Filopodia Formation Research Articles

SOST Inhibits Prostate Cancer Invasion.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Investigation of the changes of biophysical/mechanical characteristics of differentiating preosteoblasts in vitro

BackgroundTopography, stiffness, and composition of biomaterials play a crucial role in cell behaviors. In this study, we have investigated biochemical (gene markers), biophysical (roughness), and biomechanical (stiffness) changes during the osteogenic differentiation of preosteoblasts on gelatin matrices.ResultsOur results demonstrate that gelatin matrices offer a favorable microenvironment for preosteoblasts as determined by focal adhesion and filopodia formation. The osteogenic differentiation potential of preosteoblasts on gelatin matrices is confirmed by qualitative (Alizarin red, von kossa staining, immunofluorescence, and gene expression) and quantitative analyses (alkaline phosphatase activity and calcium content). The biomechanical and biophysical properties of differentiating preosteoblasts are analyzed using atomic force microscopy (AFM) and micro indentation. The results show sequential and significant increases in preosteoblasts roughness and stiffness during osteogenic differentiation, both of which are directly proportional to the progress of osteogenesis. Cell proliferation, height, and spreading area seem to have no direct correlation with differentiation; however, they may be indirectly related to osteogenesis.ConclusionsThe increased stiffness and roughness is attributed to the mineralized bone matrix and enhanced osteogenic extracellular matrix protein. This report indicates that biophysical and biomechanical aspects during in vitro cellular/extracellular changes can be used as biomarkers for the analysis of cell differentiation.

OP52THE ROLE OF DOCK4 IN CANCER CELL INVASION AND ANGIOGENESIS IN BREAST CANCER BRAIN METASTASES

INTRODUCTION: Development of brain metastatic lesions requires activation of signaling pathways for cancer cell invasion. Metastatic disease is intrinsically linked to endothelial cell activation for brain colonization and angiogenic growth for disease progression. DOCK180 family of Rho guanine nucleotide exchange factors (GEFs) are implicated in invasion, and recent studies show they control blood vessel development. The study aims to elucidate the role of the Rac GEF DOCK4 in breast cancer brain metastases. METHOD: i) Patient samples were analysed for DOCK4 expression levels which were related to histological parameters in primary tumours, and expression levels in brain metastases, ii) the role of DOCK4 in the brain vasculature was studied in heterozygous Dock4 null mice and mouse model of breast cancer brain metastasis, iii) the DOCK4 signalling pathway was delineated using biochemical assays and proteomics approaches. RESULTS: i) Significant correlation was observed between DOCK4 expression and histological grade in primary breast tumours, independent of ER and LN status. There was a trend towards reduced survival in cases with moderate/ strong DOCK4 expression. Moderate/ strong DOCK4 expression was observed in brain metastases specimens. ii) There was significant decrease in brain blood vessel lumen size in Dock4 + /- mice and number of blood vessels with dilated lumens in EO771 intracranial tumours in Dock4 + /- mice. iii) DOCK4 interacts with the Cdc42 GEF DOCK9 and RhoG effector ELMO to control filopodia formation and blood vessel lumen morphogenesis. CONCLUSION: DOCK4 and interaction partners are potential targets and biomarkers in brain metastases through regulation of cancer cell invasion and angiogenesis.

Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

BackgroundTranscription of the myristoylated alanine‐rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS‐mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo.Methods and Results MARCKS knockdown blocked platelet‐derived growth factor (PDGF)‐induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane‐associated phosphatidylinositol 4,5‐bisphosphate (PIP 2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP 2, restored membrane‐associated PIP 2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP 2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation.Conclusions MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP 2 at the plasma membrane. This study delineates a novel mechanism for MARCKS‐mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

Abstract IA06: Recycling Myc to survive stress

Abstract Missexpression of Myc family members has been linked to numerous abnormalities ranging from developmental defects to cancer. Indeed there is considerable evidence that Myc expression is controlled at every level of gene regulation, including protein degradation. Recently we found that Myc is targeted by calcium-dependent cytosolic proteases from the calpain family. Unlike proteasomal degradation resulting in complete protein destruction, calpain mediated proteolysis leads to partial cleavage of substrates. While regulated calpain activity is essential for embryonic development and terminal differentiation, aberrant calpain levels and activity have been correlated with tumor development and metastasis. We found that upon calpain cleavage, the C-terminus of Myc is degraded, while an N-terminal segment of about 300 aminoacids is preserved and gives rise to Myc-nick. The removal of the C-terminus of Myc, which is essential for nuclear translocation and to binding to DNA and to Max, renders Myc-nick transcriptionally inactive and cytoplasmically localized. The conversion of Myc into Myc-nick is significantly enhanced in cancer cells exposed to stress conditions such as hypoxia and nutrient deprivation. Importantly, we found that Myc-nick expression increases the survival of colon cancer cells following nutrient deprivation, hypoxia, and treatment chemotherapeutic agents such as etoposide, cisplatin and imatinib. Myc-nick expressing cells display an increase in autophagy and treatment with inhibitors of autophagy prevents cell survival. Therefore, we propose that activation of autophagy is the major mechanism by which Myc-nick promotes cell survival under adverse conditions and drug treatment. The ability of Myc-nick to augment autophagy and cell survival requires its binding to acetyltransferases which correlates with acetylation of cytoplasmic proteins. We find that Myc-nick augments acetylation of ATG3 and alpha-tubulin, which are required in the acetylated form for the migration of autophagosomes and their subsequent fusion with lysosomes. Overexpression of Myc-nick not only promotes the survival of colon cancer cells, but also increases their motility. Importantly, Myc-nick induces an increase in the formation of filopodia and the expression of fascin, which is a robust driver of invasiveness in solid tumors. Myc-nick expressing cells, while highly motile, do not appear to undergo epithelial to mesenchymal transition (EMT). These cells migrate while maintaining their epithelial shape by making use of very distinct filopodia. This is likely to be related to the recently described phenomenon called “collective invasion”, where cancer cells become invasive in the absence of EMT. Our results indicate that Myc-nick-induced survival and motility play important roles in promoting the progression of Myc-dependant tumors. Citation Format: Maralice Conacci-Sorrell, Celine Ngouenet, Sarah Anderson, Robert Eisenman. Recycling Myc to survive stress. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA06.

Lysine acetyltransfer supports platelet function

The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of the identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments showed that platelets express p300, which is activated in an Src kinase-dependent manner upon platelet stimulation with the platelet glycoproteinVI agonist collagen-related peptide (CRP). Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin, and cortactin, as well as F-actin polymerization, integrin activation, and platelet aggregation. Super-resolution visualization of platelet actin-rich adhesion structures revealed abundant acK protein colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodium formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiologic role for lysine acetyltransferase activity in platelet function. Together, our findings reveal lysine acetyltransfer to be a potential regulator of platelet actin dynamics, and potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A.

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

Abstract 520: MIEN1 drives breast cancer invasion by regulating cytoskeletal dynamics

Abstract Introduction: The emergence of metastases remains the primary cause of cancer mortality and morbidity. Active migration of tumour cells is a prerequisite for tumor-cell invasion and metastasis. We identified Migration and Invasion Enhancer 1(MIEN1) as a novel promoter of cell migration/invasion. Previously known as C35, C17orf37, RDX12 and MGC14832, MIEN1 is a novel gene located in the chromosomal region 17q12-21. While absent or low in normal tissues, MIEN1 is abundantly expressed in multiple cancers including breast carcinomas. A membrane-bound signaling adaptor, MIEN1 localizes to the leading edge of migrating cells and promotes migration and invasion by increasing filopodium formation. While MIEN1 is shown to promote motility through filopodia formation, little is known on the mechanisms by which MIEN1 influence the actin cytoskeleton. Exploring underlying mechanisms, we show that MIEN1 associates with the actin cytokeleton during motility and directly influence actin polymerization. Methods: We performed immunofluorescence, migration and invasion assays using breast cancer cell lines, MDA-MB 231 and MCF10CA1a to investigate the mechanisms by which MIEN1 potentiates cell motility. Results: Ablation of MIEN1 inhibited cell migration, cell adhesion and altered the phosphorylation status of motility markers. Immunofluorescence staining with FITC- conjugated-phalladoin confirmed that alteration of MIEN1 expression leads to reconfiguration of the actin cytoskeleton in breast cancer cells. Furthermore we found that MIEN1 associates with cytoskeletal proteins involved in motility processes. Conclusion: Our results show that MIEN1 is a critical regulator of cell migration and invasion. The importance of MIEN1 in tumor cell dissemination is well established; understanding the molecular mechanisms aiding the processes involved will enable the design of novel and effective treatments for metastatic tumors. Citation Format: Marilyne Kpetemey, Jamboor Vishwanatha. MIEN1 drives breast cancer invasion by regulating cytoskeletal dynamics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 520. doi:10.1158/1538-7445.AM2015-520

Abstract 1447: Functional genomics identifying a TIS1-HIF-NOTCH signaling orchestrating epithelial-mesenchymal transition for tumor invasion

Abstract Human hepatocellular carcinoma (HCC) is characterized by frequent local invasion and recurrence, which are strongly associated with poor overall survival of HCC patients. Epithelial-mesenchymal transition (EMT) is an initiating event driving tumour invasion and metastasis. However, the molecular mechanisms of EMT in HCC remains largely unknown. We have performed a human kinome/phosphatome RNAi screen to identify novel genes that are involved in invasion and metastasis of HCC cells using Hep3B, a low motile HCC cell line, and identified TIS1 (Tumor Invasion Suppressor 1) as a potential cell motility suppressor. Mechanistic studies revealed that TIS1 was a novel actin binding protein that sequestrated globular actins and prevented actin filament (F-actin) formation, thereby suppressing the formation of filopodia, lamillepodia and invadopodia. Silencing of TIS1 enhanced EMT and tumor cell migration and invasion. Notably, F-actin polymerization induced by TIS1 silencing led to activation of the NOTCH signalling via induction of HIF1A, which in turn further supports EMT for sustaining tumor invasion and metastasis. Clinically, TIS1 was frequently downregulated in many human cancers including HCC and strongly associated with tumor recurrence, local invasion, and poor prognosis. In conclusion, a novel TIS1-HIF/NOTCH signaling forms a positive feedback loop sustainingly driving mesenchymal transition of cancer cells for invasion and metastasis. This TIS1-mediated tumor invasion and metastasis signaling may serve as therapeutic targets for prevention and treatment of HCC invasion and metastasis. Citation Format: Sen-Yung Hsieh. Functional genomics identifying a TIS1-HIF-NOTCH signaling orchestrating epithelial-mesenchymal transition for tumor invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1447. doi:10.1158/1538-7445.AM2015-1447

Abstract 2644: Inhibition of PAK4 attenuates renal cell carcinoma (RCC) growth

Abstract Renal cell carcinoma (RCC) is an increasingly prevalent cancer type that is frequently asymptomatic on presentation and is associated with poor responses and resistance even to the current targeted therapies. Thus, novel therapeutic approaches to treat this disease are urgently needed. P21-activated kinase 4 (PAK4) is a mediator of filopodia formation and stabilizes β-catenin transcriptional activity. PAK4 lies in a pathway integral to both nephrogenesis and cancer. We hypothesized that inhibitors of the PAK4 signaling pathway would result in salutary effects on RCC. To test this, we evaluated the in vitro response of RCC cells to the specific PAK4 Allosteric Modulators (PAMs; KPT-8752 and KPT-9274). Similar to cells of other cancer types, 786-O (VHL-mutant RCC) showed decreases of both Phospho-PAK4 and Phospho-β-catenin after 24 h of incubation with 10 μM KPT-8752. In addition, both 786-O and ACHN (VHL-wildtype RCC cells) showed a marked time-dependent decrease in cell viability from 24 to 72 h of incubation from 2 to 10 μM of KPT-8752. KPT-9274 was orally administered at 25 and 100 mg/kg twice a day for 5 days/week to Caki-1 (VHL-wildtype RCC) and 786-O subcutaneous nude mouse xenograft models. In these studies the tumor growth rate was attenuated at both doses in the 786-O cells and at the higher dose for Caki-1 cells. There was no obvious change in the health of any of the animals suggesting manageable tolerability. We are currently evaluating combination therapy in our laboratory. In summary, PAK4 inhibitors show considerable promise as novel treatments of RCC as a single agent and warrant further investigation. Citation Format: Omran Abu Aboud, William Senapedis, Yosef Landesman, Erkan Baloglu, Robert H. Weiss. Inhibition of PAK4 attenuates renal cell carcinoma (RCC) growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2015-2644

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones.

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.

Myosin 1b functions as an effector of EphB signaling to control cell repulsion.

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.

The Formin DAAM Functions as Molecular Effector of the Planar Cell Polarity Pathway during Axonal Development in Drosophila.

Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. Here, we establish a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. We show that dDAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, we demonstrate that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of dDAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that dDAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, we propose that the Wnt5/PCP navigation system is linked by dDAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way.

SH2B1 orchestrates signaling events to filopodium formation during neurite outgrowth

Morphogenesis during development is fundamental to the differentiation of several cell types. As neurite outgrowth marks neuritogenesis, formation of filopodia precede the formation of dendrites and axons. While the structure of filopodia is well-known, the initiation of filopodia during neurite outgrowth is not clear. SH2B1 is known to promote neurite outgrowth of PC12 cells, hippocampal and cortical neurons. As a signaling adaptor protein, SH2B1 interacts with several neurotrophin receptors, and regulates signaling as well as gene expression. Our recent findings suggest that SH2B1 can be recruited to the plasma membrane and F-actin fractions by IRSp53. IRSp53 bends plasma membrane and facilitates actin bundling to set the stage for filopodium formation. We further demonstrate that SH2B1-IRSp53 complexes enhance the formation of filopodia, dendrites and dendritic branches of hippocampal and cortical neurons. While the molecular mechanism underlying filopodium initiation is not clear, we propose that SH2B1-neurotrophin interacting sites may mark the putative sites of filopodium initiation.

Possible role of cortactin phosphorylation by protein kinase Cα in actin-bundle formation at growth cone.

Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood. Immunofluorescence microscopy revealed that protein kinase C (PKC) α colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKCα directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles. Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization.

One of the key steps during tumour metastasis is tumour cell migration and invasion, which require actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are the filopodia, which act like cell sensory organs to communicate with the extracellular microenvironment and participate in fundamental cell functions such as cell adhesion, spreading and migration in the three-dimensional environment. Fascin is the main actin-bundling protein in filopodia. Using high-throughput screening, here we identify and characterize small molecules that inhibit the actin-bundling activity of fascin. Focusing on one such inhibitor, we demonstrate that it specifically blocks filopodial formation, tumour cell migration and invasion in vitro, and metastasis in vivo. Hence, target-specific anti-fascin agents have a therapeutic potential for cancer treatment.

Decrease of phosphorylated proto-oncogene CREB at Ser 133 site inhibits growth and metastatic activity of renal cell cancer

Objective: Cyclic-AMP-responsive element-binding protein (CREB) is a proto-oncogenic transcription factor. The authors’ previous reports showed that blocking the CREB binding site at Ser 133 inhibited the expression of target genes, which related to the progression of some tumors. In this study, the authors investigated the role of phosphorylated CREB (pCREB) at Ser133 in renal cell carcinoma (RCC) growth and metastases.Methods: Immunohistochemistry, xenograft model in nude mice, cell proliferation assay, cell invasion/migration assay, fluorescent immunocytochemistry and Western analysis were performed in an immortalized proximal tubule epithelial cell line and clear-cell RCC.Results: The authors’ results showed that knockdown of pCREB inhibited kidney cancer cells growth in vivo. Furthermore, suppression of the pCREB level blunted the capabilities of cell migration and invasion in vitro and was accompanied with significantly decreased expression of MMP-2 and MMP-9, the filopodia formation and epithelial-mesenchymal transition-related proteins. Surprisingly, no changes of expression or location of vimentin were revealed in the experiment. Bioinformatic software explained the possible reason for this is that the promoter of vimentin does not contain the CRE sequence.Conclusions: These data suggest that decreasing the level of pCREB inhibits the growth and metastasis of RCC by targeting the Ser 133 site.

CRMP1 Inhibits Proliferation of Medulloblastoma and Is Regulated by HMGA1.

Many facets of the tumor biology of medulloblastoma (MB) have not been fully elucidated. Collapsin response mediator protein 1 (CRMP1) is a member of cytoplasmic family of proteins that regulate the development of central nervous system. Recent studies demonstrated that CRMP1 could function as an invasion suppressor. We reported previously that high mobility group AT-hook 1 (HMGA1) contributed to development of MB and regulated its growth and migration/invasion. Transcriptional profiling and quantitative RT-PCR revealed increased expression of CRMP1 in HMGA1-depleted cells, suggesting that CRMP1 may be a downstream target of HMGA1 in MB. In this study, we showed HMGA1 can bind CRMP1 promoter by chromatin immunoprecipitation (ChIP) assay. Luciferase assay demonstrated a marked enhancement of CRMP1 transcription activity in HMGA1-depleted cells. Furthermore, quantitative RT-PCR revealed a negative correlation between HMGA1 and CRMP1 in 32 MB samples. To investigate the biological roles of CRMP1 in MB pathogenesis, we established MB clones stably expressing CRMP1. Functional analysis revealed that expression of CRMP1 significantly inhibited proliferation, migration, invasion and formation of filopodia and intense stress fiber of MB cells. Our data suggest that HMGA1 regulates CRMP1 expression and CRMP1 is implicated in MB pathogenesis.

MIEN1 promotes oral cancer progression and implicates poor overall survival

Oral squamous cell carcinoma is a highly malignant tumor with the potential to invade local and distant sites and promote lymph node metastasis. Major players underlying the molecular mechanisms behind tumor progression are yet to be fully explored. Migration and invasion enhancer 1 (MIEN1), a novel protein overexpressed in various cancers, facilitates cell migration and invasion. In the present study we investigated the expression and role of MIEN1 in oral cancer progression using an in vitro model, patient derived oral tissues and existing TCGA data. Expression analysis using immortalized normal and cancer cells demonstrated increased expression of MIEN1 in cancer. Assays performed after MIEN1 knockdown in OSC-2 cells showed decreased migration, invasion and filopodia formation; while MIEN1 overexpression in DOK cells increased these characteristics and also up-regulated some Akt/NF-κB effectors, thereby suggesting an important role for MIEN1 in oral cancer progression. Immunohistochemical staining and analyses of oral tissue specimens, collected from patients over multiple visits, revealed significantly more staining in severe dysplasia and squamous cell carcinoma compared to mildly dysplastic or hyperplastic tissues. Finally, this was corroborated with the TCGA dataset, where MIEN1 expression was not only higher in intermediate and high grade cancer with significantly lower survival but also correlated with smoking. In summary, we demonstrate that MIEN1 expression not only positively correlates with oral cancer progression but also seems to be a critical molecular determinant in migration and invasion of oral cancer cells, thereby, playing a possible role in their metastatic dissemination.

Short-term withdrawal from developmental exposure to cocaine activates the glucocorticoid receptor and alters spine dynamics

Although glucocorticoid receptors (GRs) contribute to the action of cocaine, their role following developmental exposure to the psychostimulant is still unknown. To address this issue, we exposed adolescent male rats to cocaine (20mg/kg/day) from post-natal day (PND) 28 to PND 42 and sacrificed them at PND 45 or 90. We studied the medial prefrontal cortex (mPFC), a brain region that is still developing during adolescence. In PND 45 rats we found enhanced GR transcription and translation as well as increased trafficking toward the nucleus of the receptor, with no alteration in plasma corticosterone levels. We also showed reduced expression of the GR co-chaperone FKBP51, that normally keeps the receptor in the cytoplasm, and increased expression of Src1, which cooperates in the activation of GR transcriptional activity, revealing that short withdrawal alters the finely tuned mechanisms regulating GR action. Since activation of GRs regulate dendritic spine morphology, we next investigated spine dynamics in cocaine-withdrawn rats. We found that PSD95, cofilin and F-actin, molecules regulating spine actin network, are reduced in the mPFC of PND 45 rats suggesting reduced spine density, confirmed by confocal imaging. Further, formation of filopodia, i.e. the inactive spines, is enhanced suggesting the formation of non-functional spines. Of note, no changes were found in molecules related to GR machinery or spine dynamics following long-term abstinence, i.e. in adult rats (PND 90). These findings demonstrate that short withdrawal promotes plastic changes in the developing brain via the dysregulation of the GR system and alterations in the spine network.