CRMP4 Expression Research Articles

Effects of esketamine on depression-like behavior and dendritic spine plasticity in the prefrontal cortex neurons of spared nerve injury-induced depressed mice.

The present study utilized the spared nerve injury (SNI) to create a mouse model of depression to investigate the impact of esketamine on depressive-like behaviors, on the expression of PSD-95 and CRMP2 proteins, and on changes in neuronal dendritic spine plasticity in the prefrontal cortex (PFC). Depressive-like behavioral tests were performed 1 h after esketamine treatment, and the PFC tissues were obtained on the fourth day after completing the behavioral tests. Then, dendritic spine density and morphology in the PFC were measured using Golgi staining, and CRMP2 and PSD-95 proteins were obtained from PFC tissue by western blotting. The results of this study showed that esketamine significantly increased the immobility time in the forced swimming test and tail suspension test. In the open field test, esketamine increased the time spent in the open arms, the time spent in the central area, and the total distance covered. It also increased the protein expression levels of CRMP2 and PSD-95 in addition to the total and mature dendritic spine density of the PFC in SNI-depressed mice. Esketamine can significantly improve depression-like behaviors in SNI-depressed mice and promote an increase in dendritic spine density and maturation in the PFC. These effects may be associated with changes in CRMP2 and PSD-95 expression.

Elevated Hippocampal CRMP5 Mediates Chronic Stress-Induced Cognitive Deficits by Disrupting Synaptic Plasticity, Hindering AMPAR Trafficking, and Triggering Cytokine Release

Chronic stress is a critical risk factor for developing depression, which can impair cognitive function. However, the underlying mechanisms involved in chronic stress-induced cognitive deficits remain unclear. Emerging evidence suggests that collapsin response mediator proteins (CRMPs) are implicated in the pathogenesis of psychiatric-related disorders. Thus, the study aims to examine whether CRMPs modulate chronic stress-induced cognitive impairment. We used the chronic unpredictable stress (CUS) paradigm to mimic stressful life situations in C57BL/6 mice. In this study, we found that CUS-treated mice exhibited cognitive decline and increased hippocampal CRMP2 and CRMP5 expression. In contrast to CRMP2, CRMP5 levels strongly correlated with the severity of cognitive impairment. Decreasing hippocampal CRMP5 levels through shRNA injection rescued CUS-induced cognitive impairment, whereas increasing CRMP5 levels in control mice exacerbated memory decline after subthreshold stress treatment. Mechanistically, hippocampal CRMP5 suppression by regulating glucocorticoid receptor phosphorylation alleviates chronic stress-induced synaptic atrophy, disruption of AMPA receptor trafficking, and cytokine storms. Our findings show that hippocampal CRMP5 accumulation through GR activation disrupts synaptic plasticity, impedes AMPAR trafficking, and triggers cytokine release, thus playing a critical role in chronic stress-induced cognitive deficits.

CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy

As a hallmark of epilepsy, mossy fiber sprouting was regarded as an ideal mode to study neural rewiring upon injury. The process of mossy fiber sprouting constitutes key steps for neural circuit formation, including axon collateral formation and outgrowth, reversed pathfinding and synapse connection. The canonical function of CRMP2 is to promote neurite/axon outgrowth via binding to tubulin heterodimers, which is mainly regulated by its phosphorylation state. CRMP2 expression and phosphorylation were reported to change in medial temporal epilepsy patients and animal modes of epilepsy. As a novel anti-epilepsy drug, Lacosamide is able to impair CRMP2 mediated tubulin polymerization. Previous studies suggested possible roles of CRMP2 in mossy fiber sprouting. Here, we provide direct evidence to support the role of CRMP2 in the process of mossy fiber sprouting in an animal model of epilepsy. We found that CRMP2 phosphorylation was downregulated specifically in the hippocampus during latent phase of epileptic rats. In addition, with the reduction of CRMP2 expression levels in dentate gyrus by CRMP2 shRNA, we observed decreased mossy fiber sprouting in these CRMP2 knockdown rats. Our results demonstrated that CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy.

GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3β in T-cell motility. Inhibition of GSK3β by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3β, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3β activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3β-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3β in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3β inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3β controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats

Ethnopharmacological relevanceTrillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. Aim of the studyWe aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. MethodsMale Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. ResultsTSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3β, and down-regulated phosphorylated β-catenin and CRMP-2 expression. ConclusionTaken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/β-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

Long-term maternal separation potentiates depressive-like behaviours and neuroinflammation in adult male C57/BL6J mice

Early life experience is closely related to depression caused by stress in adulthood. Early life experience, including maternal separation (MS), has been shown to evoke stress sensitivity to depression upon re-exposure to stress in adults. However, MS has also been shown to lead to resilience to stress-induced depression, which is contradictory and rarely studied. To investigate the effects of MS on depression in adults and the related mechanism, male C57/BL6J mouse pups were exposed to different MS procedures from postnatal day (PD)1 to PD21. Body weight (BW) measurements and behavioural tests (the forced swimming test (FST) and open field test (OFT)) were performed on PD41 to explore depressive and anxiety-like behaviours. Then, as adults, the mice were exposed to chronic unpredictable mild stress (CUMS) for 28 days, and then behavioural tasks were recorded. After CUMS exposure, the mice in the MS180 group (which were separated from their mothers for 3 h on PD1-PD21) showed significantly decreased time spent in the centre of the open field and reduced velocity in the OFT, a reduced latency to immobility in the FST, and decreased BW. However, the mice in the MS15 group (which were separated from their mothers for 15 min on PD1-PD21) performed similarly to NSNC mice (which were not separated from their mothers) in the behavioural tests. We further found that the expression of Iba1, a marker of neuroinflammation, was increased in the MS180 group but not in the MS15 group. In addition, our study showed decreased mRNA and protein expression of CRMP2, an important neuroprotective factor, in the MS180 group, but CRMP2 expression was unchanged in the MS15 group. This study confirmed the generation of different behavioural responses to stress exposure in adulthood due to different degrees of MS. Neuroinflammation and neuroprotection are involved, which requires further research.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

CRMP4a suppresses cell motility by sequestering RhoA activity in prostate cancer cells

ABSTRACT Objectives: Distant metastasis is a critical factor for cancer-associated death. Our previous studies identified collapsin response mediator protein 4a (CRMP4a) as a metastasis suppressor in prostate cancers. Enhancing CRMP4 expression by promoter-targeted small activating RNAs reduced cell migration in vitro and abolished distal metastasis in mouse xenograft models. In this study, we investigated the mechanism for CRMP4a-mediated suppression of cell migration. Methods: PC-3 cells were stably infected with lentiviruses expressing CRMP4a cDNA or a shRNA sequence. Cytoskeletal organization was analyzed by measuring cellular focal adhesion area and number, percentage of cell area and lamellipodia numbers after phalloidin staining or anti-vinculin immunocytofluorescent staining. Cell migration was evaluated with TranswellTM chambers coated with MatriGel. RhoA activation was determined with a Rhotekin RBD agarose bead-based assay kit. Lentiviruses harboring RhoA-Q63L or RhoA-T19N mutant constructs were used to overexpress mutant RhoA proteins. Results: CRMP4a overexpression largely reduced while CRMP4a knockdown remarkably increased cytoskeletal organization in PC-3 cells. CRMP4a immunoprecipitation pulled down RhoA but not cdc42 or Rac1 proteins. Manipulating CRMP4a expression levels reversely altered active RhoA levels. Overexpression of RhoA active (Q63L) but not inactive (T19N) mutants reversed CRMP4a-mediated reduction of cancer cell migration while RhoA inhibitor Rhosin diminished CRMP4a shRNA-induced increase of cancer cell migration. CRMP4a overexpression also largely reduced cell spreading that was abolished by overexpressing RhoA active mutant. Conclusion: Our data demonstrated that CRMP4a interacts with RhoA and sequesters its activity, resulting in suppression of cytoskeletal organization, cell migration and spreading.

Hippocampal neuronal viability via CRMP-2 expression is mediated by PI3K-mTOR-S6K signaling pathways

Hippocampal neuronal viability via CRMP-2 expression is mediated by PI3K-mTOR-S6K signaling pathways

Human CRMP4 mutation and disrupted Crmp4 expression in mice are associated with ASD characteristics and sexual dimorphism

Autism spectrum disorders (ASD) are more common among boys than girls. The mechanisms responsible for ASD symptoms and their sex differences remain mostly unclear. We previously identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sex-different expression during sexual differentiation of the hypothalamic sexually dimorphic nucleus. This study investigated the relationship between the sex-different development of autistic features and CRMP4 deficiency. Whole-exome sequencing detected a de novo variant (S541Y) of CRMP4 in a male ASD patient. The expression of mutated mouse CRMP4S540Y, which is homologous to human CRMP4S541Y, in cultured hippocampal neurons derived from Crmp4-knockout (KO) mice had increased dendritic branching, compared to those transfected with wild-type (WT) Crmp4, indicating that this mutation results in altered CRMP4 function in neurons. Crmp4-KO mice showed decreased social interaction and several alterations of sensory responses. Most of these changes were more severe in male Crmp4-KO mice than in females. The mRNA expression levels of some genes related to neurotransmission and cell adhesion were altered in the brain of Crmp4-KO mice, mostly in a gender-dependent manner. These results indicate a functional link between a case-specific, rare variant of one gene, Crmp4, and several characteristics of ASD, including sexual differences.

PI3K-mTOR-S6K Signaling Mediates Neuronal Viability via Collapsin Response Mediator Protein-2 Expression.

Collapsin response mediator protein (CRMP)-2 and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are associated with common physiological functions such as neuronal polarity, axonal outgrowth and synaptic strength, as well as various brain disorders including epilepsy. But, their regulatory and functional links are unclear. Alterations in CRMP-2 expression that lead to its functional changes are implicated in brain disorders such as epilepsy. Here, we investigate whether changes in CRMP-2 expression, possibly regulated by mTOR-related signaling, correlates with neuronal growth and viability. Inhibition of mTOR and/or phosphoinositol-3-kinase (PI3K) led to deceased p-S6K, and p-S6 signals also reduced CRMP-2 expression. These changes corresponded to inhibition of neuronal viability and proliferation in cultured hippocampal HT-22 cells under both basal serum-free and serum- or insulin-induced mTOR pathway-activated conditions. CRMP-2 expression tended to be increased by mTOR activation, indicated by an increase in p-S6/S6 level, in pentylentetrazole (PTZ)-induced epileptic rat hippocampal tissues was also significantly reduced by mTOR inhibition. Knockdown of CRMP-2 by si-RNA reduced the neuronal viability without changes in mTOR signaling, and overexpression of CRMP-2 recovered the glutamate-induced neurotoxicity and decrease of mTOR signaling in HT-22 cells. In conclusion, CRMP-2 protein expression controlled by the PI3K-mTOR-S6K signaling axis exerts its important functional roles in neuronal growth and survival.

MicroRNA-130a is an oncomir suppressing the expression of CRMP4 in gastric cancer.

Gastric cancer is one of the most common causes of death worldwide, although its incidence has steadily declined in recent years. There is strong evidence that aberrantly expressed microRNAs (miRNAs) are involved in gastric cancer tumorigenesis. Furthermore, CRMP4 is closely associated with the occurrence and development of gastric cancer, and our predictions suggest that miR-130a, which can promote gastric cancer tumorigenesis, is a potential CRMP4 regulator. In this study, we investigated the expression of CRMP4 and miR-130a in human gastric cancer cell lines by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB) examination and direct interactions between miR-130a and CRMP4 by dual-luciferase reporter assay. We also evaluated the biological roles of miR-130a and CRMP4 in gastric cancer cells by flow cytometry, MTT assay, soft agar colony formation assay, and Transwell tests and confirmed CRMP4 function in vivo, using a tumor xenograft model. Our results demonstrated that CRMP4 expression was significantly decreased at both the gene and protein levels, while miR-130a expression was notably increased, in five human gastric cancer cell lines compared with human gastric epithelial cells. Dual-luciferase reporter assays indicated that CRMP4 was the direct target of miR-130a. Moreover, an inverse regulatory relationship between miR-130a and CRMP4 was verified by qRT-PCR and WB, and overexpression of miR-130a in BGC823 cells enhanced apoptosis and cell proliferation, arrested the cell cycle in G0/G1, and facilitated cell colony formation, invasion, migration, and adhesion, while upregulation of CRMP4 had opposite effects. Finally, the growth and weight of transplanted tumors derived from BGC823 cells in which CRMP4 was knocked down were remarkably reduced. These data indicate that miR-130a is an oncomir targeting CRMP4 and could be developed as a potential prognostic factor and a novel therapeutic target in gastric cancer.

Suppression of Prostate Cancer Metastasis by DPYSL3-Targeted saRNA.

Metastasis is the sole cause of cancer death and there is no curable means in clinic. Cellular protein CRMP4 (DPYSL3 gene) was previously defined as a metastasis suppressor in human prostate cancers since its expression is dramatically reduced in lymphatic metastatic diseases and DPYSL3 overexpression in prostate cancer cells significantly suppressed cancer cell migration and invasion. To develop a CRMP4-based antimetastasis therapeutic approach, the small activating RNA (saRNA) technique was utilized to enhance CRMP4 expression in prostate cancer cells. A total of 14 saRNAs were synthesized and screened in multiple prostate cancer cell lines. Two saRNAs targeting the isoform-2 promoter region were determined to have significant activating effect on DPYSL3 gene expression at the mRNA and protein levels. These saRNA also largely reduced prostate cancer cell migration and invasion in vitro and in vivo. Most significantly, PSMA aptamer-mediated prostate cancer cell homing of these saRNAs blocked distal metastasis in an orthotopic nude mouse model. In conclusion, our data demonstrated that saRNA-based DPYSL3 gene enhancement is capable of suppressing tumor metastasis in prostate cancer, which provides a potential therapeutic approach for cancer management.

VEGF promotes gastric cancer development by upregulating CRMP4.

This study aimed to investigate the precise role of CRMP4 in gastric tumor growth and patient survival. The mRNA and protein expression levels of CRMP4, VEGF and VEGFR2 were validated by qRT-PCR and immunohistochemistry. We investigated the effects on tumor growth of overexpression and knockdown of CRMP4 both in vitro and in vivo by constructing stable gastric cell lines using lentiviral-mediated transduction and shRNA interference-mediated knockdown of CRMP4 expression. We further validated the role of the ERK/AKT signaling pathways in VEGF and CRMP4 expression using ERK and PI3K inhibitors. Increased expression of VEGF and CRMP4 were observed in gastric cancer tissues compared with tumor-adjacent tissue. We found that higher CRPM4 expression was associated with lymph node metastasis, TNM stage, tumor differentiation and poorer prognosis in gastric cancer patients. In HGC27 and SGC7901 gastric cancer cells, VEGF upregulated CRMP4 in time and dose-dependent manners. Overexpression of CRMP4 increased cell proliferation, migration and invasion, whereas knockdown of CRMP4 expression had opposite effects. VEGF activated CRMP4 expression in gastric cancer cells, and this effect was significantly inhibited by MAPK and PI3K inhibitors (PD98059 and LY294002). In mice, CRMP4 overexpression also resulted in increased tumor growth. These results suggest that increased CRMP4 expression mediated by the activation of VEGF signaling facilitates gastric tumor growth and metastasis, which may have clinical implications associated with a reduced survival rate in gastric cancer patients.

Effects of sevoflurane on dendritic development and the expression of collapsin response mediator proteins in the hippocampus of developing rats

Objective To investigate the effects of sevoflurane (Sevo) on dendritic development and the expression of collapsin response mediator proteins (CRMP) in the hippocampus of developing rats. Methods Twenty-four neonatal Sprague Dawley (SD) rats at postnatal day 7 (P7) were randomly divided into control group or sevoflurane group (12 rat pups for each group). Rats in the control group were exposed to air for 4 h, whereas rats in the sevoflurane group were exposed to 2.8% sevoflurane for 4 h. The hippocampus of some rats were collected, and the expressions of CRMP1, CRMP2 and CRMP4 proteins and phosphorylation of CRMP2 protein at Ser522, Thr514 and Thr555 were detected by Western blot 6h after exposure (n=6). The rest rats were housed till P30, the expression of CRMP1, CRMP2 and CRMP4 proteins in the hippocampus were detected by Western blot (n=6) and the morphology changes of dendrites in the dentate gyrus (DG) of hippocampal neurons were detected by Golgi-Cox Staining (n=6). Results The expression of CRMP1, CRMP2 and CRMP4 proteins of rats at P7 in the sevoflurane group was decreased by 35.0% (P=0.004), 27.5% (P=0.015) and 12.0% (P=0.003), respectively, and the phosphorylation of CRMP2 protein at Ser522 and Thr514 in the sevoflurane group were increased by 68.3% (P<0.01), 74.5% (P<0.01), respectively, 6 h after exposure compared with control rats. However, the phosphorylation of CRMP2 protein at Thr555 was not significantly changed after sevoflurane exposure. At P30, both total dendrite length (P=0.001) and the dendrites length at level 2 and 3 (P=0.033, P<0.01, respectively) were shorter and the dendritic branching at 120, 140 and 160 μm rings in Sholl analysis were less (P=0.009, P=0.028, P=0.048, respectively) for rats in the sevoflurane group, compared with control rats. There were no significant changes at the expressions of CRMP1, CRMP2 and CRMP4 proteins. Conclusion Sevoflurane inhibits the development of dendrites in the hippocampal DG area of developing rats, which may be related to inhibition of CRMP1, CRMP2 and CRMP4 proteins expression and hyperphosphorylation of CRMP2 Ser522 and Thr514. Key words: Sevoflurane; Dendrite; Collapsin response mediator protein; Hippocampus

The Role of p53 in the Proliferation and Differentiation of Neural Stem Cells in Organotypic Cultures of Mouse Hippocampus

We report here our studies of the effects of p53 on the proliferation and differentiation of neural stem cells (NSC) in organotypic hippocampal cultures from adult mice. The role of p53 was assessed experimentally by using the p53 inhibitor pifi thrin-alpha (PFT) and the p53 activator tenovin-1 (TEN). The results show that administration of PFT had no effect on the number of phosphohistone H3-immunopositive cells in the subgranular zone of the hippocampus. This is evidence that inhibition of p53 did not alter the level of NSC proliferation. Administration of TEN, conversely, increased the proliferative activity of NSC. Analysis of Pim-1 and Phb1 proteins, which are involved in regulating the cell cycle, showed that activation of p53, which activated proliferation, also increased the Pim-1 level. These results are consistent with published data and provide evidence that Pim-1 positively regulates the cell cycle in NSC. In contract to Pim-1, Phb1 had antiproliferative activity. Our results indicated that TEN inhibits Phb1 expression. Administration of PFT initially led to an increase in the Phb1 level, which was followed by a sharp drop, with no change in the level of proliferation. Thus, the increase in the level of proliferation seen here in response to administration of TEN was partially dependent on inhibition of the antiproliferative function of Phb1. These studies demonstrate that both TEN and, to a greater extent, PFT stimulated neuronal differentiation, activating CRMP-2 expression, but had no effect on gliogenesis. These results show that p53 is an important factor in the regulation of neuronal differentiation, which is probably mediated by proteins associated with cell cycle regulation, such as Pim-1 and Phb1.

Abstract 4063: Potential targets of PTP4A3 involved in uveal melanoma migration

Abstract Uveal melanoma (UM) is the most frequent eye cancer in the adulthood and it represents 4-5% of all melanomas. Although at diagnosis over 95% of patients have disease limited to the eye, about 50% will develop metastases after a median time of three years. From transcriptomic study, we identified PTP4A3/PRL3 level as an efficient predictor of patient's survival. Overexpression of PTP4A3 in UM OCM-1 increased both cell migration and invasiveness. We then got interested in identifying downstream targets of PTP4A3 and more precisely proteins linked to cytoskeleton. We first highlighted collapsin Response Mediator Protein (CRMP2) by 2-D electrophoresis of phosphorus-enriched UM OCM1 stably-expressing PTP4A3 or an inactive catalytic mutant of PTP4A3 (C104S). CRMP2 was dephosphorylated in presence of PTP4A3. CRMP2 expression in UM is inversely correlated with that of PTP4A3, suggesting an antagonistic role of these proteins in tumor progression. Through the use of shRNA in OCM-1, the downregulation of CRMP2 led to an increase in cell migration of PTP4A3-expressing OCM1. In addition, we focused on ATIP3, a protein encoded by the gene MTUS1, which binds to microtubules. In breast cancer, MTUS1 expression was shown to be downregulated in highly proliferative breast carcinomas of poor clinical outcome. Similarly, our transcriptomic analysis showed that expressions of PTP4A3 and MTUS1 were anti-correlated, suggesting that the reduction of MTUS1 gene and ATIP3 expression in UM would be of bad prognosis. In our OCM1 cells, we demonstrated that ATIP3 overexpression blocks proliferation however effect of ATIP3 overexpression on cell migration still need to be elucidaded.In conclusion, CRMP2 and ATIP3 may play a role in the metastatic process of UM tumors expressing high levels of the phosphatase. Citation Format: Geraldine Liot, Oceane Anezo, Cecile Laurent, Selma Maacha, Nathalie Planque, Simon Saule. Potential targets of PTP4A3 involved in uveal melanoma migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2014-4063

Collapsin response mediator protein 2 is involved in regulating breast cancer progression

Altered expression of collapsin response mediator proteins (CRMPs) has been reported in several malignant tumors, including downregulation of CRMP1 in lung cancer and upregulation of CRMP2 in colorectal cancer. This study aimed to investigate the relationship between CRMP expression and clinicopathological characteristics in patients with breast cancer. Twenty-two breast cancer and four normal breast tissues were used to assess CRMP mRNA expression. The average expression level of each CRMP (CRMP1-5) mRNA was analyzed in a subset of breast cancer specimens and compared with that in normal breast tissue by real-time quantitative reverse-transcription polymerase chain reaction. Furthermore, 173 breast cancer specimens and matching normal breast controls were used for immunohistochemistry based on the tissue microarray technique. Levels of CRMP2 and phosphorylated CRMP2 protein were assessed, and possible correlations between the clinicopathological characteristics were evaluated. The expression of CRMP2 mRNA was significantly decreased in breast cancer tissues, while that of the other CRMPs was similar between normal and breast cancer tissues. Immunohistochemistry revealed that CRMP2 protein expression was also decreased in breast cancer tissues (P < 0.001). Phosphorylated CRMP2 was observed in the nuclei of breast cancer cells but not in normal mammary cells (P < 0.001). Furthermore, nuclear phosphorylated CRMP2 expression was increased in proportion to the histological grade and triple-negative subtype. Reduced CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 may be associated with breast cancer progression.

Targeting of CRMP-2 to the Primary Cilium Is Modulated by GSK-3β

CRMP-2 plays a pivotal role in promoting axon formation, neurite outgrowth and elongation in neuronal cells. CRMP-2′s role in other cells is unknown. Our preliminary results showed CRMP-2 expression in cilia of fibroblasts. To localize CRMP-2, define its role and study the regulation of CRMP-2′s expression in cilia we carried out the following experiments. We find that in fibroblasts CRMP-2 localizes to the centrosome and is associated with the basal body and -at a low level- is present in primary cilia. Phosphorylated pCRMP-2 can only be detected at the basal body. RNAi knockdown of CRMP-2 interfered with primary cilium assembly demonstrating a critical requirement for CRMP-2. Deletion analysis of CRMP-2 identified a 51 amino acid sequence in the C-terminus that is required for targeting to the basal body and primary cilium. This domain contains GSK-3β phosphorylation sites as well as two repeats of the VxPx motif, previously identified as a cilium targeting signal in other primary cilium proteins. To our surprise, mutation of the CRMP-2 VxPx motifs did not eliminate primary cilium targeting. Instead, mutation of the GSK-3β phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3β inhibitor, or with two specific GSK-3β inhibitors (the L803-mts peptide inhibitor and CHIR99021) resulted in cilium elongation and decreased basal body levels of pCRMP-2 as well as increased levels of total CRMP-2 at the primary cilium. In summary, we identified CRMP-2 as a protein critically involved in primary cilia formation. To our knowledge this is the first demonstration of modulation of primary cilium targeting by GSK-3β.

Collapsin Response Mediator Protein 4 Expression is Associated with Liver Metastasis and Poor Survival in Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with one of the worst mortality rates of all cancers. Recently, collapsin response mediator proteins (CRMPs) were reported to be associated with proliferation, apoptosis, differentiation, and invasion in several cancers. However, CRMP expression and their role in pancreatic cancer have not been investigated. This study aimed to clarify the clinical significance of CRMPs in pancreatic cancer. Expression of crmp genes in 11 pairs of pancreatic cancer and corresponding noncancerous pancreas tissues were examined by real-time RT-PCR. Knockdown of CRMP4 expression using siRNA was examined in pancreatic cancer cell lines to determine whether CRMP4 regulates cell proliferation and invasion in vitro. Furthermore, CRMP4 protein levels in primary tumors of pancreatic cancer (n = 53) were examined by immunohistochemistry and compared with the clinicopathological features of the tumors. Of all the CRMPs, only CRMP4 was differentially expressed in pancreatic cancer tissues (p = 0.008). CRMP4 knockdown using siRNA reduced cellular invasion, but did not affect proliferation. The expression of CRMP4 was detected immunohistochemically in 34 (64.2 %) of the 53 pancreatic cancer samples, and CRMP4 expression was correlated with severe venous invasion (p = 0.044), stage (p = 0.019), and liver metastasis (p = 0.021). Multivariate analyses suggested that venous invasion and CRMP4 overexpression were prognostic factors for survival. Our results suggested that CRMP4 is significantly associated with poor prognosis by promoting liver metastasis and can serve as a novel therapeutic target for pancreatic cancer.