM). Based on microarray data of 63 UM patients that were downloaded from Gene Expression Omnibus database, the authors found that AHNAK2 expression is higher in UM primary tumor tissues from patients who developed metastases after enucleation than that in UM primary tumor tissues from patients without metastasis after enucleation. On the basis of the data obtained from The Cancer Genome Atlas database, they found that high AHNAK2 expression is closely associated with shorter overall survival time in UM patients. From quantitative reverse transcription polymerase chain reaction analyses, they revealed that the mRNA expression level of AHNAK2 was significantly upregulated in M17 and SP6.5 cell lines compared with that in D78. Functionally, knockdown of AHNAK2 using small interfering RNA in M17 and SP6.5 cells dramatically suppressed cell proliferation, migratory and invasive abilities, as well as inhibited the activation of phosphatidylinositol 3-kinase (PI3K) signaling pathway. Taken together, their results illustrated that AHNAK2 was upregulated in UM and plays a promoting role in the proliferation and migration of UM cells possibly via regulating PI3K signaling pathway.

Damage and Phenotype Change in PC12 Cells Induced by Lipopolysaccharide Can Be Inhibited by Antioxidants Through Reduced Cytoskeleton Protein Synthesis.

The present study investigated changes in cellular phenotype and oxidative stress during the inflammatory response in PC12 cells stimulated by lipopolysaccharide (LPS) and assessed the effects of minocycline, astragalus (AST), and baicalin on inflammation. PC12 cells were exposed to LPS with or without minocycline, AST, or baicalin. Cell viability was measured by a thiazolyl blue tetrazolium bromide (MTT) assay. Contrast and laser confocal microscopy were used to analyze changes in cellular phenotype and cytoskeleton synthesis. Western blotting tested the expression of α7nAChR and vimentin. Inhibitory ratio of superoxide dismutase (SOD) activity and leakage of lactate dehydrogenase (LDH) were detected to evaluate cellular oxidative stress. Results showed that LPS could attenuate PC12 cell viability in a time- and dose-dependent manner, which could be rescued by minocycline. In addition, minocycline could reverse PC12 cell phenotypic change and the synthesis of the mesenchymal cytoskeleton protein vimentin, both induced by LPS. During LPS-initiated inflammation, α7nAChR and vimentin expression were obviously inhibited by minocycline, AST, or baicalin. The inhibitory rate of SOD activity and LDH leakage in PC12 cells were increased by LPS and attenuated significantly when exposed to minocycline, AST, or baicalin. These findings suggest phenotype change, altered cytoskeleton protein synthesis, and oxidative stress are all involved in the inflammatory response in PC12 cells during which α7 nicotinic acetylcholine receptor (α7nAChR) is induced by LPS stimulation. Minocycline, AST, and baicalin have a protective effect against PC12 cell injury, acting as antioxidants and inhibitors of mesenchymal proteins.

Characterization and Transcriptional Activation of the Immediate Early Gene ARC During a Neural Correlate of Classical Conditioning.

Plasticity and learning genes require regulatory mechanisms that have the flexibility to respond to a variety of sensory stimuli to generate adaptive behavioral responses. The immediate early gene (IEG) activity-regulated cytoskeleton-associated protein (ARC) is rapidly induced not only by neuronal stimulation but also during a variety of learning tasks. How ARC is regulated in response to complex stimuli during associative learning remains to be fully detailed. Here, we characterized the structure of the ARC gene in the pond turtle and mechanisms of its transcriptional activation during a neural correlate of eyeblink classical conditioning. The tARC gene is regulated in part by the presence of paused polymerase (RNAPII) that is poised at the promoter for rapid gene induction. Conditioning induces permissive chromatin modifications in the tARC promoter that allows binding by the transcription factor cAMP response element-binding protein (CREB) within 5 min of training. During learning acquisition, the pausing factor negative elongation factor (NELF) dissociates from the promoter thereby releasing RNAPII for active transcription. Data additionally suggest that the DNA insulator protein CCCTC-binding factor (CTCF) is required for transcription by mediating a learning-induced interaction of the ARC promoter with an enhancer element. Our study suggests that the learning-inducible IEG tARC utilizes both paused RNAPII and rapid chromatin modifications that allow for dynamic gene responsiveness required when an organism is presented with a variety of environmental stimuli.

Cortical network dynamics are coupled with cognitive aging in rats.

Changes in neuronal network activity and increased interindividual variability in memory are among the most consistent features of growing older. Here, we examined the relationship between these hallmarks of aging. Young and aged rats were trained on a water maze task where aged individuals reliably display an increased range of spatial memory capacities relative to young. Two weeks later, neuronal activity was induced pharmacologically with a low dose of pilocarpine and control animals received vehicle. Activity levels were proxied by quantifying the immediate early gene products Arc and c-Fos. While no relationship was observed between basal, resting activity, and individual differences in spatial memory in any brain region, pilocarpine-induced marker expression was tightly coupled with memory in all areas of the prefrontal cortex (PFC) and hippocampus examined. The nature of this association, however, differed across regions and in relation to age-related cognitive outcome. Specifically, in the medial PFC, induced activity was greatest in aged rats with cognitive impairment and correlated with water maze performance across all subjects. In the hippocampus, the range of induced marker expression was comparable between groups and similarly coupled with memory in both impaired and unimpaired aged rats. Together the findings highlight that the dynamic range of neural network activity across multiple brain regions is a critical component of neurocognitive aging.

Expression of Protein 4.1 Family in Breast Cancer: Database Mining for 4.1 Family Members in Malignancies.

BACKGROUND The protein 4.1 family is a family of cytoskeletal proteins that play an important role in maintaining normal cell morphology and cell adhesion, migration, division, and intercellular signaling. The main aim of this study was to explore the prognostic significance of the protein 4.1 family in breast cancer (BC) patients and to provide new biomarkers and therapeutic targets for the diagnosis and treatment of BC. MATERIAL AND METHODS The expression of 4.1 family members in various tumor types was compared to normal controls using the ONCOMINE and GOBO databases. The prognostic significance of the 4.1 family in BC patients was determined by Kaplan-Meier Plotter. RESULTS EPB41L2 (4.1G) was expressed at higher levels in normal tissues compared with BC patients for all 4.1 family members. In survival analysis, 4.1G and EPB41 (4.1R) mRNA high expressions were associated with better survival in BC patients. Moreover, 4.1G high expression was significantly associated with longer overall survival (OS) in luminal A and protracted relapse-free survival (RFS) in luminal B subtype BC patients who received Tamoxifen treatment. In addition, high expression of each 4.1 family member also showed better prognostic value in different molecular subtypes of BC. CONCLUSIONS These results indicate that the protein 4.1 family can be regarded as novel biomarkers and potential therapeutic targets for BC. Further research is needed to explore the detailed biological functions.

Upregulation of LASP2 inhibits pancreatic cancer cell migration and invasion through suppressing TGF-ß-induced EMT.

LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-ß (TGF-ß). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-ß-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.

Ezrin Promotes Stem Cell Properties in Pancreatic Ductal Adenocarcinoma.

Self-renewal maintains the long-term clonogenic growth that is required for cancer relapse and progression, but the cellular processes regulating this property are not fully understood. In many diseases, self-renewal is enhanced in cancer stem cells (CSC), and in pancreatic ductal adenocarcinoma (PDAC), CSCs are characterized by the surface expression of CD44. In addition to cell adhesion, CD44 impacts cell shape and morphology by modulating the actin cytoskeleton via Ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family of linker proteins. We examined the expression of Ezrin in PDAC cells and found higher levels of both total and activated Ezrin in CSCs compared with bulk tumor cells. We also found that the knockdown of Ezrin in PDAC cells decreased clonogenic growth, self-renewal, cell migration, and CSC frequency in vitro as well as tumor initiation in vivo. These effects were associated with cytoskeletal changes that are similar to those occurring during the differentiation of normal stem cells, and the inhibition of actin remodeling reversed the impact of Ezrin loss. Finally, targeting Ezrin using a small-molecule inhibitor limited the self-renewal of clinically derived low-passage PDAC xenografts. Our findings demonstrate that Ezrin modulates CSCs properties and may represent a novel target for the treatment of PDAC. IMPLICATIONS: Our findings demonstrate that Ezrin modulates CSCs' properties and may represent a novel target for the treatment of PDAC.

ELMO3 - a Negative Prognostic Marker in Minor Salivary Gland Carcinoma.

Engulfment and cell motility 3 protein (ELMO3) is a protein that is involved in cell migration and promotes the remodeling of the cytoskeleton. Moreover, it is described as a prognostic marker in several cancers. The aim of this study was to evaluate ELMO3 expression in patients with minor salivary gland carcinoma. The expression of ELMO3 was examined by immunohistochemistry. The intensity of staining was evaluated and data was correlated to clinical outcome. Forty-six patients with complete clinical data were included into statistical analysis. ELMO3 expression was observed in 85% of the cases. High staining intensity of ELMO3 correlated with a significantly worse disease free survival (p = .0495) and a higher recurrence rate (p = .0071). In conclusion, it is still difficult to predict the clinical outcome of patients with minor salivary gland carcinoma. Evaluation of ELMO3 might serve as a clinical prognostic marker in future.

Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene.

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF-regulated neuronal IEGs, activity-regulated cytoskeleton-associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain-derived neurotrophic factor (BDNF)-induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity-responsive element (SARE). Regardless of the requirement for the SRF-binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF-mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF-mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation.